biosynthesis of 4-aminobutyrate aminotransferase

Eur. J. Biochem. 161,289-294 (1986) 0 FEBS 1986

Biosynthesis of 4-Aminobutyrate aminotransferase Soo-Young CHOI and Jorge E. CHURCHICH Department of Biochemistry, University of Tennessee, Knoxville

(Received May 27/August 20,1986) - EJB 86 0489

Mitochondria1 4-aminobutyrate aminotransferase was synthesized in a cell-free reticulocyte lysate using polysomal RNA isolated from pig brain. Its primary translation product has a higher molecular mass than the mature enzyme. The difference in relative molecular mass is approximately 2000 as revealed by SDS/polyacrylamide gel electrophoresis.

The precursor of 4-aminobutyrate aminotransferase recognizes polyvalent antibodies raised against the mature enzyme. The precursor of 4-aminobutyrate aminotransferase binds pyridoxal-5-P and displays catalytic activity.

Enzymatic activity was detected using a sensitive fluorimetric method, which is based on the formation of condensation products between succinic semialdehyde and cyclohexane-l,3-dione.

It is concluded that removal of an extra peptide from the precursor is not an obligatory first step in the production of biological active species.

The mitochondrial enzyme 4-aminobutyrate aminotransferase catalyzes the reversible transamination of 4- aminobutyrate with the active-site pyridoxal-5-P to yield succinic semialdehyde and pyridoxamine-5-P. Pyridoxal-5-P is reformed by transamination with 2-oxoglutarate to yield glutamate and enzyme-bound pyridoxal-5-P. The funda- mental catalytic step of transamination is considered to be the loss of hydrogen from the carbon atom bearing the amino group [l]. 4-Aminobutyrate aminotransferase, isolated from pig brain tissues, is made up of two subunits of equal size and 50000 relative molecular mass. The amino acid sequence of a peptide (20 amino acids) carrying the cofactor pyridoxal-5-P has been elucidated, together with the amino acid sequence of the N-terminal portion of the macromolecule (16 amino acids) [2 ] . Only one N-terminal sequence was detected by automatic Edman degradation, indicating that the dimeric protein is made up of two homologous polypeptide chains.

Further studies, using physicochemical methods, have permitted one to define the mode of binding of the cofactor as well as the stability of the dimeric structure in solution [3, 41. Despite those studies, little is known about the biosynthesis of 4-aminobutyrate aminotransferase and its translocation to the mitochondria matrix.

It is the purpose of the present work to answer two questions: (a) is the enzyme synthesized by in vitro translation systems as a precursor of higher molecular mass than the native enzyme? and (b) if the enzyme is synthesized as a higher- molecular-mass precursor, then is the extra peptide removal an obligatory first step in the production of biologically active protein?

EXPERIMENTAL PROCEDURE

Purification of the holoenzyme 4-Aminobutyrate aminotransferase, a mitochondrial en-

zyme, was purified from brain tissues by procedures developed Correspondence to J . Churchich, Department of Biochemistry,

University of Tennessee, Knoxville, Tennessee, USA-37916 Abbreviations. SDS-PAGE, sodium dodecyl sulfate/polyacryl-

amide gel electrophoresis; PJNaCI, 50 mM phosphate-buffered saline (pH 7.4).

in our laboratory [5 ] . Inactivation of 4-aminobutyrate aminotransferase was attained by preincubating the holoenzyme with excess pyridoxal-5-P (tenfold molar excess), followed by reduction with NaBH4 and exhaustive dialysis against water. The reduced enzyme does not regain activity upon addition of pyridoxal-5-P.

Enzymatic assays

A fluorimetric method [6], based on fluorescence measurements of the condensation product of cyclohexane-l,3-dione with succinic semialdehyde, was used for activity assays. This method permits the detection of succinic semialdehyde (0.05 nmol) without interference by the reagent cyclohexane- 1,3-dione.

The reagent was a solution containing 0.25 g cyclohexane- 1,3-dione, 10 g ammonium acetate, 5 ml glacial acetic acid in 100 ml water. The substrate solution contained 20 mM 4- aminobutyrate and 10 mM 2-oxoglutarate adjusted to pH 8.4 with NaOH. For enzymatic assays the substrate solution (0.5 ml) was incubated with either precursor or mature protein at 37°C for 40 min. Aliquots (100 pl), withdrawn from the incubation mixture at several time intervals, were mixed with 100 p1 of the reagent solution, heated for 15 min in a water bath at 60°C, diluted to 2 ml by addition of water, and the fluorescence intensity was recorded at 460 nm (excitation 365 nm).

