enzymatic synthesis of glycerol l-phosphate (d-a=glycerophosphate)

THE JOURNAL OF Bm~oorca~ CI~EMISTRV Vol. 243, No. 8, Issue of April 25, pp. 1823-1832, 1968

Prinied in U.S.A.

Enzymatic Synthesis of Glycerol l-Phosphate

(D-a=Glycerophosphate)

INORGANIC PYROPHOSPHATE-GLYCEROL PHOSPHOTRANSFERASE AND INORGANIC PYROPHOS- PHATE-GLUCOSE PHOSPHOTRANSFERASE COMPARED*

(Received for publication, October 19, 1967)

MARJORIE R. STETTEN AND DAVID ROUNBEHLER

From the Department of Medicine, Rutgers Medical School, New Brunswick, New Jersey 08903

SUMMARY

“Microsomal” particulate fractions of rat organ homogenates are capable of catalyzing the synthesis of glycerol l-phosphate’ from inorganic pyrophosphate and glycerol. The product has been isolated and characterized by elementary analysis, specific rotation, and chemical and enzymatic studies. PPi-glycerol phosphotransferase is pre- sumably specific for the primary carbinol group of glycerol opposite to that which is phosphorylated by ATP-glycerol phosphotransferase (glycerol kinase).

The distribution and some of the kinetic properties of PPi-glycerol phosphotransferase have been studied and compared with those of PPi-glucose phosphotransferase, which catalyzes the synthesis of glucose 6-phosphate. The two activities are roughly parallel in liver, kidney, and intestinal mucosa, and glucose is a competitive inhibitor of the synthesis of glycerol l-phosphate by these organ preparations. Spleen, brain, and lung preparations catalyze the phosphorylation of glycerol from PPi while they are completely incapable of glucose 6-phosphate synthesis. In these organs glucose does not inhibit the synthesis of glycerol l-phosphate. This suggests that there are at least two transferase enzymes which phosphorylate glycerol, one of which may be the same as the liver PPi-glucose phospho-

* This investigation was supported by Grant AM-07279 from the National Institutes of Health, United States Public Health Service.

1 In accordance with the recommendations made in the report of the ITJPAC-IUB Commission on Biochemical Nomenclature of __ _..- --~~~~ Lipids (10) the stereospecific numbering for glycerol derivatives is used in this paper. Thus glycerol l-phosphate (equivalent to sn-glycerol I-(dihydrogen)hosphate or sn-glycero l-phosphoric acid, where the prefix “sn” means stereospecifically num- bered) is used instead of the older designation of the compound as Da-glycerophosphate. Glycerol 3-phosphate = the commonly occurring Le-glycerophosphate; glycerol a-phosphate = @-glyc- erophosphat,e; rat-glycerol l(3)-phosphate = DLa-glycerophosphate; glycerol l(3)-phosphate = a-glycerophosphate (an un- determined mixture of glycerol-l-P and glycerol-3-P) ; glycerol S-phosphate dehydrogenase = L-a-glycerophosphate dehydrogenase .

transferase that is probably identical with glucose 6-phosphatase.

Liver PPi-glycerol phosphotransferase has a pH optimum of about 5.0, is activated by preliminary treatment with deoxycholate or by NH4OH at pH 9.5, has no requirement for Mg++ or other added cofactor, and has a K, for PPi of 5 X low3 and for glycerol of about 3 M.

The rate of hydrolysis of inorganic pyrophosphate, which proceeds simultaneously with synthetic transferase activity, is inhibited by glucose and accelerated by glycerol. This phenonemon is observed also when studied as a function of pH or of PPi, glucose, or glycerol concentration.

Cytidine triphosphate or glucose-6-P can replace PPi as an e5cient donor, while ATP is relatively inactive in the synthesis of glycerol l-phosphate by the liver microsomal enzyme.

Glycerol l-phosphate is hydrolyzed at a much faster rate than is glycerol J-phosphate by the liver microsomal preparations.

This enzymatic synthesis provides a convenient method for the preparation of glycerol l-phosphate (D-ar-glycerophosphate).

The ability of mammalian liver particulate fractions to catalyze the utilization of inorganic pyrophosphate for the activation of glucose, first reported by Rafter (l), has been the subject of a number of studies. Inorganic pyrophosphate-glucose phosphotntnsferase activity has been found to occur primarily in microsomal membrane fractions (2) and to have properties which suggest its identity with microsomal inorganic pyrophosphatase and glucose 6-phosphatase (2-6). A similar activity occurs in kidney microsomes (2, 7) but is absent in most other organs and tissues. A number of nucleoside di- and triphos- phates can serve in place of inorganic pyrophosphate as donors of a phosphoryl group (8), and a variety of sugars and related compounds can substitute for glucose (9).

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1824 Enzymatic Synthesis of Glycerol l-Phosphate Vol. 243. No. 8

Using experimental conditions which had been found to be optimal for glucose 6-phosphate formation, we have carried out acceptor specificity studies in which phosphorylation was followed by measuring incorporation of radioactive phosphorus from azI’a2Pi into stable organic phosphorus compounds (9). Under these conditions many hexoses, pentoses, and heptoses, as well as sugar alcohols, were phosphorylated in varying de- grees. The presence of a primary alcohol group was the chief requirement for reactivity.

Among the alcohols effectively phosphorylated was glycerol. Incorporation of a2Pi into glycerol was about 36% as effective as incorporation into glucose under comparable conditions (9). It was found, by enzymatic assay, that very little of the well known biologically important compound, glycerol 3-phosphate, had been formed. In this paper we report the isolation and characterization of the product as glycerol l-phosphate1 and describes some of the properties of the enzyme responsible for its formation. A brief report of some of the results has been published (11).

EXPERIMENTAL PROCEDURE

Preparation and Assay of Enzymes-Liver microsomal fractions were prepared from rats fasted for 24 hours and were stored frozen as previously described (4). Homogenation and fractionation of the other organs studied were carried out in approximately the same way. The “microsomal” fraction used was that which precipitated in 1 hour between 12,000 and 100,000 x g. Immediately prior to use the freshly thawed fractions were treated at 0” with 0.1 volume of 1 N NHIOH. This treatment, at about pH 9.7, has been found to produce an instantaneous increase in vitro of between 50 and 300% in the level of microsomal glucose 6-phosphatase and its related enzymatic activities while avoiding the thermal instability introduced by detergent-like activators such as deoxycholate and Triton X-100 (12, 13). Alternative treatments of the enzyme preparations are indicated with the reports of results. Assay methods for glucose 6-phosphatase, inorganic pyrophosphatase, and inorganic pyrophosphate-glucose phosphotransferase activities were those previously used (6).

