VOL. 2 ~ (1957) FRACTIONATION OF IMMUNE RABBIT SERUM y-(;I.OBULIN 75

4.5, however, the antibody was shown to be present only in the most rapidly migrating subfraction (top fraction), while it was practically lacking in the others. These results agree substantially with those of SalvE1~ .\Nl) CORVV and of HUMPHREY AND PORTHt. The impossibility of separating y-globulin subfractions from normal rabl)it serum by the method used here is perhaps due to the fact that our method is relatively less sensitive compared with those used by the above authors. The fact that the separation was possible in the case of sera from rabbits submitted to long-time immunization probably shows that the "microheterogeneity'" is more marked under these conditions. The hypothesis of HUMPHREY AND PORTER that in the immunization process different types of cells produce different types of ~,-globulin is not in disagreement with the results obtained in these experiments, but the presence of the antibody in only one subfraction suggests that each cell produces antibodies with relatively identical

properties.

S I !M M AR Y

R a b b i t s e rum y-globul in was s u b m i t t e d to paper e lec t rophores is in two different buffers of p H 8.6 and 4.5, and of ionic s t r eng t h o.15 and o.o 4, respect ively. Only one spo t was de tec ted for t he y-globul in of no rma l an ima l s , b u t the y-g lobul in of an i ma l s t rea ted for m a n y m o n t h s wi th gu inea pig e ry th rocy t e s was separa ted into 3 -4 subf rac t ions . T he specific an t i body was presen t only in t h e mos t rap id ly mig ra t i ng subf rac t ion and was comple te ly absen t in the others .

R E F E R E N C E S

I A. ,~AIFER AND J. CORE'C, Proc. Soc. Exptl. Biol. Med., 86 (1954) 46. 2 A. SAII"ER AND J. COREr, J. Biol. Chem., 217 (I9.5.5) 23. 3 A. SAIFER AND J. COREY, J. Biol. Chem., 222 (19.56) 803. 4 j . H. HUMPHREY AND R. R. PORTER, Biochem. J., 62 (t956) 93. s R. A. KEKWlCK, Biochem. J., 34 (I94 °) 1248"

Received January 9th, 1957

B O R A T E A N D P H O S P H O G L U C O S E I S O M E R A S E IN T H E

ASSAY OF P H O S P H O M A N N O S E I S O M E R A S E

F R A N C I S C O A L V A R A D O * AND A L B E R T O SOLS

Department o[ Enzymology, Centro Ca]al de Investigaciones Biol6gicas C.S.I.C., Madrid (.Spain)

Phosphomannose isomerase (PMI) and phosphoglucose isomerase (PGI) are widely distributed, but in every case so far examined the activity of PGI greatly exceeds that of PMI x,2,3. Moreover, PMI is usually far less stable than PGI. Consequently, fractionation procedures applied to several tissues have been successful in giving preparations of PGI free of PMI but not vice versa.

In the assay of PMI by the direct method of estimation of ketose formation (fructose-6-phosphate, F-6-P) from mannose-6-phosphate (M-6-P), the presence of

" Fellow of the 1;undaci6n Marquesa de Pelayo.

Re/erences p. 77.

76 r. ALVARADO, A. SOLS VOL. 25 (1957)

PGI tends to decrease the amount of ketose accumulated because of partial trans- formation to another aldose ester, i.e., glucose-6-phosphate (G-6-P) (reaction 3 in the scheme below).

I • ~ ,~.

M-6-P PMI F-6-P PGI G-6-P

\ 2 \ 4

SLEIN 4, working with rabbit muscle preparations, reported that an estimation of PMI activity without interference by PGI was possible at pH 5.5 "the optimum for PMI". This procedure cannot be generalized, since we have found that the pH optima for PMI of intestinal mucosa and testicle are in th.e pH range 7-8', ~. Moreover, an interference with the activity of contaminating PGI, unless it were strictly selective and complete, would be inadvisable. A partial interference would make the assay less reliable, since such an interference would tend to be irregular (see below). To decrease variability we have rather preferred to add a large excess of a purified preparation of PGI to approach the theoretical limit of the equilibrium between F-6-P and G-6-P catalyzed by this enzyme. Ketose accumulation in these conditions would be about 1/3 of ketose formation; but this procedure obviously decreases the

lOO

Bo

2o

SUBSTRATE :

r

/ . " / / .