Controls, containing substrate without enzyme and the reagent cyclohexane-l,3-dione were run in parallel. A stan- dard curve for succinic semialdehyde reacted with cyclohexane-l,3-dione was determined for each set of enzymatic assays.

Determination of pyridoxal-5-P A method, based on fluorescence measurements of

pyridoxal-5-P reacted with aminooxyacetate, was used for determination of the cofactor of reticulocyte lysates.

The reagent was a solution containing aminooxyacetate (1 M) in 0.1 M potassium phosphate (pH 7.4).

290

The reagent solution (0.5 ml) was allowed to react with various concentrations of reticulocyte lysate (2 ml) in 0.1 M potassium phosphate (PH 7.4) at 37°C for 1 h. At the end of the reaction the mixtures were centrifuged and the fluorescence of the supernatant read at 440 nm (excitation 340 nm).

Controls, containing reticulocyte lysates without reagent, were run in parallel. A srandard curve, i. e. fluorescence emit- ted by known concentrations of pyridoxal-5-P reacted with aminooxyacetate, was used to evaluate the concentration of pyridoxal-5-P in the reticulocyte lysate samples.

Preparation of antiserum

Rabbit antiserum against porcine 4aminobutyrate aminotransferase was prepared as described by Isemura et al. [7]. A New Zealand white rabbit received three subcutaneous injections at 2-week intervals of an emulsified mixture (1.5 ml) of 4-aminobutyrate aminotransferase (1.5 mg/ml) and Freund's complete adjuvant. Blood was drawn from the ear vein 3 weeks after the final injection and thereafter.

The presence of anti-aminotransferase antibody was detected by immunodiffusion using the method of Ouchter- lony and Nilsson [8].

Purification of immunoglobulin G

Serum collected from the immunized rabbit was adjusted to 40% saturation in ammonium sulfate. The precipitate was dissolved in 50 mM potassium phosphate containing 0.15 M NaCl at pH 7.4, and dialyzed against the same buffer. The immunoglobulin G fraction of immune serum was purified by chromatography on DEAE-cellulose according to the procedure of Fahey and Horbett [9].

Western blots

Proteins were transferred from polyacrylamide gels to nitrocellulose membranes electrophoretically using a modification of the procedure of Britten et al. [lo]. Transfer to nitrocellulose was conducted at a constant voltage of 100 V, 3 h at 4°C.

Nitrocellulose membranes were rinsed four times with phosphate-buffered saline containing 0.3% Tween-20 (Pi/ NaCl/Tween buffer) and the proteins probed with the anti- aminotransferase antibody. The incubation was allowed to proceed at 22 "C four 2 h with gentle rocking.

After rinsing four times with Pi/NaC1/Tween buffer at 22 "C, the nitrocellulose membrane was incubated with horseradish peroxidase coupled to a goat antibody against rabbit IgG. The incubation time was 1 h at 22°C. The nitrocellulose membrane was rinsed four times with Pi/NaCl/ Tween buffer at 22"C, and incubated with 1.4 mM 3,3'- diaminobenzidine containing 0.1 % H20z in Pi/NaC1 buffer. The incubation was allowed to proceed at 22°C for 5 min. The reaction was stopped by washing the nitrocellulose blots with distilled water.

In vitro protein synthesis

Polysomal RNA was prepared by the method of Glisin et al. [l I]. Polyadenylated RNA was purified by affinity chromatography on oligo(dT) cellulose [12]. Both polysomal RNA and polyadenylated RNA were used independently in translation experiments.

The cell-free mRNA-dependent translation system from rabbit reticulocyte lysate was used in these experiments (BRL products). Conditions for the translation and measurements of incorporated radioactivity were exactly as described by Pelham and Jackson [13].

Translations were performed in the presence of either polysomal RNA (1 mg) or polyadenylated RNA (80 pg), together with 1000 pCi [35S]methionine and tRNA from calf liver (50 pg)/ml rabbit reticulocyte lysate. The incubation was allowed to proceed for 60 min at 30"C, then the samples were frozen for further studies.