Measurement of Phosphorylated ProductsGlucose-6-P was measured by oxidation catalyzed by TPN-specific glucose-6-P dehydrogenase (14). Glycerol S-phosphate was determined by means of DPN-specific glycerol 3-phosphate dehydrogenase (L-cr-glycerophosphate dehydrogenase) (15). These enzymes were obtained from Sigma.

Glycerol l-phosphate was determined by measurement of incorporation of the radioactive phosphorus by a modification of the method used in earlier acceptor specificity studies (9) or by measurement of incorporation of glycerol-i4C into the organic phosphorus compound enzymatically synthesized. Sodium pyrophosphate containing radioactive phosphorus, prepared by The Radiochemical Centre, Amersham, England, and uni- formly labeled glycerolJC from the International Chemical and Nuclear Corporation were used. Enzymatic reactions in the presence of isotopically labeled precursors, usually carried out at 30” for 10 min at pH 5.4, were stopped by the addition of an equal volume of 10% trichloracetic acid prior to inorganic phosphate or isotope determination. When enzymatic measurement of the products was to be performed, the reaction was terminated by heating the mixture at 100’ for 3 min. It was found that no significant hydrolysis of glucose-6-P or glycerol

l(3)-phosphate occurred under these conditions. Corrections were made, where applicable, for a very small amount of ruc- glycerol l(3)-phosphate which was formed nonenzymatically when high concentrations of glycerol and PPi were heated at slightly acid pH.

Quantitative aliquots (2 or 4 ~1) of the deproteinized radioactive reaction mixtures were developed on paper by descending chromatography for 24 hours at 3” with CH30H-NH40H-H20 (6:1:3) to separate the organic phosphate products from inorganic phosphate and pyrophosphate. Radioactivity in the clearly defined product areas was measured by means of a paper strip scanner coupled to a recorder and digital integrator (Nuclear-Chicago). Along with each experiment in which glycerol 1(3)-P was to be measured by the isotope method, a parallel enzymatic reaction was run with glucose as the acceptor and the same sample of a2Pa2Pi. Glucose-6-P formed was measured enzymatically as well as isotopically. We have previously established that quantitatively similar results can be obtained by either of these methods (9). From these simultaneously determined glucose-6-P measurements and the quantity of radioactivity incorporated into the glycerol 1(3)-P, t,he total concentration of glycerol 1(3)-P was calculated. The isotopically determined total glycerol 1(3)-P was corrected when necessary for small amounts of contaminating enzymatically determined glycerol a-phosphate to estimate the concent,ration of glycerol l-phosphate.

It was found possible to determine quantitatively both the glucose-6-P and the glycerol 1(3)-P formed simultaneously in enzymatic reaction mixtures in which both glucose and glycerol were present as acceptors of phosphoryl transfer from a2Pa2Pi. For example, in 30 hours of development at 3” of a carefully spotted aliquot of the products formed enzymatically from 0.04 M a2Pa2Pi, 2 M glycerol, and 0.4 M glucose in 0.08 M cacodylate buffer, glucose-6-P had migrated 28 cm and glycerol 1(3)-P, 34.5 cm. A separation of about 2 cm with no overlap was observed between the two compounds.

Isolation of Glycerol-l-P-From the reaction mixtures produced by incubation of liver “microsomes” with high concentrations of glycerol and inorganic pyrophosphate at pH 5.2, glycerol-l-P was isolated as a barium salt and converted to a lithium salt by modifications of methods used in the preparation of chemically (16) or enzymatically (17) synthesized glycerol- 3-P.

To 80 ml of a solution containing 2 M glycerol and 0.04 M

inorganic pyrophosphate in 0.1 M sodium acetate buffer at pH 5.2 were added 8 ml of an isotonic sucrose suspension of a liver microsomal preparation containing about 100 mg of protein, pretreated with 0.1 volume of 1 N NHIOH. After incubation at 30” for 60 min, the reaction was stopped by heating at 100” for 5 min. Coagulated protein was removed by centrifugation. Aliquots of the reaction mixture were analyzed for free inorganic phosphate, organically bound phosphorus stable to 1 N H$SO, at 100” for 10 min, total phosphorus, and glycerol 3phosphate. It was estimated that 17% of the total phosphorus was organically bound, of which only a trace was in the form of glycerol 3-phosphate. An excess of pulverized solid Ba(OH)* was added to remove inorganic phosphate and pyrophosphate. The precipitate was extracted several times with water before being discarded. After the removal of excess Ba+f with CO*, the combined supernatant solution was concentrated under reduced pressure to a small volume. Barium-glycerol l-phosphate was


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Issue of April 25, 1968 M. R. Stetten and D. Rounbehler 1825

precipitated by the addition of 2 volumes of ethanol and the mixture was refrigerated overnight. The alcohol-insoluble, colloidal barium salt, washed well with aqueous ethanol to remove glycerol and acetate, was redissolved in water at room temperature. Cations were removed by passing the solution through an ion exchange column of AG-50W-X8 (hydrogen form, Calbiochem) resin. The acid effluent was adjusted to pH 7.4 with LiOH.and concentrated under reduced pressure to about 7 ml; it was then filtered and treated with about 5 volumes of ethanol. The flocculent precipitate was centrifuged, freed of adhering ethanol, redissolved in water, and reprecipitated with ethanol. The reprecipitated lithium salt, washed with alcohol, 50% alcohol-ether, and finally ether, was dried under reduced pressure over PsOh; yield, 119 mg. Based upon the analysis of phosphorus compounds organically bound, and the assumption that glycerol l-phosphate is the only such compound formed, this represents a yield of 58% in the isolation.

RESULTS

Characterizatim of Glycerol I-Phosphate-Analysis of the isolated anhydrous dilithium salt (Schwarzkopf Microanalytical Laboratory) was as follows.

LizCsH~POe(183.95)

Calculated: C 19.59, H 3.84, P 16.84, Li 7.55 Found : C 19.34, H 3.92, P 16.80, Li 7.48

Optical rotation, [a& = +1.71” (5y0 solution of lithium salt in 2 N HCl); literature values (16) for glycerol 3-phosphate: [a],, = - 1.45” (10% solution of barium salt in 2 N HCI) ; glycerol 3-phosphate by enzymatic assay: 4.5%.

