Interconversion of multiple forms of tyrosine aminotransferase in vitro and in vivo in cultured hepatoma cells.

Corticosteroid-induced tyrosine aminotransferase (EC 2.6.1.5) from cultured hepatoma cells was separated by carboxymethyl-Sephadex chromatography into three molecular forms resembling those described previously in the rat liver. Enzyme forms were isolated and used as purified substrates to examine their in vitro interconversion by various subcellular fractions. Isolated form III was converted to forms II and I, and isolated form II was converted to form I by the coarse particulate fraction sedimenting at 1000 X g. This activity was inhibited by the serine enzyme inhibitor phenylmethane sulfonyl fluoride or by raising the pH to 8.7. Conversion of enzyme forms in vitro in the opposite direction (I leads to II leads to III) could not be detected. The distribution of enzyme forms in vivo was examined by the use of experimental conditions that prevent their in vitro interconversion during cell extraction. Tyrosine aminotransferase extracted from cell subjected to various treatments that affect the rates of enzyme synthesis or degradation existed always predominantly as form III. It appears, therefore, that multiple forms of tyrosine aminotransferase are not related to the turnover of this enzyme in vivo.     

Studies on the conversion of multiple forms of tyrosine aminotransferase in rat liver.

Studies from several laboratories have demonstrated the existence of at least three separable forms of the hepatic enzyme, tyrosine aminotransferase. The significance of these separable forms of the enzyme isolated in vitro for the nature and regulation of the enzyme in vivo has been the subject of some controversy. The studies reported in this paper demonstrate the existence of a heat-labile, pH- and temperature-dependent, nondialyzable component associated predominantly with the lysosomal and mitochondrial fraction of rat liver which catalyzes the conversion of form II to forms III and IV of the enzyme. The activity of this conversion factor is not significantly affected by F^?, molybdate ions, or two inhibitors of proteases. On the other hand, the cyanate ion completely inhibits the conversion of form II to forms III and IV of tyrosine aminotransferase, as do iodoacetate and oxidized glutathione. p-Chloromercuribenzoate also markedly inhibits the conversion. Kinetic studies suggest that the shift from one form to another follows the pathway: II to III to IV. Titration of the available sulfhydryl groups of the three forms of the enzyme demonstrates that form II possesses between 16 and 17 titratable SH groups per mole, while forms III and IV possess 15 and 13 or 14, respectively. The possible catalytic mechanism by which the conversion of the multiple forms of tyrosine aminotransferase is accomplished is discussed.     

Biochemical and biological properties of cortexillin III,a component of Dictyostelium DGAP1–cortexillin complexes

Cortexillins I–III are members of the α-actinin/spectrin subfamily of Dictyostelium calponin homology proteins. Unlike recombinant cortexillins I and II, which form homodimers as well as heterodimers in vitro, we find that recombinant cortexillin III is an unstable monomer but forms more stable heterodimers when coexpressed in Escherichia coli with cortexillin I or II. Expressed cortexillin III also forms heterodimers with both cortexillin I and II in vivo, and the heterodimers complex in vivo with DGAP1, a Dictyostelium GAP protein. Binding of cortexillin III to DGAP1 requires the presence of either cortexillin I or II; that is, cortexillin III binds to DGAP1 only as a heterodimer, and the heterodimers form in vivo in the absence of DGAP1. Expressed cortexillin III colocalizes with cortexillins I and II in the cortex of vegetative amoebae, the leading edge of motile cells, and the cleavage furrow of dividing cells. Colocalization of cortexillin III and F-actin may require the heterodimer/DGAP1 complex. Functionally, cortexillin III may be a negative regulator of cell growth, cytokinesis, pinocytosis, and phagocytosis, as all are enhanced in cortexillin III–null cells.     

Endogenous factor in rat liver that converts tyrosine aminotransferase form III to form I: a rapid assay of this converting factor.

After induction by cortisol, tyrosine aminotransferase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) present in rat liver homogenates can be resolved into three peaks of activity by CM-Sephadex chromatography. Based on differential elution of these forms by a linear KCl gradient, a three-tube assay was developed that quantitates the amount of form III relative to total enzyme. The assay was used to determine the presence of a factor in the liver that converts tyrosine aminotransferase form III to form I. Definitive evidence for the liberation of such a factor is presented.     