Immunoprecipitat ion and SDSlpolyacrylamide gel electrophoresis

The pooled lysates (600 pl) were diluted with five volumes of 50 mM Tris/HCl (PH 7.2) containing 150 mM NaCI, 5 mM EDTA and 1% Triton X-100 (buffer A). After centrifugation (I50000 x g for 90 min at O'C), the supernatant was mixed with 300 ~ 1 2 0 % (v/v) suspension of protein-A - Sepharose in 50 mM Tris/HC1 (pH 7.2). After incubation at 4°C for 1 h the sample was centrifuged and the supernatant was mixed with 200 pg anti-(aminobutyrate aminotransferase) antibody and allowed to incubate at 4°C for 14 h. 500pl 20% (v/v) suspension of protein-A-Sepharose were added; and 2 h later the immunoprecipitated samples were collected by centrifugation. Subsequently the Sepharose-protein-A - antibody-antigen complexes were resuspended in 200 p1 of 50 mM Tris/HCl (pH 7.2). The suspension was divided in two portions of 100 p1 each. One portion was used for enzymatic assays the other portion was centrifuged and the precipitate washed five times with buffer A.

After the last wash the pellet was resuspended in 100 pl electrophoresis buffer (60 mM Tris/HCl pH 7.5,5% SDS, 5% 2-mercaptoethanol) and held for 3 min at 100°C.

After centrifugation the resulting supernatant was analyzed by SDS/polyacrylamide gel electrophoresis following the procedure of Laemmli [14]. Gels were stained with Coomassie blue and dried under reduced pressure for autoradiography. Kodak-X-Omat XRP-1 film was exposed for 4 weeks at - 80°C.

Materials

Nitrocellulose membranes were obtained from Bio-Rad. Guanidinium isothiocyanate, sodium dodecyl sarconate, ce- sium chloride, RNAse inhibitor, oligo-(dT)-cellulose, and in vitro translation kit were purchased from BRL, and [35S]methionine (1132 Ci/mmol) from NEN.

Protein-A - Sepharose CL4B, CM-Sephadex, DEAE- Sephadex were purchased from Pharmacia, and Tween-20, 3,3'-diaminobenzidine (3,3',4,4'-tetraaminobiphenyl tetra- hydrochloride), 30% H202, anti-(goat IgG) - peroxidase conjugate were purchased from Sigma.

RESULTS

Immunological purification of the precursor of 4-aminobutyrate aminotransferase

Polysomal RNA, isolated from pig brain, directs protein synthesis in a rabbit reticulocyte lysate system as revealed by the incorporation of [35S]methionine (4.8 x lo7 cpm/ml lysate) in the synthesized proteins.

29 1

0.2 0 L 0.6 0 8 1 0 Relative mobility

Fig. 1. SDSIpolyacrylamide gel electrophoresis and autoradiography of the in vitro translated 4-aminobutyrate aminotransferase. Polysomal RNA from pig brain was translated in vitro in a reticulocyte lysate in the presence of [35S]methionine as described in Experimental Procedure. Final reaction mixtures were subjected to immunoprecipitation with anti-(4aminobutyrate aminotransferase) antibody and separated by SDS/polyacrylamide gel electrophoresis (lane A). Autoradiography after SDS-PAGE of the translation products immunologically extracted with anti-(4aminobutyrate aminotransferase) antibody (lane B). Kodak X-Omat XRP-1 film was exposed at - 80°C for 4 weeks. The apparent M. of this newly synthesized protein was 52000 whereas purified mature 4-aminobutyrate aminotransferase exhibits a M, of 50000. The molecular mass markers (Fig. 1) were bovine serum albumin (l), catalase (2), lactate dehydrogenase (3) and mature 4-aminobutyrate aminotransferase (m). A protein band was not detected by autoradiography when non-immune serum was uscd for precipitation of the in vitro translation products

Products synthesized in vitro were purified by binding to the antibody of mature 4-aminobutyrate aminotransferase coupled to protein-A - Sepharose, and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography. As shown in Fig. 1, proteins of the in vitro translation system were precipitated by the specific antibody, but only one major radioactive band was detected by autoradiography.