Elementary analyses of the isolated product are in excellent agreement with theoretical values for a dilithium salt of glycero- phosphoric acid. One equivalent of formaldehyde was produced by periodate oxidation (18) ; thus, the possibility of appreciable contamination of the product by glycerol 2-phosphate was eliminated. These results, together with the positive value for the specific rotation determined on the free acid, are taken as proof that the compound formed enzymatically from glycerol and PPi was glycerol l-phosphate, the opposite enantiomer from that usually synthesized biologically. We are indebted to Dr. Eric Baer, who devised the original chemical synthesis of both glycerol l-phosphate and glycerol 3-phosphate (D- and L-W

glycerophosphate) (16, 19), for a gift of reference samples of these compounds. The 4.5% of glycerol 3-phosphate which contaminated the isolated product is a maximum value and probably resulted from a small amount of nonenzymatic synthesis of glycerol 1(3)-P, which occurred under the conditions of very high concentrations of substrate found to be optimal for the enzymatic reaction. Less glycerol 3-phosphate was always found when the enzymatic reaction was terminated with trichloracetic acid than when heat was used. Any slight enzymatic synthesis of the 3-isomer or intramolecular equilibra- tion with glycerol 2-phosphate during the isolation procedure could also have contributed to the content of the a-isomer.

Properties of Liver Enzyme-The isotope incorporation method, with either a*PaPi or glycerol-%, was used in a series of experiments in which the properties of the particulate enzyme which synthesizes glycerol l-phosphate were studied. Many of the properties were found to resemble closely those of liver and kidney microsomal PPi-glucose phosphotransferase. No added

MP- ion or other cofactor is required for activity. A cor-

0.4

0.3

V

0.2

0.1

glucose-$-P _----

.’ -xc

/ glycerol- I-P -0

50 100 2! I

P Pi

r I , r 1 I I I

.Ol .02 .04M .08

c yl FIG. 1. Effect of PPi concentration on rate of glycerol l-phos-

phate and glucose g-phosphate format,ion. ‘*P’*Pi was varied between 0.01 and 0.08 M in a 0.08 M sodium cacodylate buffer of pH 5.4 in the presence of either 2 M glycerol or 0.4 M glucose. The enzyme used was a preparation of liver “microsomes,” activated by pretreatment with NHhOH, equivalent to 0.54 mg of protein per ml of reaction mixture. Incubation was for 10 min at 30”. The enzymatic reaction was stopped by the addition of trichloracetic acid, and the amount of product formed was determined by the isotopic method described in “Experimental Procedure.” V, micromoles of product formed per min per mg of protein. -, glycerol-l-P formed; - - -, glucose-6-P formation determined at the same t.ime wit,h the same J’P**Pi, enzyme, buffer, and incubation conditions. See Fig. 5 for the pyrophosphatase activity in the same experiment.

respondingly low pH optimum of about 5.0 was observed for the two synthetic activities. Activation of the enzyme preparations by pretreatment with NH40H, deoxycholate, or Triton X-100 caused a slight shift in the optimum to about pH 5.2 to 5.4 (13).

Effect of Concentration of Inorganic Pyrophosphate-When the formation of glycerol l-phosphate was studied as a function of the pyrophosphate concentration, with glycerol constant at 2 M, a concentration of about 0.04 M PPi was found to be optimal (Fig. 1, solid line). From a double reciprocal plot of this data,

the apparent Michaelis constant for PPi in the pyrophosphate- glycerol phosphotransferase reaction was found to be 5.3 x 1OW M. This is close to the value that had been observed for

PPi in the pyrophosphate-glucose phosphotransferase reaction (2). Approximately the same results were obtained when glycerolJC and nonisotopic PPi were used in a similar experiment.

E$ect of Concentratim of Glycerol-Very high concentrations of glycerol are required to saturate the enzyme site involved in the synthesis of glycerol l-phosphate. The reaction rate was nearly linearly related to glycerol concentration up to about 2 M

glycerol. From a study of the effect of glycerol concentration upon the reaction rate at a constant PPi concentration of 0.04 M,

an apparent K, of about 3 M glycerol was obtained (Fig 2). Comparison of e$ects of Glucose and Glycerol on Hydrolysis of

Inorganic Pyrophosphate-

PPi + HtO -t 2 Pi

PPi + glucose + Pi + glucose-6-P

PPi + glycerol + Pi + glycerol-l-P

(1)

(2)

(3)


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1826 Enzymatic Synthesis of Glycerol 1 -Phosphate Vol. 243, No. 8

I 2 3 4 5M

[GLYCEROL]

FIG. 2. Effect of glycerol concentration on rate of glycerol l- phosphate formation. The 32P’*Pi concentration was constant at 0.04 M, and glycerol was varied between 0.2 and 5 M. Buffer, pH, enzyme, incubation, and assay conditions were the same as in the experiment reported in Fig. 1. V, micromoles of glycerol-l-P formed per rn& per mg of prott ?in

t A. Tronsferase actiwty

?G----- 60 mmutes

IO 30 60 12c

FIG. 3. PPi-phosphotransferase reactions and PPi hydrolysis measured simult,aneously, as a function of time. Three parallel reactions were carried out with the same samples of activated enzyme, 0.04 M 32P3zPi, pH 5.4 cacodylate buffer, and either 2 M glycerol (0), 0.4 M glucose (O), or no acceptor other than water (X). Total orthophosphate formation was measured colori- metrically, and formation of glucose-6-P (= PPi reacting with glucose) or glycerol-l-P (- PPi reacting with glycerol) was measured by isotope incorporation. PPi reacting with water was calculated from these values (total Pi formed minus glucose-6-P (or glycerol-l-P) formed/2 = PPi reacting with water).

In a series of experiments the hydrolysis of PPi by microsomal acid pyrophosphatase alone (Reaction 1) was compared with this hydrolysis in the presence of either glucose or glycerol. With glucose present Reactions 1 and 2 proceed simultaneously while Reactions 1 and 3 proceed in the presence of glycerol. Parallel experiments were run at the same time with the same samples of radioactive PPi and identical enzyme preparations and assay conditions.

With PPi concentration and other conditions constant, it was found that approximately the same molar quantities of phosphorylated products, glycerol l-phosphate or glucose-6-P, were formed from 2 M glycerol and 0.4 M glucose. An example of this

equivalence, measured as a function of reaction time, is shown in Fig. 3A. Whereas the formation of organic phosphate compounds was here nearly identical with glucose and glycerol, orthophosphate production was found to differ markedly in the two cases. Glucose inhibited Pi formation, as noted earlier (2), while glycerol stimulated a production of orthophosphate much greater than was observed with no added acceptor. I f the assumption is made that the only reactions that produce significant amounts of orthophosphate are Reactions 1 and either 2 or 3, the quantity of PPi hydrolyzed (Reaction 1) in each case can be calculated from the total Pi and the quantities of glucose- 6-P or glycerol l-phosphate formed. This has been done and the rate of the hydrolase reaction is plotted in Fig. 3B.