Studies on the different molecular forms of tyrosine aminotransferase from rat liver and hepatoma cells

In an attempt to clarify the significance of the separable forms of tyrosine aminotransferase, the enzyme from rat liver and from cultured hepatoma cells was studied by carboxymethyl-Sephadex chromatography. Our studies of the form conversion during the purification procedure of the enzyme, where all cellular components were quickly discarded, do not allow us to invoke a specific "converting factor", the existence of which in the particulate fraction has been suggested. Moreover the addition of serine protease inhibitors is not sufficient to prevent the classical conversion. More probably, several factors depending on the environmental conditions might influence different reactions which lead to a preferential conformation of the enzyme in vitro. The difference in the PO4- content of the various enzyme forms and the consecutive differences in negative charge may be the determining factor in the elution pattern of the three forms of the isolated soluble enzyme. This observation raises the possibility that phosphorylation might play a specific role in the regulation of tyrosine aminotransferase synthesis.     

Tryptophan degradation in Saccharomyces cerevisiae: Characterization of two aromatic aminotransferases

Tryptophan was found to be degraded in Saccharomyces cerevisiae mainly to tryptophol. Upon chromatography on DEAE-cellulose two aminotransferases were identified: Aromatic aminotransferase I was constitutively synthesized and was active in vitro with tryptophan, phenylalanine or tyrosine as amino donors and pyruvate, phenylpyruvate or 2-oxoglutarate as amino acceptors. The enzyme was six times less active with and had a twenty times lower affinity for tryptophan (K _m=6 mM) than phenylalanine or tyrosine. It was postulated thus that aromatic aminotransferase I is involved in vivo in the last step of tyrosine and phenylalanine biosynthesis. Aromatic aminotransferase II was inducible with tryptophan but also with the other two aromatic amino acids either alone or in combinations. With tryptophan as amino donor the enzyme was most active with phenylpyruvate and not active with 2-oxoglutarate as amino acceptor; its affinity for tryptophan was similar as for the other aromatic amino acids (K _m=0.2–0.4 mM). Aromatic aminotransferase II was postulated to be involved in vivo mainly in the degradation of tryptophan, but may play also a role in the degradation of the other aromatic amino acids.A mutant strain defective in the aromatic aminotransferase II (aat2) was isolated and its influence on tryptophan accumulation and pool was studied. In combination with mutations trp2 ^fbr, aro7 and cdr1-1, mutation aat2 led to a threefold increase of the tryptophan pool as compared to a strain with an intact aromatic aminotransferase II.     

Subcellular distribution of a factor inactivating tyrosine aminotransferase. Study of its mechanism and relationship to different forms of the enzyme.

The subcellular distribution of a tyrosine aminotransferase inactivating factor in rat liver has been investigated. Most of its activity is associated with plasma membranes, with minor amounts in mitochondria and endoplasmatic reticulum. The factor is also found in kidney and inactivates the enzyme reversibly in presence of cysteine, most likely by modification of -SH groups. ATP counteracts this inactivation only, when crude enzyme extracts are inactivated by purified subcellular fractions or when the purified enzyme is inactivated in presence of liver or kidney cortex homogenates. The relationship of this inactivation to reported different forms of the enzyme has been investigated. Form I of three different forms, that can be obtained by hydroxyl-apatite chromatography, is readily inactivated, form III can be partly converted to form I by incubation in presence of purified plasma membranes. The relationship of these findings to a possible multistep mechanism in the turnover of the enzyme discussed.     

A lysosomal factor that interconverts multiple forms of rat liver tyrosine aminotransferase.

A particulate factor of rat liver is described which interconverts three forms of rat liver cytosolic tyrosine aminotransferase $\text{in}$$\text{vitro}$ with no alteration of enzyme activity. The factor appears to be a heat- and pH-sensitive lysosomal protein. The interconversion process is stimulated $\text{in}$$\text{vitro}$ by 2.5 mM MgCl₂ and 2.5 mM ATP. Asparate aminotransferase multiple forms are also susceptible to $\text{in}$$\text{vitro}$ interconversion by the lysosomal factor. The properties of the factor explain several anomalous effects of $\text{in}$$\text{vitro}$ manipulation on the tyrosine aminotransferase forms which have been reported in the literature and implicate the form interconversion in the degradation of tyrosine aminotransferase.     