The apparent molecular mass of this newly synthesized protein was determined by SDS-PAGE to be approximately 52 000, whereas purified mature 4-aminobutyrate aminotransferase exhibits a relative molecular mass of 50000 (Fig. 2). A protein band was not detected by autoradiography when non-immune serum was used for precipitation of the in vitro translation products.

The total radioactivity obtained from polypeptides precipitated by addition of trichloroacetic acid was 4.8 x lo7 cpm/ ml translation mixture, whereas the radioactivity precipitated by the antibody approached a value of 3 x lo3 cpm. Thus, approximately 0.006% of the total protein synthesized using polysomal RNA corresponds to the putative precursor of 4-aminobutyrate aminotransferase.

We have attempted to estimate the amount of precursor 4-aminobutyrate aminotransferase produced in a typical ex- periment using 60 pg polysomal RNA/60 p1 reticulocyte lysate.

Based on the specific activity of [35S]methionine, the number of methionines in the synthesized protein (1 51 100 kDa), the amount of radioactivity in trichloroacetic-acid- precipitable material and the ratio of 4-aminobutyrate aminotransferase to total synthesized products (gel density of autoradiography), a value of about 20 ng/60 p1 translation mixture is obtained.

Fig. 2. The immunoblottingpatterns of theprecursor of 4-aminobutyrate aminotransferase. The immunoprecipitate precursor and mature 4- aminobutyrate aminotransferase, transferred from SDS-PAGE to nitrocellulose membrane, were probed with the anti-(4-aminobutyrate aminotransferase) antibody and incubated with horseradish peroxidase coupled to goat antibody against rabbit IgG. After addition of the substrate of the horseradish peroxidase, followed by incubation at 22°C for 5min. and washing with water, the immunoblotting patterns show the presence of two protein bands differing in electrophoretic mobility. The immunoblot of precursor (P) (lane A), mature enzyme (m) (lane B), and the mixture of precursor and mature (lane C) 4-aminobutyrate aminotransferase are shown in the figure

Immunological identification of the precursor For verifying the identity of the putative precursor of

mitochondria1 4-aminobutyrate aminotransferase, the in vitro

292

translation products were analyzed by immunoblotting tech- niques.

To this end, proteins transferred from SDS-PAGE to nitrocellulose membrane were probed with the specific antibody and incubated with horseradish peroxidase coupled to goat antibody against rabbit IgG. After addition of the substrate of horseradish peroxidase, followed by incubation at 22°C for 5 min and washing with water, the results included in Fig. 2 were obtained.

The immunoblotting patterns show the presence of two protein bands differing in electrophoretic mobility. Thus, the antibody against mature 4-aminobutyrate aminotransferase recognizes both protein bands, a finding which is consistent with the hypothesis that mitochondria1 4-aminobutyrate aminotransferase is synthesized in the cytosol as a precursor of higher molecular mass than that of the mature form of the enzyme.

Determination of catalytic activity of the precursor of 4-uminobutyrute minotransferase

The fluorimetric method used to detect the formation of succinic semialdehyde in the reaction catalyzed by the aminotransferase was standardized with known concentrations of mature enzyme.

From 5 ng to 50 ng mature 4-aminobutyrate aminotransferase were incubated with the substrates (4- aminobutyrate and 2-oxoglutarate) at pH 8.4 for 40 min at 37 "C. The reaction was stopped by addition of cyclohexane- 1,3-dione as outlined in Experimental Procedure, and the fluorescence recorded at 460 nm (excitation 365 nm). Fig. 3 illustrates the results obtained for the assays of the mature enzyme.

The same method was applied to the evaluation of the catalytic activity of the precursor of 4-aminobutyrate aminotransferase in the translation mixtures as well as in the proteins immunoprecipitated by the specific antibody.

Rabbit reticulocyte lysate did not show any 4-aminobutyrate aminotransferase activity prior to the addition of polysomal RNA. However, following preincubation with polysomal RNA for 1 h at 30 "C, the catalytic activity of the lysate corresponded to the presence of approximately 25 ng protein/60 p1 lysate (Fig. 3).

The enzymatic assays were repeated five times in the absence and presence of exogenous pyridoxal-5-P (1 pM). In both cases the same level of catalytic activity was detected, indicating that the concentration of pyridoxal-5-P in rabbit reticulocyte lysate (0.05 pM) is enough to saturate the precursor of the aminotransferase. The catalytic activity of the newly synthesized proteins immunoprecipitated with the specific antibody were also tested using the fluorimetric method standardized with known concentrations of mature enzyme.