This inhibition of pyrophosphatase activity by glucose and acceleration by glycerol was found under ,a11 conditions studied. The difference was particularly evident when studied as a function of acceptor concentration. Parallel experiments in which only the glucose or glycerol concentrations differed are recorded in Tables I and II. Glucose-6-P formation increased with increasing glucose concentration to a maximum at about 0.4 M glucose, while Pi formation was decreased (2). With increasing glycerol concentrations up to 3 M, both the glycerol l-phosphate and Pi formations were increased. When expressed as percentage of total reacting PPi which is involved in the transfer reaction, the percentage transfer continually increased with increasing concentrations of both glucose and glycerol (Tables I and II, fourth column). However, when the data were calculated to measure the residual PPi involved in Reaction 1 alone (Fig. 4), it could be seen that glucose greatly inhibited the hydrolysis of PPi, while glycerol had a stimulating effect on this reaction.

Again, as a function of PPi concentration, with glucose and glycerol concentrations which produced approximately equimolar quantities of either glucose-6-P or glycerol l-phosphate (Fig. l), the hydrolysis of PPi was inhibited by glucose and accelerated by glycerol (Fig. 5). This as yet unexplained difference was also observed over a wide range of hydrogen ion concentrations (Fig.

TABLE I

Effect of glycerol concentration on PP;-phosphotransferase and PPi-hydrolase reactions

Reaction mixtures contained 40 Mmoles of “P”Pi and activated rat liver microsomes equivalent to 0.56 mg of protein, and the indicated concentrations of glycerol in 1.0 ml of 0.08 M cacodylate buffer at pH 5.4. Incubation was for 10 min at 30”. Reaction was stopped by the addition of trichloracetic acid.

Glycerol concentration

16

4.8 3 2 1 0.8 0.6 0.4 0.2 0

A, Pi B, Glycerol-l-P

fimoles/min/mg polein

1.29 0.382 1.41 0.358 1.38 0.308 1.19 0.181 1.15 0.151 1.04 0.116 1.00 0.095 0.93 0.051 0.83 0

TransfeP

%

46 41 37 26 23 20 17 10 0

a Percentage transfer = pmoles glycerol-l-P

fimoles PPi


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6). Substrate concentrations were used which at all pH values produced glucose-6-P at somewhat faster rates than glycerol l-phosphate in the transferase reactions (Fig. 6, inset). PPi

hydrolysis, calculated from these values and the quantities of

total Pi formed in the same reaction mixtures, was depressed by glucose and stimulated by glycerol at all pH levels used. The optimum pH for the hydrolase was somewhat higher than for

the transfemse when both were determined in the same reaction

mixture, just as was found when both were separately determined (4).

Synthesis of Glycerol l-Phosphate from Phosphmyl Donors Other Than PPi-A preliminary study has been made of alterna-

tive phosphoryl donors with the use of enzyme and substrate

conditions favorable for synthesis of glycerol l-phosphate and

glucose-6-P from PPi. Use of liver microsomal preparations

free of supernatant proteins, in the absence of added Mg++ and

TABLE II

Egecl of glucose concentration on PPi-phosphotransferase

and PPi-hydrolase reaclions

Reaction mixtures contained 40 rmoles of “P’*Pi and activated rat liver microsomes equivalent to 0.56 mg of protein, and the

indicated concentrations of glucose in 1.0 ml of 0.08 M cacodylate buffer at pH 5.4. Incubation was for 10 min at 30”. Reaction was stopped by the addition of trichloracetic acid.

GlUCOSe concentration

N

1 0.4

0.2 0.08 0.04

0

” Percentage

A Pi B, Glucose-6-P

jmol~ss/min/nrg profcin

0.513 0.333 0.690 0.382 0.741 0.311 0.818 0.252 0.828 0.152 0.832 0

.ansfer = pmoles glucose-B-P

pmoles PPi

TransfeP

%

79 71 59 47

31 0

0

in presence of glycerol

[PPi] = 0.4M

L in presence of glucose

1 1 0.5 I 2 3M

GLUCOSE or GLYCEROL

FIG. 4. Effect of glucose and glycerol concentrations on microsomal inorganic pyrophosphatase reaction. PPi reacting with water in the presence of constant PPi (0.04 M) and different glucose or glycerol concentrations has been calculated from the experiments described in Tables I and II. PPr reacting with H20 = total Pi formed minus glucose-6-P (or glycerol-l-P) formed/a.

X PPi with Hz0 alone

1 0 in presence of glucose (0.4M )

I I I 1

.Ol .02 .04M .08 PPi CONCENTRATION

FIG. 5. Effect of glucose and glycerol on microsomal inorganic pyrophosphatase, as a function of PPi concentration. From the data plotted in Fig. 1 and the simultaneously determined total Pi formed in each case, the PPi involved in Reaction 1 was calculated.

i 4

x Pq alone o with glycerol ( 2M 1 v l with glucose (0.4M)

n4 5 DH = ’

-----I

0 0

1 5

PH 6 7

FIG. 6. Effect of pH on microsomal acid pyrophosphatase, PPi- glycerol phosphotransferase, and PPi-glucose phosphotransferase activities, assayed simultaneously. Three parallel experiments were carried out with the same series of solutions, with a final concentration of 0.04 M a*Ps*Pi in approximately 0.08 M sodium cacodylate-acetate buffers at pH values between 4 and 7. Reac- tion mixtures contained 2 M glycerol (O), or 0.4 M ghCOSe (a), or no added acceptor except water (X). Reaction was started by the addition of NHdOH-activated microsomes equivalent to 0.7 mg of protein per ml. Incubation was at 30” for 10 min. pH values were measured on parallel reaction mixtures containing nonisotopic PPi. Reaction was stopped by addition of trichloracetic acid, and Pi, glucose-6-P, and glycerol-l-P levels were determined in the solutions after removal of precipitated protein. The transferase results are given in the inset, in which V = micromoles of glucose-6-P or glycerol-l-P formed per min per mg of protein, while the calculated values for the pyrophosphatase are shown on the main portion of the graph.


