S-adenosylmethionine: protein-arginine methyltransferase. Purification and mechanism of the enzyme.

Protein methylase I (S-adenosylmethionine: protein-arginine methyltransferase, EC 2.1.1.23) has been purified from calf brain approximately 120-fold with a 14% yield. The final preparation is completely free of any other protein-specific methyltransferases and endogenous substrate protein. The enzyme has an optimum pH of 7.2 and pI value of 5.1. The Km values for S-adenosyl-L-methionine, histone H4, and an ancephalitogenic basic protein are 7.6 X 10(-6), 2.5 X 10(-5), and 7.1 X 10(-5) M, respectively, and the Ki value for S-adenosyl-L-homocysteine is 2.62 X 10(-6) M. The enzyme is highly specific for the arginine residues of protein, and the end products after hydrolysis of the methylated protein are NG,NG-di(asymmetric), NG,N'G-di(symmetric), and NG-monomethylarginine. The ratio of [14C]methyl incorporation into these derivatives by enzyme preparation at varying stages of purification remains unchanged at 40:5:55, strongly indicating that a single enzyme is involved in the synthesis of the three arginine derivatives. The kinetic mechanism of the protein methylase I reaction was studied with the purified enzyme. Initial velocity patterns converging at a point on the extended axis of abscissas were obtained with either histone H4 or S-adenosyl-L-methionine as the varied substrate. Product inhibition by S-adenosyl-L-homocysteine with S-adenosyl-L-methionine as the varied substrate was competitive regardless of whether or not the enzyme was saturated with histone H4. On the other hand, when histone H4 is the variable substrate, noncompetitive inhibition was obtained with S-adenosyl-L-homocysteine under conditions where the enzyme is not saturated with the other substrate, S-adenosyl-L-methionine. These results suggest that the mechanism of the protein methylase I reaction is a Sequential Ordered Bi Bi mechanism with S-adenosyl-L-methionine as the first substrate, histone H4 as the second substrate, methylated histone H4 as the first product, and S-adenosyl-L-homocysteine as the second product released.     

Enzymatic methyl esterification of Escherichia coli ribosomal proteins.

Enzymatic methyl ester formation in Escherichia coli ribosomal proteins was observed. Alkali lability of the methylated proteins and derivatization of the methyl groups as methyl esters of 3,5-dinitrobenzoate indicate the presence of protein methyl esters. The esterification reaction occurs predominantly on the 30S ribosomal subunit, with protein S3 as the major esterified protein. When the purified 30S subunit was used as the methyl acceptor, protein S9 was also found to be esterified. The enzyme responsible for the esterification of free carboxyl groups in proteins, protein methylase II (S-adenosyl-L-methionine:protein carboxyl methyltransferase, EC 2.1.1.24), was identified in E. coli Q13. This enzyme is extremely unstable when compared with that from mammalian origin. By molecular sieve chromatography, E. coli protein methylase II showed multiple peaks, with a major broad peak around 120,000 daltons and several minor peaks in the lower-molecular-weight region. Rechromatography of the major enzyme peak showed activities in several fractions that are much lower in molecular weight. The substrate specificity of the E. coli enzyme is similar to that of the mammalian enzyme. The Km value for S-adenosyl-L-methionine is 1.96 X 10(-6) M, and S-adenosyl-L-homocysteine was found to be a competitive inhibitor, with a Ki value of 1.75 X 10(-6) M.     

Human placenta S-adenosylmethionine: protein carboxyl O-methyltransferase (protein methylase II). Purification and characterization

1. Protein methylase II was purified from human placenta approx. 8700-fold with a yield of 14%. 2. Unlike protein methylase II from other sources, the activity of human placenta enzyme was completely inhibited by 2 mM Cu2+. Other divalent ions were without effect. 3. Human chorionic gonadotropin (HCG), immunoglobulin A and calf thymus histones served as good in vitro substrates for the enzyme, particularly HCG. 4. The Km for S-adenosyl-L-methionine and Ki for S-adenosyl-L-homocysteine were 2.08 x 10(-6) and 5.8 x 10(-7) M, respectively. 5. The protein methylase II activity in human placenta changed with gestational age, the activity at 1st and 2nd trimester being approximately twice that of term placenta.     

Purification and properties of S-adenosyl-L-methionine: caffeic acid O-methyltransferase from leaves of spinach beet (Beta vulgaris L).

1. An enzyme catalysing the methylation of caffeic acid to ferulic acid, using S-adenosyl-L-methionine as methyl donor, has been extracted from leaves of spinach beet and purified 75-fold to obtain a stable preparation. 2. The enzyme showed optimum activity at pH 6.5, and did not require the addition of Mg2+ for maximum activity. 3. It was most active with caffeic acid, but showed some activity with catechol, protocatechuic acid and 3,4-dihydroxybenzaldehyde. The Km for caffeic acid was 68 muM. 4. 4. The Km for S-adenosyl-L-methionine was 12.5 muM. S-Adenosyl-L-homocystein (Ki = 4.4 muM) was a competitive inhibitor of S-adenosyl-L-methionine. 5. The synthesis of S-adenosyl-L-homocysteine from adenosine and L-homocysteine and its consequent effect on caffeic acid methylation were demonstrated with a partially-purified preparation from spinach-beet leaves, which possessed both S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) and adenosine nucleosidase (EC 3.2.2.7) activities. This preparation was also able to catalyse the rapid breakdown of S-adenosyl-L-homocysteine to adenosine and adenine; the possible significance of this reaction in relieving the inhibition of caffeic acid methylation by S-adenosyl-L-homocystein is discussed.     