The hemoglobin of the crossopterygian fish, Latimeria chalumnae (Smith). Subunit structure and oxygen equilibria

There are five oxidation-reduction states of horseradish peroxidase which are interconvertible. These states are ferrous, ferric, Compound II (ferryl), Compound I (primary compound of peroxidase and H₂O₂), and Compound III (oxy-ferrous). The presence of heme-linked ionization groups was confirmed in the ferrous enzyme by spectrophotometric and pH stat titration experiments. The values of pK were 5.87 for isoenzyme A and 7.17 for isoenzymes (B + C). The proton was released when the ferrous enzyme was oxidized to the ferric enzyme while the uptake of the proton occurred when the ferrous enzyme reacted with oxygen to form Compound III. The results could be explained by assuming that the heme-linked ionization group is in the vicinity of the sixth ligand and forms a stable hydrogen bond with the ligand.The measurements of uptake and release of protons in various reactions also yielded the following stoichiometries: Ferric peroxidase + H₂O₂ → Compound I, Compound I + e^? + H⁺ → Compound II, Compound II + e^? + H⁺ → ferric peroxidase, Compound II + H₂O₂ → Compound III, Compound III + 3e^? + 3H⁺ → ferric peroxidase.Based on the above stoichiometries and assuming the interaction between the sixth ligand and heme-linked ionization group of the protein, it was possible to picture simple models showing structural relations between five oxidation-reduction states of peroxidase. Tentative formulae are as follows: [Pr·Po·Fe-(II) $\text{\$}\text{?}$PrH⁺·Po·Fe(II)] is for the ferrous enzyme, Pr·Po·Fe(III)OH₂ for the ferric one, Pr·Po·Fe(IV)OH^? for Compound II, Pr(OH^?)·Po⁺·Fe(IV)OH^? for Compound I, and PrH⁺·Po·Fe(III)O₂^? for Compound III, in which Pr stands for protein and Po for porphyrin. And by Fe(IV)OH^?, for instance, is meant that OH^? is coordinated at the sixth position of the heme iron and the formal oxidation state of the iron is four.     

Isolation and properties of three forms of phosphorylase and phosphorylase kinase from human skeletal muscle]

Three forms of phosphorylase (I, II and III), two of which (I and II) were active in the presence of AMP and one (III) was active without AMP, were isolated from human skeletal muscles. The pI values for phosphorylases b(I) and b(II) were found to be identical (5.8-5.9). During chromatofocusing a low molecular weight protein (M(r) = 20-21 kDa, pI 4.8) was separated from phosphorylase b(II). This process was accompanied by an increase of the enzyme specific activity followed by its decline. During reconstitution of the complex the activity of phosphorylase b(II) returned to the initial level. Upon phosphorylation the amount of 32P incorporated into phosphorylase b(II) was 2 times as low as compared with rabbit phosphorylase b and human phosphorylase b(I). It may be supposed that in the human phosphorylase b(II) molecule one of the two subunits undergoes phosphorylation in vivo. This form of the enzyme is characterized by a greater affinity for glycogen and a lower sensitivity to allosteric effectors (AMP, glucose-6-phosphate, caffeine) compared with phosphorylase b(I). Thus, among the three phosphorylase forms obtained in this study, form b(II) is the most unusual one, since it is partly phosphorylated by phosphorylase kinase to form a complex with a low molecular weight protein which stabilizes its activity. A partially purified preparation of phosphorylase kinase was isolated from human skeletal muscles. The enzyme activity necessitates Ca2+ (c0.5 = 0.63 microM). At pH 6.8 the enzyme is activated by calmodulin (c0.5 = 15 microM). The enzyme activity ratio at pH 6.8/8.2 is equal to 0.18.     

Characterization of calcium-independent forms of protein kinase C-beta in phorbol ester-treated rabbit platelets