The immunoprecipitate, obtained as outlined in Ex- perimental Procedure, was centrifuged, washed twice with 10 mM potassium phosphate (PH 7.5), resuspended in the same buffer (0.5 ml) and mixed with 10 pg inactivated 4- aminobutyrate aminotransferase to displace the precursor protein from the antibody - protein-A - Sepharose complex.

After incubation for 1 h at 30°C, followed by centrifugation, the immunoprecipitated and supernatant were tested for radioactivity. Most of the radioactivity (90%) was recovered in the supernatant, indicating that displacement of the newly synthesized protein has taken place. The supernatant was then allowed to incubate with pyridoxal-5-P (1 pM) to ensure

I 400

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200

100

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0 10 20 30 LO 50 60 L-Arninobutyrote arninotransferase ingl

Fig. 3. Detection of the formation of succinic semialdehyde catalyzed by the precursor of 4-aminobutyrate aminotransferase. 5 - 50 ng mature 4-aminobutyrate aminotransferase (0) were incubated with the substrates (4-aminobutyrate and 2-oxoglutarate) at pH 8.4 for 40 min at 37°C. The reaction was stopped by addition of cyclohexane-l,3-dione as described in Experimental Procedure and the fluorescence recorded at 460 nm (excitation at 365 nm). The formation of succinic semialdehyde in the reaction catalyzed by the precursor of 4-aminobutyrate aminotransferase in the absence (0) and presence (0) of exogenous pyridoxal-5-P (1 pM) are given

$ 300 0 W - I .l

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0 10 20 30 40 5 0 Incubation time h in l

Fig. 4. Determination of catalytic activity of the precursor of 4- aminobutyrate aminotransferase. The in vitro translation products, immunoprecipitated with anti-(4-aminobutyrate aminotransferase) antibody as described in Experimental Procedure, were centrifuged, washed with 10 mM potassium phosphate buffer (pH 7.4), resuspended in the same buffer (0.5 d). This resuspension was incubated with 100 pg inactivated mature 4-aminobutyrate aminotransferase. After incubation for 1 h at 30"C, followed by centrifugation, the supernatant was used to measure catalytic activity. Results shown were obtained when samples of the precursor were incubated with the substrates of various lengths of time

binding of the cofactor at low protein concentrations, and tested for catalytic activity.

Fig.4 shows the results obtained when samples of precursor were tested for catalytic activity at various incubation times in the presence of substrates 4-aminobutyrate and 2-oxoglutarate. Similar results were obtained in five different experiments.

DISCUSSION Recently the subject of biosynthesis of aspartate amino-

transferase, a vitamin-B6-dependent enzyme, has attracted the

293

attention of several laboratories [ 15 - 181. In summary, the results reported on the biosynthesis of aspartate aminotransferase indicate that the mitochondrial enzyme is synthesized by free polysomes as a precursor of higher molecular mass than the mature protein; and it is translocated into mitochondria by a post-translational and energy-dependent process [19, 201. Several mitochondrial enzymes have been shown to be synthesized in the cytosol as larger precursors [21-251, but a few studies have been designed to test the catalytic behavior of the precursor enzymes. One of the few mitochondrial enzymes investigated is glutamate dehydrogenase, were it was demonstrated that the release of the extra peptide of the precursor is not obligatory first step for the formation of catalytically active species [26].

In the present studies we have demonstrated that another aminotransferase, i. e. 4-aminobutyrate aminotransferase, is synthesized in a cell-free system as a precursor of larger molecular mass.

The precursor of the aminotransferase was isolated by its specific immunoprecipitation with anti-(4-aminobutyrate aminotransferase) antibody. The precursor has an apparent relative molecular mass of around 52000 as compared to purified 4-aminobutyrate aminotransferase characterized by a M , of 50000.

An interesting aspect of the present work is the finding that the precursor of the aminotransferase binds pyridoxal- 5-P to generate a catalytically active species. Using a sensitive fluorimetric method, it was shown that rabbit reticulocyte lysates, incubated with polysomal RNA isolated from pig brain, yield newly synthesized proteins endowed with 4- aminobutyrate aminotransferase activity. Catalytic activity was detected without addition of exogeneous pyridoxal-5-P because the concentration of t h s cofactor in rabbit reticulocyte lysates is enough to saturate the binding sites of the precursor of the aminotransferase.