GLUCOSE - 6 - P N A N N O S E - G - P

Fig. I. Pe rcen tage of hexose p h o s p h a t e as ketose a t equi l ibr ium ca ta lyzed by phosphohexose isomerases , in t he absence (open) and in the presence (shaded) of o. i M borate . The va lues w i thou t bora te are t aken f rom expe r imen t s carr ied ou t wi th p repara t ions f rom several t i ssues of m a m m a l s as well as f rom yeas t s and Ascaris suis t, a. The equi l ibr ium for P M I alone, which could no t be observed exper imenta l ly , is based on SL~ZN'S d a t a 4. The e x p e r i m e n t s wi th bora te were carr ied ou t wi th a purified prepa- ra t ion of P G I f rom yeas t free of P M I and a p repa ra t ion of bull test icle con ta in ing bo th PMI and PGI. Ke tose was e s t ima ted wi th re-

sorcinol I or cys te ine-carbazolL

Re/emnces p. 77.

2O

o

E

) '

o

2 4 g $ lO

TW4E (M~UTES)

Fig. 2. Assay of PMI activity in the presence of var iable a m o u n t s of PGI, wi th and wi thou t borate . A purified p repara t ion of bull test icle con ta in ing PMI and PGI in the rat io i : 4 o, i pmo l e M-6-P, xo pmo le s po t a s s ium p h o s p h a t e p H 7.2, and add i t ions as follows: O no ad- d i t ions; • a purified p repara t ion of PGI s to change the P M I : P G I rat io to I : 1,2oo; /k 7 2 p m o l e s of bora te ; • bo th the P G I p repa ra t ion a n d borate , in a to ta l vo lume of o.6 ml. Ketose was e s t ima t ed ~ a f te r i ncuba t ion a t 3o ° for t he s t a t ed t imes. The do t t ed line r ep resen t s one

th i rd of t he m a x i m a l PMI ac t iv i ty .

VOL. 25 (1957) ASSAY OF PHOSPttOMANNOSE ISOMER.\SE 77

sensi t ivi ty of the assay and its val idi ty is open to question if assay conditions art~ changed.

To obviate the difticulties related to the presence of P(]I it appeared highly desirable to find a method which could entirely el iminate its interference. This has been accomplished on the basis of the abi l i ty of borate to complex with F - 6 - P . Boratc had been utilized by COHE.~ 5 and others to displace the equil ibr ium in the enzymic isomerization of pentoses.

With in the usual range of pH and substrate concentrat ion o . I M borate se- lectiveh, complexes with the F-6-P so comph:tely that the isomerase reaction goes essentially to lOO% ketose ester formation from the aldose ester added (Fig. I). F-6-P is not appreciably isomerized by the phosphohexose isomerases in the presence of borate. Only reactions I or 4 in the scheme above are possible in these conditions.

\ \ ' hen applied to the assay of PMI activity, borate eliminates interference by any accompanying PGI and gives maximal sensitivity. Both aspects are i l lustrated by the experiments presented in Fig. 2, which corresponds to the early course of F-6-P appearance from M-6-P catalyzed by PMI, in the presence of variable amounts of PGI with and without borate. The fact that the effect of a 4o-fold excess of PGI is not maximal can be a t t r ibu ted to lack of saturat ion with substrate and perhaps competi t ive inhibi t ion by M-6-P. On the other hand, borate, by prevent ing any backward reaction, can give a ketose accumulat ion appreciably greater than that which could be expected with PMI alone. This fact eliminates the nece.ssity for correction to init ial rate as recommended by BOl)aNSKV s.

It appears that a systematic util ization of borate could be very useful in the

assay of isomerases.

SUMMARY

The i.somerization catalyzed by phosphohexose isomerases is essentially irreversible in the presence of excess borate, which selectively complexes with ffuctose-6-phosphate. This trapping effect gives zero order kine.tics as long as there is enough substrate to saturate the enzyme. It also makes it possible to assay phosphomannose isomerase without interference by any contaminating phosphoghmose isomerase, and vice versa. A systematic utilization of borate in the assay o isomerases is recommended.

REFERENCES

i .XI. W. Sz.EIY, J . Biol. Chem., 186 (195 o) 753- 2 A. SoLs A,XD F. ALVARADO, 2a Reuni6n Sot:. Espafi. Cienc. Fisiol., r955, p. 35 I.

F. AI.VARADO, unpublished experiments. M. \V. SLEIN, Federation Proc., 13 (x954) "298.

5 S. S. COHEN, J . Biol. Chem., 2or (I953) 78. n j . H. RoE, J. II. [".PSTEIN AND N. P. GOLDSTV:IN, J . Biol. Chem., ~78 (I949) 839. v Z. I)mCHE AND E. BORENFR~:OND, .[. Biol. Chem., t92 (195 I) 583 .

O. BODANSKV, J . Biol. Chem., 2o5 (I953) 73 I.

Received February 5th, I957