The subcellular distribution, size, and activation state of protein kinase C (PKC) were studied after short term exposure of rabbit platelets to a saturating dose of 12-O-tetradecanoylphorbol 13-acetate (TPA). Cytosolic and Nonidet P-40-solubilized particulate extracts prepared from TPA-treated platelets were subjected to analytical column chromatography on Mono Q, hydroxylapatite, and Superose 6/12. PKC activity was assayed according to the ability of the enzyme to phosphorylate (i) histone H1 in the presence of the activators calcium, diacylglycerol, and phosphatidylserine; (ii) histone H1 after proteolytic activation of PKC with trypsin; and (iii) protamine in the absence of calcium and lipid. Within 1 min of TPA treatment of platelets, greater than 95% of the PKC activity was particulate associated, as assessed by all three methods. The particulate PKC activity from 1-min TPA-treated cells eluted from Mono Q with approximately 0.35 M NaCl (peak I), and it was highly dependent upon Ca2+ and lipid for optimal histone H1 phosphorylation. With longer exposure times of platelets to TPA, the disappearance of the Mono Q peak I form of PKC was correlated with the production of new PKC species that were released from Mono Q with approximately 0.4 M NaCl (peak II), approximately 0.5 M NaCl (peak III), and approximately 0.6 M NaCl (peak IV). These last forms of PKC were still lipid activated but exhibited little Ca2+ dependence. The Mono Q peak III form displayed a particularly high level of histone H1 phosphorylating activity in the absence of lipid and Ca2+. All of these forms behaved as approximately 65-kDa proteins on Superose 6/12, but on sodium dodecyl sulfate-polyacrylamide gels, Western blotting with anti-PKC-beta antibodies revealed immunoreactive polypeptides of approximately 79 kDa (Mono Q peaks I, II, and IV) and approximately 100-kDa (Mono Q peak III). Hydroxylapatite column chromatography permitted partial resolution of the Mono Q peaks I and II forms, which were eluted within a concentration range of potassium phosphate (100-150 mM) which was typical of the beta isozyme of PKC. Treatment of the Mono Q peak III and IV PKC forms with alkaline phosphatase resulted in the production of the peak I form, which implicated protein phosphorylation in the interconversion of the various PKC forms.     

Stimulation of collagenase production by collagen in mammalian cell cultures.

In this study, we investigated the possible regulatory role of collagen in collagenase production by cultured human skin fibroblasts and human and rabbit synovial cells. Addition of types I, II or III collagen in solution to the culture media markedly stimulated trypsin-activable collagenase activity in these cultures. In the human cell cultures the stimulatory effect of collagen was further enhanced by a soluble factor isolated from human monocyte culture media (Dayer, Russell and Krane, 1977). Both native and denatured forms of collagen stimulated enzyme production; their relative efficacy varied among the different types. The native form of both types I and II collagen showed a greater effect on collagenase production than the corresponding denatured form, whereas with type III collagen the denatured form was more effective.     

Absence of tyrosine aminotransferase multiple forms in several mammalian animals

Tyrosine aminotransferase multiple forms occurring in rat liver are not present in all mammalian species. Among animals examined only rat and mouse liver possesses multiple forms of tyrosine aminotransferase; in guinea-pig, rabbit, bovine and sheep liver the enzyme occurs in a single form. The presence of lysosomal converting factor (cathepsin T), responsible for arising of multiple forms of tyrosine aminotransferase in rat liver, has been checked in another species lacking enzyme subforms. Lysosomal extracts of guinea-pig liver interconverts tyrosine aminotransferase from rat liver; lysosomal extracts of rat liver does not generate multiple forms of the enzyme from guinea-pig liver. It has been concluded that in some animals hepatic tyrosine aminotransferase is resistant to the proteolytic cleavage by lysosomal cathepsin T.     

Multiple forms of rat-liver dihydropteridine reductase identified by their differing isoelectric points

Purified rat-liver dihydropteridine reductase is homogeneous by gel filtration (Mr approximately 51,000), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately 25,500), and native polyacrylamide gel electrophoresis, suggesting that the enzyme is composed of two identical subunits. However, analysis by isoelectric focusing has revealed three enzyme forms with approximate isoelectric points of 6.5, 5.9, and 5.7 (designated forms, I, II, and III, respectively). The three forms, isolated in 65% yield by preparative chromatofocusing, are stable in 0.05 M phosphate buffer, pH 6.8, containing 1 mM beta-mercaptoethanol and exhibit similar kinetic constants when the catalytic activities of the isolated forms are compared with quinonoid dihydrobiopterin as substrate. All forms generate complexes with the enzymatic cofactor NADH which are also detectable by IEF. When examined further by IEF under denaturing conditions in 6 M urea the enzyme demonstrates a differing subunit composition for its three forms. Two distinct subunits, designated alpha and beta, can be identified, and additional evidence suggests that the native enzyme forms I, II, and III represent the three differing dimeric combinations alpha alpha (form I), alpha beta (form II), and beta beta (form III).     

New crystal forms of glutamine synthetase and implications for the molecular structure.