After immunoprecipitation with the specific antibody, followed by displacement with inactive enzyme, the newly synthesized protein was found to be catalytically competent.

Since the estimate of total precursor in the translation mixture from radioactivity incorporation into the immunoprecipitated protein (20 ng/60 pl) is comparable to the amount calculated from enzymatic assays (18 ng/60 pl), it seems rea- sonable to conclude that the specific activity of the precursor could be approaching that of the mature enzyme.

In considering the overall processing of the precursor of 4-aminobutyrate aminotransferase, it can be envisaged that two distinctive covalent chemical modification events have taken place: (a) the binding of pyridoxal-5-P leading to the formation of active species; and (b) the proteolytic removal of the extra peptide from the NH,-terminal portion of the precursor.

We consider it likely that extra peptide removal is relevant only to the translocation of the precursor into mitochondria; perhaps that extra peptide is needed for recognition of specific binding sites on the mitochondrial membrane as suggested for the translocation of aspartate aminotransferase [19] and other mitochondrial proteins [20]. However, it should be noted that recent work by Doonan et al. [21] indicates that mature mitochondrial aspartate aminotransferase is imported into mitochondria, whereas the cytosolic form of the enzyme failed to bind to mitochondria or to be imported into the organelle.

Based on the enzymatic results it appears that the step associated with the binding of pyridoxal-5-P is not dependent upon the proteolytic removal of the extra peptide of approximately 2000 M , . Thus, extra peptide removal from the

precursor is not an obligatory first step in the production of biologically active protein.

Although the complete amino acid sequence and structure of 4-aminobutyrate aminotransferase remains to be elucidated, we suggest that the extra peptide portion of the precursor sequence exists as an independent domain, quite sepa- rate and without interaction with the remainder of the macromolecule.

In this connection it is worthy to note that mature 4- aminobutyrate aminotransferase is cleaved by trypsin to yield an enzymatically active species, which can be separated from the split peptides by gel filtration [27]. The shortened enzyme derivative, endowed with activity, gives one band (M, = 95 000) on polyacrylamide gradient gel electrophoresis. Hence, it appears that a large portion of the NH2-terminal sequence belongs to a different domain, which does not in- teract with the catalytic domain of the protein. The results reported in this paper have some bearing of the biosynthesis of aminotransferases and other pyridoxal-5-P-dependent enzymes under in vivo conditions. It is conceivable that binding of pyridoxal-5-P to the precursor proteins occurs in the cytosol, where the cofactor is generated by the catalytic action of the cytosolic enzymes, pyridoxal kinase and pyridoxine- 5-P oxidase [28, 291. Alternatively there is the possibility that pyridoxal-5-P produced in the cytosol is transported into mitochondria prior to the binding of proteins located in both the intermembrane and matrix compartments.

While this explanation seems attractive, it may be disputed on the basis that most of the cofactor produced by the catalytic action of pyridoxal kinase and pyridoxine-5-P oxidase is immediately bound to vitamin-B,-dependent enzyme located in the cytosol[30].

The concentration of free pyridoxal-5-P in cytosol has been estimated to be approximately 0.1 pM [28], and if free pyridoxal-5-P is transported into the mitochondria matrix it is evident that this concentration of pyridoxal-5-P is consider- ably lower than the concentration needed to saturate vitamin- B6-dependent enzymes located in the mitochondrial matrix.

As an example it should be noted that the total concentration of aspartate and 4-aminobutyrate aminotransferase in mitochondria reaches a value of 10 pM [31]. Furthermore, it has been shown [31] that concentrations of free pyridoxal- 5-P of 10 pM in the mitochondria matrix brings about inactivation of other proteins; i. e. glutamate dehydrogenase [31].

This work was supported by a grant from National Institutes of Health (GM 27639-04A3) and by a grant from National Science Foundation (85- 1023 7).

REFERENCES 1. Braunstein, A. E. (1960) Theenzymes22, 113-181. 2. Kim, D. S., Churchich, J. E. & Schiltz, E. (1984) Chemical and

biological aspects of vitamin Bs catalysis, part B, pp. 161 - 167, Alan R. Liss, New York.