Human placental protein methylase--I. Purification and characterization.

1. Protein methylase I (S-adenosylmethionine[:]protein-arginine N-methyltransferase; EC 2.1.1.23) which methylates protein-bound arginine residues has been purified from human term placenta 400-fold with an approximate yield of 6%. 2. When histone was used as in vitro substrate, the methylation products were found to be NG-mono-, NG, NG-di- and NG, N'G-dimethylarginine. The enzyme was found to be sensitive toward Cu2+ with Ki value of 8 x 10(-5) M. The Km value for S-adenosyl-L-methionine was 5 x 10(-6) M. 3. When this partially purified protein methylase I was incubated with isolated human placental nuclei and S-adenosyl-L-[methyl-3H]methionine, the major endogenous [methyl-3H]-labeled proteins were protein species of 23, 38, 45 and 68 kDa, the 23 kDa species being the most predominant. 4. The endogenous enzyme activity during the pregnancy increased significantly, reaching more than 4 times the initial activity at the end of term.     

Self-methylation of BspRI DNA-methyltransferase.

The DNA (cytosine-5)-methyltransferase (m5C-MTase) M.BspRI is able to accept the methyl group from the methyl donor S-adenosyl-L-methionine (AdoMet) in the absence of DNA. Transfer of the methyl group to the enzyme is a slow reaction relative to DNA methylation. Self-methylation is dependent on the native conformation of the enzyme and is inhibited by S-adenosyl-L-homocysteine, DNA and sulfhydryl reagents. Amino acid sequencing of proteolytic peptides obtained from M.BspRI, which had been methylated with [methyl-3H]AdoMet, and thin layer chromatography of the modified amino acid identified two cysteines, Cys156 and Cys181 that bind the methyl group in form of S-methylcysteine. One of the acceptor residues, Cys156 is the highly conserved cysteine which plays the role of the catalytic nucleophile of m5C-MTases.     

Purification of protein methylase II from human erythrocytes

Protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC. 2.1.1.24) which methyl esterifies free carboxyl groups of protein substrate using S-adenosyl-L-methionine as the methyl donor, has been purified from human erythrocytes approximately 13000-fold with a yield of 12%. The purified enzyme was over 95% pure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A bulk of hemoglobin present in the erythrocyte lysate, which severely limited the use of affinity chromatography for purification, was effectively removed by ammonium sulfate precipitation and by the subsequent salt washing of the precipitates followed by molecular sieve chromatography on Sephadex G-75. This preparation can be further purified by affinity chromatography, in which S-adenosyl-L-homocysteine is covalently linked to Sepharose-4B, followed by Sephadex G-75 chromatography to yield an enzyme with an activity of 27000 pmol methyl group transferred/mg/min at 37 degrees C.     

PRMT1 is the predominant type I protein arginine methyltransferase in mammalian cells

Type I protein arginine methyltransferases catalyze the formation of asymmetric omega-N(G),N(G)-dimethylarginine residues by transferring methyl groups from S-adenosyl-L-methionine to guanidino groups of arginine residues in a variety of eucaryotic proteins. The predominant type I enzyme activity is found in mammalian cells as a high molecular weight complex (300-400 kDa). In a previous study, this protein arginine methyltransferase activity was identified as an additional activity of 10-formyltetrahydrofolate dehydrogenase (FDH) protein. However, immunodepletion of FDH activity in RAT1 cells and in murine tissue extracts with antibody to FDH does not diminish type I methyltransferase activity toward the methyl-accepting substrates glutathione S-transferase fibrillarin glycine arginine domain fusion protein or heterogeneous nuclear ribonucleoprotein A1. Similarly, immunodepletion with anti-FDH antibody does not remove the endogenous methylating activity for hypomethylated proteins present in extracts from adenosine dialdehyde-treated RAT1 cells. In contrast, anti-PRMT1 antibody can remove PRMT1 activity from RAT1 extracts, murine tissue extracts, and purified rat liver FDH preparations. Tissue extracts from FDH(+/+), FDH(+/-), and FDH(-/-) mice have similar protein arginine methyltransferase activities but high, intermediate, and undetectable FDH activities, respectively. Recombinant glutathione S-transferase-PRMT1, but not purified FDH, can be cross-linked to the methyl-donor substrate S-adenosyl-L-methionine. We conclude that PRMT1 contributes the major type I protein arginine methyltransferase enzyme activity present in mammalian cells and tissues.     