New crystal forms of glutamine synthetase from Escherichia coli are reported. Two of these (II A and II B) demand that the dodecameric molecule contains a 2-fold axis of symmetry perpendicular to the apparent hexagonal face.Whereas forms II A and II B and others reported previously (I and III A) were grown from enzyme containing covalently bound AMP groups, a third new form (III C) was grown from enzyme lacking covalently bound AMP groups. Form III C is isomorphous with form III A. This demonstrates that the addition of AMP groups, which profoundly affect the catalytic and regulatory properties of glutamine synthetase, does not alter the dimensions of the molecular envelope. Thus adenylylation of the enzyme does not seem to cause a quaternary structural transition, though small changes of intensities suggest that there may be tertiary structural changes within the subunits.Other new forms include form III B, a low symmetry polymorph, closely related to form III A, and form IV, a trigonal polymorph with large asymmetric unit. All crystal forms are consistent with a symmetry of 622 for the glutamine synthetase molecule.     

Control of ketogenesis from amino acids. III. In vitro and in vivo studies on ketone body formation lipogenesis and oxidation of tyrosine by rats.

Ketone body formation from tyrosine was studied in rat liver in vitro with special references to the activities of tyrosine aminotransferse (EC 2.6.1.5) and p-hydroxyphenylpyruvate hydroxylase (EC 1.14.2.2). Liver was obtained from rats which had been given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase. The enzyme activities of the preparations were plotted against the amounts of ketone body formed from tyrosine. It was found that over a low range of tyrosine aminotransferase activities, activity was proportional to the amount of ketone body formed. However, above this range, ketone body formation ceased to increase and p-hydroxyphenylpyruvate started to accumulate. This inhibition of ketone body formation and accumulation of the p-hydroxyphenylpyruvate could be prevented by addition of ascorbate. These results suggest that the primary factor regulating metabolism of tyrosine in vitro is tyrosine aminotransferase and when the activity of this is high so that it is no longer rate limiting, p-hydroxyphenylpyruvate hydroxylase becomes the rat limiting step because its activity is inhibited by the accumulation of p-hydroxyphenylpyruvate. For in vivo studies rats were given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase and then injected with a tracer dose of [U- or 1- 14C]tyrosine. Then their respiratory 14CO2 and the incorporation of 14C into total lipids of liver were measured. The amounts of radioactivity in CO2 and lipids were found to be proportional to the tyrosine aminotransferase activity and were not affected by the free tyrosine concentration in the liver. After injection of [U- 14C]acetate the radioactivities in CO2 and lipids were not proportional to the tyrosine aminotransferase activity. These results indicate that the enzyme activity also regulates tyrosine metabolism in vivo. In vivo studied gave no evidence of the participation of p-hydroxyphenylpyruvate hydroxylase in regulation of tyrosine metabolism.     

Phenylalanine- and tyrosine-auxotrophic mutants of Saccharomyces cerevisiae impaired in transamination

This paper reports the first isolation of Saccharomyces cerevisiae mutants lacking aromatic aminotransferase I activity (aro8), and of aro8 aro9 double mutants which are auxotrophic for both phenylalanine and tyrosine, because the second mutation, aro9, affects aromatic aminotransferase II. Neither of the single mutants displays any nutritional requirement on minimal ammonia medium. In vitro, aromatic aminotransferase I is active not only with the aromatic amino acids, but also with methionine, α-aminoadipate, and leucine when phenylpyruvate is the amino acceptor, and in the reverse reactions with their oxo-acid analogues and phenylalanine as the amino donor. Its contribution amounts to half of the glutamate:2-oxoadipate activity detected in cell-free extracts and the enzyme might be identical to one of the two known α-aminoadipate aminotransferases. Aromatic aminotransferase I has properties of a general aminotransferase which, like several aminotransferases of Escherichia coli, may be able to play a role in several otherwise unrelated metabolic pathways. Aromatic aminotransferase II also has a broader substrate specificity than initially described. In particular, it is responsible for all the measured kynurenine aminotransferase activity. Mutants lacking this activity grow very slowly on kynurenine medium.     