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1828 Enzymatic Synthesis of Glycerol l-Phosphate Vol. 243, No. 8

TABLE III

Synlhesis of glycerol 1 -phosphate and glucose B-phosphale by alternative donor compounds

Each reaction mixture contained 0.05 M PPi, ATP, CTP, or glucose-G-P with either.2 M glycerol-Ci4 or 0.4 M glucose and acti-

vated microsomes containing 0.112 mg of protein, in a total volume of 0.1 ml of cacodylate buffer of pH 5.4. Incubation was at 30” for 20 min. Reaction was stopped by heating at 100” for 3 min.

mixtures of very small total volume. Since, even with these very favorable synthetic conditions, only about 0.5% of the total glycerol-W used was incorporated into glycerophosphate, the low i4c levels in the samples counted introduced a greater possible error in these results than in those in which “Pi could be used as the label.

Glucose-6-P synthesis from the same phosphoryl donors and enzyme was studied at the same time with nonisotopic glucose as acceptor and TPN-specific glucose 6-phosphate dehydrogenase to measure the product formed. The concentration of total glycerol 1(3)-P formed was calculated from the ‘4c incorporation relative to radioactive phosphorus incorporation from 32Pa2Pi into glycerol and glucose in a parallel experiment. Glycerol 3-phosphate formation, enzymatically measured, was found to be negligible, which is proof of the absence of significant glycerokinase activity under the assay conditions used.

Results (Table III) indicate that CTP and glucose-6-P are excellent donors for the synthesis of glycerol l-phosphate by the PPi-glycerol phosphotransferase system, although slightly less efficient than PPi. ATP is a very much less efficient donor. Approximately the same relative efficiencies were obtained for PPi-glucose phosphotransferase and associated nucleoside triphosphate-glucose phosphotransferase activities in the synthesis of glucose-6-P in our parallel experiment (Table III) and in the experiments of Nordlie and Arion (8).

Transfmase Activities of Subcellular Fractions of Liver-En- zymatic assays of crudely separated subcellular fractions of liver homogenates indicated that the PPi-glycerol phosphotransferase activity was concentrated in the microsomal fraction and was completely lacking in the soluble portion (Table IV). Its distribution was found to be exactly parallel to that of PPi- glucose phosphotransferase activity, which is known to occur only in those particulate fractions of liver which have glucose

TABLE V

Distribution of lransjerase activities in rat organs and tissues

Portions of organs and tissues of a rat fasted for 24 hours were homogenized at 0” in 5 volumes of sucrose solution containing 1 mM EDTA. Aliquots of freshly prepared whole homogenates,

minus only that crude debris which settled without centrifugation, were used for assays. Enzyme fractions were not activated with NHIOH or detergent. Assay mixtures contained 0.04 M

‘*P’*Pi and either 2 M glycerol or 0.4 M glucose in cacodylate buffer

of pH 5.4. Incubation was at 30” for 30 min. Reactions were stopped by heating at 100” for 3 min. Glucose-6-P and glycerol-1(3)-P were determined isotopically. Glucose-6-P and glyc-

erol-3-P were determined enzymatically.

Products formed Donor (0.05 M) Glucose-6-P rl

;lycerol-3-P Glycerol-l-P b

pmoksfmin/mg protein

Glycerol-i4C (2 M) as

acceptor PPi ATP

CTP Glucose-6-P

Glucose (0.4 M) as

acceptor PPi ATP

CTP

0.015 0.307 0.006 0.020

0.012 0.209 0.012 0.289

0.380

0.045 0.210

a Determined by enzymatic analysis. b Determined by isotope analysis, corrected for nonenzymatic

control.

TABLE IV

Distribution of transjerase activities in rat liver

Subcellular fractions of rat liver homogenate in 0.25 M sucrose

containing 1 mM EDTA, crudely fractionated by ultracentrifuga- tion (22), were simultaneously assayed for PPi-glycerol phosphotransferase and PPi-glucose phosphotransferase activities. En-

zyme fractions were activated with NHIOH just prior to assay. Incubation was at 30” for 30 min with 0.08 M “Pa*Pi and 2 M glycerol or 0.4 M glucose.

Product formed

Subcellular fraction Protein Ratio of A:B

-

-

n&g liner pmoks/min/g prokin

124 40 36 9 23 22

33 102 96

77 0 0

Whole homogenate (mi-

nus debris and nuclei) Mitochondrial Microsomal.

Soluble (105,000 X g supernatant)

1.11 1.05

1.06

-

-_

Products formed - Homogenate D Corrected for a small amount of nonenzymatically formed

rat-glycerol-1(3)-P. Glycerol-3-P IG1yce;1-1(3)-/ G?ycerol-1-P /Glucose-O-P

floles/min/g protein

at low pH, eliminated essentially all hexokinase or glycerokmase activity, since these enzymes are found in the soluble fractions of liver and require divalent cations and higher pH values for their activity (15, 20, 21).

Because of the very high concentrations of glycerol required for significant phosphorylation, glycerol-W is a much less desirable tracer for the reaction than is a2Pa2Pi. In the experiment recorded in Table III, ATP, CTP, and glucose-6-P were compared with PPi with 2 M glycerol-W as acceptor in reaction

Liver................ 1.6 38 Kidney. 2.2 46 Intestinal mucosa.. 1.5 17.1

Spleen 0.5 5.3 Brain. 2.1 7.0 Lung, 0.7 7.1 Heart muscle. 0.8 1.7 Skeletal muscle. 0.5 1.0

No enzyme control. . ~0.6 G1.2

37 39 44 46

15 6 5 0 5 0 6 0

0 0 0 0


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Issue of April 25, 1968 M. R. Stetten and D. Rounbehler

6-phosphatase activity (%4). Like glucose 6-phosphatase activity, the transferase activities probably occur only in the endoplasmic reticular membranes, and such activity as is found in the crude fractions is probably due to microsomal contamination.

Occurrence of PPi-Glycerol Phosphotransjerase and PPi- Glucose Phosphotransjerase Activities of Rat Organs and Tis- suesIn contrast to the identical subcellular distribution within liver, the occurrence of the two transferase activities was different in the various organs tested (Table V). The same whole homogenate preparation of each organ and the same level of radioactive pyrophosphate were used for the two assays. The ability to synthesize glucose-6-P was completely lacking in all organs and tissues tested except liver, kidney, and intestinal mucosa. The same results were obtained with both the enzymatic and the isotope methods. On the other hand, formation of glycerol l-phosphate, in significant excess over the small amounts of glycerol 3-phosphate present or of rue-glycerol 1(3)-P formed in the nonenzymatic control, was found with all homogenates tested except muscle. Liver and kidney were most effective, and intestinal mucosa had appreciable PPi-glycerol phosphotransferase activity. Spleen, brain, and lung homogenates were less effective but gave clearly positive results with glycerol.