3. Churchich, J. E., Kim, D. S. & Schnackerz, K. D. (1983) FEBS Lett. 160, 221 -225.

4. Kirby, N., Fowler, L. J., Edwardson, J. M. &Phillips, N. I. (1985) Biochem. J . 230,481 -488.

5. Churchich, J . E. & Moses, U. (1981) J. Biol. Chern. 256, 1101 - 1104.

6. Beeler, T. & Churchich, J. E. (1978) Eur. J . Biochem. 85, 365- 371.

7 . Isemura, M., Sato, N. & Yoszawa, Z. (1982) J. Biol. Chem. 257,

8. Ouchterlony, M. & Nilsson, L. A. (1973) Handbook of experimental immunology (Weir, D. W., ed.) 2nd edn, Blackwell, Oxford.

14054- 14057.

294

9. Fahey, J. L. & Horbett, P. (1959) J. B id . Chem. 234,2645-2651. 10. Brittnen, M., Kupffer, P. & Morris, C. F. (1980) Anal. Biochem.

11. Glisin, V., Crkvenjakov, R. & Byus, C. (1974) Biochemistry 13,

12. Edmonds, M. (1971) in Procedures in nucleic acid research (Cantoni, G. L. & Davis, D. R., eds) vol. 2, p.924, Harper and Row, New York.

13. Pelham, H. R. B. & Jackson, R. J. (1976) Eur. J . Biochem. 67,

14. Laemmli, U. K. (1970) Nature (Lond.) 227,680-685. 15. Sonderegger, P., Jaussi, R., Chisten, P. & Gehring, H. (1982)

J. Biol. Chem. 257,3339-3343. 16. Sakakibara, R., Kamisaki, Y. & Wada, H. (1981) Biochem. Biu-

phys. Res. Commun. 102,235-239. 17. Sannia, G., Abrescia, P., Colomo, M., Giardina, P. & Marino,

G. (1982) Biochem. Biophys. Res. Commun. 105,444-449. 18. Barra, D., Bossa, F., Doonan, S., Fahmy, H. M. A., Hughes, G.

J., Martini, F., Petruzelli, R. & Wittman-Beibold, B. (1980) Eur. J. Biochem. 108,405-412.

102,459-471.

2633-2637.

247-256.

19. Behra, R. 8z Christen, P. (1986) J . Biol. Chem. 261,257-263. 20. Bohni, P., Gasser, S., Leawer, C. & Schatz, G. (1980) in The

organization and the expression of the mitochondria1 genome

(Kroon, A. M. & Saccone, C., eds) pp.423 -433, Elsevier Scien- tific, Amsterdam.

21. O’Donovan, C. K. M., Doonan, S., Marra, E., Passarella, S. & Quagliarello, E. (1985) Biochem. J. 228,609-614.

22. Morito, T., Mori, M., Tatibana, M. & Cohen, P. P. (1981) Bio- chem. Biophys. Res. Commun. 99,623 - 629.

23. Conboy, J. G., Kalousek, R. & Rosenberg, L. E. (1979) Proc. Natl Acad. Sci. USA 76, 5724- 5727.

24. Shore, G. C., Carignan, P. & Raymond, Y. (1979) J. Biol. Chem.

25. Maccecchini, M. L., Rudin, Y., Blobel, G. & Schatz, G. (1979)

26. Felipo, V., Miralles, V., Knecht, E., Hernandez-Yago, J. &

27. Kim, D. S. & Churchich, J. E. (1983) J. Biol. Chem. 258,11768 -

28. Kwok, F. & Churchich, J. E. (1979) J. Bid . Chem. 254, 1488-

29. Churchich, J. E. (1984) Eur. J . Biochem. 138, 327-332. 30. Lawrence, A. L. & Li, T. K. (1982) J. Bid. Chem. 257, 14903-

31. Choi, Soo-Young & Churchich, J. E. (1985) Biochim. Biophys.

254,3141 -3144.

Proc. Natl Acad. Sci. USA 76,343 - 347.

Grisolia, S. (1983) Eur. J. Biochem. 133,641 -644.

11 773.

1495.

14906.

Act0 830, 120-126.

biosynthesis of 4-aminobutyrate aminotransferase

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