Control of ketogenesis from amino acids: III. In vitro and in vivo studies on ketone body formation, lipogenesis and oxidation of tyrosine by rats

Ketone body formation from tyrosine was studied in rat liver in vitro with special references to the activities of tyrosine aminotransferase (EC 2.6.1.5) and p-hydroxyphenylpyruvate hydroxylase (EC 1.14.2.2). Liver was obtained from rats which had been given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase. The enzyme activities of the preparations were plotted against the amounts of ketone body formed from tyrosine. It was found that over a low range of tyrosine aminotransferase activities, activity was proportional to the amount of ketone body formed. However, above this range, ketone body formation ceased to increase and p-hydroxyphenylpyruvate started to accumulate. This inhibition of ketone body formation and accumulation of the p-hydroxyphenylpyruvate could be prevented by addition of ascorbate. These results suggest that the primary factor regulating metabolism of tyrosine in vitro is tyrosine aminotransferase and when the activity of this is high so that it is no longer rate limiting, p-hydroxyphenylpyruvate hydroxylase becomes the rate limiting step because its activity is inhibited by the accumulation of p-hydroxyphenylpyruvate.For in vivo studies rats were given a high protein diet or cortisol to induce various levels of tyrosine aminotransferase and then injected with a tracer dose of [U- or 1-¹⁴ C]tyrosine. Then their respiratory ¹⁴CO₂ and the incorporation of ¹⁴C into total lipids of liver were measured. The amounts of radioactivity in CO₂ and lipids were found to be proportional to the tyrosine aminotransferase activity and were not affected by the free tyrosine concentration in the liver. After injection of [U-¹⁴C] acetate the radioactivities in CO₂ and lipids were not proportional to the tyrosine aminotransferase activity. These results indicate that the enzyme activity also regulates tyrosine metabolism in vivo. In vivo studies gave no evidence of the participation of p-hydroxyphenylpyruvate hydroxylase in regulation of tyrosine metabolism.     

Structure of the core regions in lipopolysaccharides from Escherichia coli K12 W2252-11U−, the Ter-15 mutant,and Ter-15 (F′-lac) and Ter-15 (F+) cells

From Escherichia coli K12 W2252-11U^? cells, the Ter-15 mutant, the Ter-15 (F′-lac) and the Ter-15 (F⁺) cells, lipopolysaccharides were isolated and the primary structure of its core oligosaccharides was elucidated. When the F′-lac episome is transferred to the Ter-15 mutant by conjugation, the structure of the glucose III(1 → 3)glucose II(1 → 3)glucose I residue and the galactose I(1 → 2)-linked to the glucose I residue in the core oligosaccharide from the Ter-15 mutant changes into the structure of the glucose IV(1 → 6)glucose III(1 → 2)glucose II(1 → 3)glucose I residue and the galactose I (1 → 6)-linked to the glucose I residue in the core oligosaccharide from the Ter-15 (F′-lac) cells, but the core oligosaccharide in the Ter-15 (F⁺) cells is the same structure with that of the core oligosaccharide from the Ter-15 mutant when F⁺ episome is transferred to the Ter-15 mutant. Also, the core oligosaccharide from the Ter-15 (F′-lac) cells shows the same structure with that of the core oligosaccharide from E. coli K12 W2252-11U^? cells (the parent cells). As the result, the ability to produce the structure of the core oligosaccharide in E. coli K12 W2252-11U^? cells is recovered in the Ter-15 (F′-lac) cells by the dominant expression of lac gene or its containing DNA segment in F′-lac episome.     

Translation of poly-A RNA from rat liver in vitro. Evidence for a high molecular weight subunit of tyrosine aminotransferase

Poly-A RNA extracted from the rat liver was translated in a cell-free wheat germ system and a rabbit reticulocyte lysate. The subunit of tryptophan pyrrolase precipitated by specific antiserum after synthesis in vitro has the same molecular weight as the corresponding subunit derived from the rat liver. With specific antiserum prepared against tyrosine aminotransferase, however, a radioactive protein from both the in vitro assays was precipitated with an about 5% higher molecular weight than the tyrosine aminotransferase subunit precipitated from rat liver. The immunological evidence and the comparison of the specific peptide patterns prepared by cyanogen bromide treatment showed that the in vitro product corresponds to tyrosine aminotransferase. Various concentrations of potassium or spermidine used in the wheat germ translation system did not alter the size of the enzyme subunit synthesized. The run of the tyrosine aminotransferase purified form the rat liver in the SDS-polyacrylamide gel electrophoresis was not influenced by treatment with Escherichia coli alkaline phosphatase. The possibility is discussed that the larger enzyme synthesized in vitro represents a precursor molecule which is cleaved proteolytically in vivo.