Because whole unfractionated, unactivated homogenates were assayed in this experiment, the absolute values obtained were low. Therefore, microsomal fractions were isolated from several organs, and the two transferase activities were measured under

0.03-

0.02 -

t mp. protein 3

w I I 0.25 0.50 1.0

mp. protein

FIG. 7. Transferase activities of organ microsomes. Depend- ence upon protein concentration. The enzymes assayed consisted of freshly prepared microsomal fractions isolated by differential centrifugation of homogenates of liver, spleen, lung, and brain of a rat fasted for 24 hours. Parallel experiments were run with increasing concentrations of enzyme added to isotopic substrate mixtures as in Table V. Incubation was for 10 min at 30’. Glyc- erol-1-P was determined by the isotope method, and glucose-6-P by both isotope and enzymatic assay. Glucose-6-P formed by spleen, lung, and brain microsomes was zero when measured by either method. Note the lo-fold difference in the scales of the ordinates.

25

20 I

T 15

IO

5

L 1 I I I

,

0.5 1.0 1.5 2.0 2.5

C GLYdEROL 1

FIG. 8. Inhibition of liver PPi-glycerol phosphotransferase activity by glucose. a*PrePi was constant at 0.04 M, while glucose was varied between 0 and 0.4 M, and glycerol was varied between 0 and 2 M at each glucose level. Enzymatic reactions were carried out for 10 min at 30” in cacodylate buffer at pH 5.3. Liver microsomes were activated by pretreatment with 0.1 M NHdOH. The phosphorylated products were separated by paper chromatography of aliquots, and the quantities were determined as described in “Experimental Procedure.” V = micromoles of glycerol-l-P formed per min per mg of protein.

more favorable conditions as a function of protein concentration (Fig. 7). Formation of glycerol l-phosphate, when catalyzed by spleen, lung, or brain microsomes, proceeded at only about one-tenth the rate obtained with liver microsomes, but in all cases the rate was approximately linearly related to the protein concentration. No glucose-6-P was formed from PPi by spleen, lung, or brain microsomes at any enzyme concentration.

Inhibition of Liver Microsomal PP;-Glucose Phosphotransjemse Activity by Glycerol and of PPi-Glycerol Phosphobansjerase Activity by Glucose-When liver microsomes were the source of the enzyme used, it was observed that glycerol inhibited glucose- 6-P formation, and glucose inhibited the synthesis of glycerol l-phosphate from PPi. In order to study the nature of the inhibition reactions, a series of experiments were carried out with identical a2P82Pi concentrations, pH, and assay conditions; glycerol concentrations were constant at 0,0.4, 1, and 2 M, while glucose concentrations were varied between 0 and 0.4 M. The quantities of glucose-6-P and glycerol l-phosphate simultaneously formed in the reaction mixtures were determined by the isotope incorporation method described in “Experimental Procedure.” In the inset graphs of Figs. 8 and 9 the reaction rates are plotted as a function of substrate concentration at different constant levels of inhibitor. It can be seen in Fig. 8 that the af%nity of the enzyme for glycerol is so low that V,., is not nearly reached with concentrations up to 2 M glycerol, even in the absence of glucose as inhibitor. With the still higher glycerol concentrations used in Fig. 2, a maximum velocity was attained. From the double reciprocal plots it appears that glucose acts as a competitive inhibitor of glycerol in the formation of glycerophosphate (Fig. 8). When the slopes from Fig. 8 were


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FIG. 9. Inhibition of liver PPi-glucose phosphotransferase activity by glycerol. to Fig. 8.

The experiment is described in the legend

of protein. V = micromoles of glucose-6-P formed per min per mg

TABLE VI

Glucose inhibition of PPi-glycerol phosphotransferase

of different organs

Enzyme samples were freshly thawed microsomal fractions of

various organ homogenates in 0.25 M sucrose containing 1 rnM EDTA, activated with NHdOH. Substrates were 0.04 M ‘*P”Pi and glucose or glycerol, or both, as acceptors in cacodylate buffer at pH 5.4. Incubation was at 30” for 10 min. Glucose-6-P and glycerol-l-P were determined isotopically. A clear separation of the two products was obtained on paper chromatograms when both products were present in the same solution.

Source of microsome!

Liver

Kidney

Spleen

Brain

--

-

Substrate Products formed

GlUCOSC

.M

0 0.4 0.4

0 0.4 0.4

0 0.4 0.4

0 0.4 0.4

Glycerol

M

2 0 2

2 0 2

2 0 2

2 0 2

Pi I I

Glwxe- Gly$&

pmolcs/min/g prokin

845 236 453 300 496 158 85

1110 312 580 390 582 213 101

109 0 28 100 0 0 101 0 24 60 0 12 57 0 0 58 0 12

replotted versus glucose concentration, a straight line was obtained, an indication that the inhibition is linear. From a Dixon plot (23) of the data the graphically determined Kc = 0.13 M glucose. The inhibition effect of glycerol on the format.ion of glucose-6-P from glucose and PPi appears to be noncompetitive (Fig. 9), with a graphically determined Ki of about 1.5 M

glycerol. The apparently noncompetitive nature of the glycerol inhibition of glucose-6-P formation was confirmed in a.separate experiment in which nonisotopic PPi was used and in which the glucose-6-P product was measured enzymatically.

Eject of Glucose upon PP;-Glycerol Phosphotransferase of

Various Organs-In view of the findings that the PPi-glucose phosphotransferase activity occurred only in liver, kidney, and intestinal mucosa while the glycerol l-phosphate-synthesizing activity was found in a number of other organs, it was of interest to study the inhibition effects of glucose on microsomal preparations obtained from different organs. Formation of glycerol l-phosphate and glucose-6-P from a2Pa2Pi was measured in the presence of glucose or glycerol, or both acceptors, simultaneously under conditions which were identical escept for the source of the microsomes (Table VI). Kidney preparations resembled those of liver in showing a marked inhibitory effect of glucose upon glycerol l-phosphate formation as well as a simultaneous inhibition by glycerol of glucose-6-P synthesis from PPi.

On the other hand, with spleen and brain microsomes, which are entirely lacking in PPi-glucose phosphotransfera?e activity, the same high concentration of glucose had no inhibitory effect upon glycerol l-phosphate formation. The absolute levels of activity in spleen and brain are about an order of magnitude less than those of liver and kidney, but the enzymatic nature of the reaction has been clearly shown (Fig. 7). An extension of this study with different levels of glycerol as substrate showed that the phosphorylation of glycerol with the spleen enzyme is independent of the level of glucose present (Table VII), in contrast to the results obtained with the liver enzyme (Fig. 8).

Activation and Stability of Liver Transferases-Conditions fol activation of the catalytic properties of the membrane enzymes and the heat stability of variously treated preparations have been studied with the use of “PatPi as substrate with glycerol or glucose. It is well known that the apparent level of activity in vitro of many enzymes associated with lipoprotein membranes is greatly elevated by treatment with detergents, such as deoxycholate and Triton X-100; these enzymes are thus rendered considerably more heat-labile in the abrence of substrate. We have shown that pretreatment of these microsomal enzymes at about pH 9.8 with NH,OH or amino acid buffers causes an activation as great or greater than that produced by deosy- cholate, but the enzymes remain relatively stable (12). .4 study of the glycerol l-phosphate synthesizing activity of liver microsomes under similar conditions gave results parallel, in most regards, to those obtained for glucose 6-phosphate synthesis.

The PPi-glucose phosphotransferase activity has previously been shown to be parallel to glucose 6-phosphatase and acid pyrophosphatase activities under the conditions of activation and inactivation studied (cf. Fig. 1 in Reference 13). Like microsomal glucose 6-phosphatase and its associated enzyme activities, an activation of PPi-glycerol phosphotransferase

TABLE VII

Eflect of glucose on glycerol-l-P formation by spleen n~icrosomes

Experimental conditions were like those of Table VI, but a different preparation of spleen microsomes was used.

Inhibitor Glycerol l-phosphate formed

Glycerol, 0.4 M Glycerol, 1.0 Y Glycerol, 2.0 Y

None. Glucose, 0.16 M..

Glucose, 0.4 M..

JLmoles/ninlg p*ok?in

14.4 62.0 14.5 62.7 15.5 61.7


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with stabilization was produced by ammonia pretreatment (Fig. 10, Curve B), and an activation with accompanying instability at 30” was caused by deoxycholate (Fig. 10, Curve C). The two activities differed somewhat, however, in the rate of inactivation at 30” after deoxycholate pretreatment. Studied with the same deoxycholate-treated enzyme preparation, all activity relative to glucose was lost somewhat earlier than was that related to glycerol. In anot.her experiment microsomal preparations, not pretreated with detergents, were heated for 6 min at various temperatures prior to assay at 30”. Inactivation of the two transferase activities under these conditions was nearly parallel, essentially all activity being lost at 50” in 6 min.

Hydrolysis of Glycerol 1 (S)-Phosphates by Microsomd Frac- tims-A preliminary comparison of the hydrolysis of enzymatically prepared glycerol l-phosphate with that of the 3 enantiomer and of the racemic mixture has been carried out. The liver microsomal membrane enzyme preferentially hydrolyzes the 1 enantiomer, the 3 being only slightly affected, and the rate of hydrolysis of the rat-glycerol 1(3)-P falls in between (Fig. 11). This is in agreement with results obtained in 1940 by Baer and Fischer (19), who used synthetically prepared glycerol 1-P and glycerol 3-P with pig kidney homogenates. In contrast, muscle extracts preferentially hydrolyzed the 3 enantiomer (24).

200 1 I ---------x-------- ---_ _ _________ Pretreated with N H,OH

B

Acceptor: Glycerol - Glucose -I----

lb - 30 rninut~s SO

PREINCUBATION TIME at 300

FIG. 10. Activation and stability of transferases. A freshly thawed sample of liver microsomes at 0”, in 0.25 M sucrose and 1mM EDTA, containing about, 10 mg of protein per ml, was divided into t,hree portions. To aliquot A 0.1 volume of Hz0 was added; to B, 0.1 volume of 1 N NHlOH; to C, 0.1 volume of 2% deoxycholate. The treated enzyme samples were incubated at 30” for various time intervals in the absence of substrate prior to enzymatic assay. Assays were carried out with 0.04 M “P3’Pi and either 2 M

glycerol or 0.4 M glucose in cacodylate buffer of pH 5.4 with about 1 mg of protein per ml of reaction mixture. Incubation was at 30” for 10 min. Reaction was stopped by the addition of trichloracetic acid. Pairs of assays with either glucose or glycerol were run at the same time with identical enzyme samples. The quantity of phosphorylated product was determined by the isotope method.

HYDROLYSIS OF I(3)-GLYCEROPHOSPHATES by Microsomal fractions

h ti6- E \

I I I 1 30 minutes 120

INCUBATION TIME

FIG. 11. Hydrolysis of l(3)-glycerophosphates by liver microsomal fractions. The hydrolysis of 0.02 M solutions of enzymatically prepared glycerol-l-P and commercially obtained rat- glycerol-1(3)-P and glycerol-3-P (Sigma), catalyzed by the same sample of NHrOH-activated liver microsomes, was followed by measuring Pi formation at 30” and pH 5.9.

DISCUSSION

Glycerol, although optically inactive, possesses a “meso” carbon atom (25), and it has been shown that the primary carbinol groups of glycerol are differentiated in biological sys- tems (26, 27). Glycerokinase (15) acts stereospecifically on glycerol to form glycerol 3-phosphate, which is the metabolic precursor of the phosphatidic acid portion of triglycerides, phosphatides, and complex lipids (28). So far as we are aware, neither the biological occurrence of free glycerol l-phosphate nor the existence of an enzyme which specifically phosphorylates the opposite primary carbon01 group from that on which glycerokinase acts has been reported previously. This enantiomer, of course, does potentially exist in such complex lipids as cardio- lipins and certain teichoic acids in which glycerol is phosphorylated at both primary hydroxyl groups. Selective hydrolysis either in vitro or in wiuo could yield glycerol l-phosphate. The action of phospholipase C on phosphatidyl glycerol has been shown to yield glycerol l-phosphate, which arises from the unesterified glycerol portion of the molecule (29). This po- tential glycerol l-phosphate portion of phosphatidyl glycerol is not formed from free glycerol but from CDP-diglyceride and glycerol 3-phosphate followed by enzymatic dephosphorylation (30).

Enzymatic phosphorylation of free glycerol in mammalian liver and kidney probably occurs ordinarily, by the action of glycerokinase, to yield the metabolically important glycerol 3-phosphate. Glycerokmase is a soluble enzyme, requiring Mg* or Mn+, and having reasonable K, values of about 4 x lo-’ M for glycerol and 2.8 X IO+ M for ATP (15, 20). In contrast, PPi-glycerol phosphotransferase, which specifically catalyzes formation of the 1 enantiomer, is found in the insoluble


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particulate fraction derived from the endoplasmic reticular membranes of cells, has no specific metal or other cofactor requirement, and has K, values for the liver enzyme of about 3 M

for glycerol and 5 x 10ea M for PPi. The very high concentrations of glycerol required for significant phosphorylation, when catalyzed by this particulate mammalian organ enzyme, are similar to the concentrations of glycerol used in early studies of the synthetic action of a variety of acid and alkaline phosphatases with organic phosphate compounds as donors (31-33). In these cases the glycerophosphate formed was generally assumed to be DL and the possible stereospecificity of the synthesis was not recognized or studied. The low affinity of the PPi-glycerol phosphotransferase for glycerol makes it appear unlikely that the synthesis of glycerol l-phosphate by this method can have great physiological significance.

The enzymatic synthesis of glycerol l-phosphate provides a method of preparation of this compound which is more convenient than those previously available. It has been prepared chemically from optically active precursors (19) and by selective enzymatic destruction of the 3 enantiomer from a racemic mixture (30).

The observation that glycerol accelerates the hydrolysis of PPr, while glucose has an inhibitory effect on this hydrolysis, does not have an obvious explanation. The possibility that the phosphorylation of glycerol proceeds by way of a glycerol di- phosphate, with rapid hydrolysis of one of the phosphate groups, is considered unlikely since no isotopic evidence of the formation of such a compound could be obtained. A more likely possibility is that glycerol produces a physical change in the membrane enzyme which allows accelerated reaction. High concentrations of glycerol may readily be pictured as modifying the water structure associated with the active site on the enzyme and altering the accessibility of the site to various acceptors. The puzzling observation, that whereas glucose is a strictly competitive inhibitor of glycerol l-phosphate synthesis, glycerol appears to affect the formation of glucose-6-P from PPi in a noncompetitive fashion, may also be attributable to alterations of the enzyme structure by the very high concentrations of glycerol used.

Many properties, such as subcellular distribution, pH optimum, stability, and mutual inhibition by glycerol and glucose, make it appear possible that PPi-glucose phosphotransferase and PPi- glycerol phosphotransferase activities may be properties of the same protein in liver. The two activities, as well as other similar reactions resulting in the formation of a variety of phosphorylated products (9), are certainly closely related and appear to be properties of the same lipoprotein membranes in liver and kidney. On the other hand, the occurrence of a PPi-glycerol phosphotransferase activity, not inhibited by glucose, in such organs as spleen, brain, and lung, which completely lack glucose-6-P- synthesizing ability, favors the assumption that the transferase are different enzymes. No definitive answer can be given to this question at present, but it is possible that a least two distinct transferase enzymes for glycerol occur, only one of which may be the same as that PPi-glucose phosphotransferase which is related to liver glucose 6-phosphatase.

The same liver microsomal preparations which synthesize glycerol l-phosphate from inorganic pyrophosphate also catalyze

the preferential hydrolysis of the 1 enantiomer. By analogy with the relationship between glucose 6-phosphatase and PPi- glucose phosphotransferase (2, 3), one may predict that the synthesis of glycerol l-phosphate and the hydrolysis of this compound may be properties of the same membrane enzyme. In fact, the synthetic action in all cases may well be biologically of secondary importance to a primary hydrolytic function of the enzymes.

1. 2. 3.

4.

5.

6.

7.

8.

9. 10.

11.

12.

13.

14.

15.

16.

17.

18. 19.

20. 21.

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(1964). STETTEN, M. R., AND TAFT, H. L., J: Biol. Chem., 239, 4041

(1964). ARION, W. J., AND NORDLIE, R. C., J. Biol. Chem., 239, 2752

(1964). FISHER, C. J., AND STETTEN, M. R., Biochim. Biophys. Acta,

121, 102 (1966). NORDLIE, R. C., AND SOODSMA, J. F., J. Biol. Chem., 241,

1719 (1966). NORDLIE. R. C.. AND ARION. W. J.. J. Biol. Chem.. 240. 2166

(1965). ’ STUTTER, M. R., J. Biol. Chem., 240, 2248 (1965). IUPAC-IUB Commission on Biochemical Nomenclature, J.

Biol. Chem., 242, 4846 (1967). STETTEN, M. R., AND ROUNBEHLER, D., Fed. Proc., 26, 606

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126, 344 (1966j. _ _

STETTEN, M. R., AND BURNETT, F. F., Biochim. Biophys. Acta, 139, 138 (1967).

HORECKER, B. L., AND KORNBERG, A., J. Biol. Chem., 176, 385 (1948).

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STEWART, H. B., AND STRICKLAND, K. P., Can. J. Biochem. Physiol., 39, 1133 (1961).

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WIELAND, O., AND SUYTER, M., Biochem. Z., 329, 320 (1957). CRANE, R. K., AND SOLS, A., in S. P. COLOWICK AND N. 0.

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206, 875’(1954). ’ 27. SWICK, R. W., AND NAKAO, A., J. Biol. Chem., 209, 883 (1954). 28. KENNEDY, E. P., Fed. Proc., 16. 847 (1957); 20, 934 (1961). 29. HAVERKATE, F., AND VAN DEENEN, L. L. M., Biochim.

Biophys. Acta, 84, 106 (1964). 30. KIYASU, J. Y., PIERINGER, R. A., PAULUS, H., AND KENNEDY,

E. P., J. Biol. Chem., 228, 2293 (1963). 31. AXELROD, B., J. Biol. Chem., 172, 1 (1948). 32. MEYERHOF, O., AND GREEN, H., J. Biol. Chem., 178,655 (1949). 33. MORTON, R. K., Biochem. J., 70, 139 (1958); 70, 156 (1958).


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Marjorie R. Stetten and David RounbehlerPHOSPHOTRANSFERASE COMPARED

AND INORGANIC PYROPHOSPHATE-GLUCOSEINORGANIC PYROPHOSPHATE-GLYCEROL PHOSPHOTRANSFERASE

-Glycerophosphate) :αEnzymatic Synthesis of Glycerol 1-Phosphate (d-

1968, 243:1823-1832.J. Biol. Chem.

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enzymatic synthesis of glycerol l-phosphate (d-a=glycerophosphate)

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