Partial purification and characterization of a protein lysine methyltransferase from plasmodia of Physarum polycephalum.

Plasmodia of Physarum polycephalum have an active protein lysine methyltransferase (S-adenosylmethionine:protein-lysine methyltransferase, EC 2.1.1.43). This enzyme has been purified 40-fold with a 13% yield, and it catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to the epsilon-amino group of lysine residues with formation of N epsilon-mono-, N epsilon-di-, and N epsilon-trimethyllysines in a molar ratio of 4:1:1 based on [14C]methyl incorporation into the methylated lysines. The ratio remains unchanged at all stages of the partial purification, as well as after fractionation by sucrose density gradient centrifugation and gel electrophoresis. The rate of protein methylation is time dependent, enzyme concentration dependent, and requires the presence of a sulfhydryl reducing agent for optimal activity. The enzyme has optimal activity at pH 8 and is inhibited by S-adenosyl-L-homocysteine and EDTA. Lysine-rich and arginine-rich histones serve as the most effective exogenous protein acceptors; P. polycephalum actomyosin is inactive, and chick skeletal myofibrillar proteins are 25% as effective as exogenous mixed histones as substrates. Lysine, polylysine, ribonuclease A, cytochrome c, and bovine serum albumin are not methylated.     

Myelin basic protein inhibits histone-specific protein methylase I

Bovine brain myelin basic protein, free of associated proteolytic activity, was found to be a specific inhibitor of histone-specific protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23) purified from bovine brain. 50% of the methyl group incorporation into the histone substrate catalyzed by the methylase I was inhibited by myelin basic protein at a concentration of 0.326 mM. However, neither of the peptide fragments (residues 1-116 and residues 117-170) generated by the chemical cleavage of myelin basic protein at the tryptophan residue retained the inhibitory activity for histone-specific protein methylase I. Proteins such as gamma-globulin, bovine serum albumin, bovine pancreatic ribonuclease and polyarginine did not exhibit significant inhibitory activity toward the enzyme. The Ki value for myelin basic protein was estimated to be 3.42 X 10(-5) M for histone-specific protein methylase I and the nature of the inhibition was uncompetitive toward histone substrate.     

Purification and characterization of S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase, the enzyme responsible for biosynthesis of the volatile ester methyl benzoate in flowers of Antirrhinum majus

S-Adenosyl-L-methionine:benzoic acid carboxyl methyltransferase (BAMT) catalyzes the transfer of the methyl group of S-adenosyl-L-methionine (SAM) to the carboxyl group of benzoic acid to make the volatile ester methyl benzoate, one of the most abundant scent compounds of snapdragon, Antirrhinum majus. The enzyme was purified from upper and lower petal lobes of 5- to 10-day-old snapdragon flowers using DE53 anion exchange, Phenyl-Sepharose 6FF, and Mono-Q chromatography. The purified protein has a pH optimum of 7.5 and is highly specific for benzoic acid, with no activity toward several other naturally occurring substrates such as salicylic acid, cinnamic acid, and their derivatives. The molecular mass values for native and denatured protein were 100 and 49 kDa, respectively, suggesting that the active enzyme is a homodimer. The addition of monovalent cations K+ and NH4+ stimulates BAMT activity by a factor of 2, whereas the addition of Fe2+ and Cu2+ has a strong inhibitory effect. Plant-purified BAMT has Km values of 28 microM and 1.1 mM for SAM and benzoic acid, respectively (87 microM and 1.6 mM, respectively, for plant BAMT expressed in Escherichia coli). Product inhibition studies showed competitive inhibition between SAM and S-adenosyl-L-homocysteine (SAH), with a Ki of 7 microM, and noncompetitive inhibition between benzoic acid and SAH, with a Ki of 14 microM.     

Protein methylases in Trypanosoma brucei brucei: activities and response to DL-alpha-difluoromethylornithine

Protein methylases I, II and III were detected in extracts of Trypanosoma brucei brucei, and characterized according to the specific amino substituent methylated. Only protein methylase II activity was elevated by difluoromethylornithine treatment of T. b. brucei, and hence this enzyme was characterized further. Protein methylase II transferred methyl groups from S-adenosyl-L-methionine (S-AdoMet) to the carboxyl residues of several protein substrates, exhibiting highest activity with histone VIII-S (arginine-rich subgroup f3). The crude enzyme had an apparent Km for histone VIII-S of 28 mg ml-1 (11.4 mM-aspartyl and 18.4 mM-glutamyl residues methylated), and an apparent Km for S-AdoMet of 8.4 microM. T. b. brucei protein methylase II was sensitive to inhibition by S-adenosyl-L-homocysteine and its analogue sinefungin with apparent Ki values of 12.9 and 1.6 microM, respectively. Using a partially purified preparation, analysis of kinetic data in the presence and absence of sinefungin indicated that this analogue acts as a competitive inhibitor of the S-AdoMet binding site, and as a non-competitive inhibitor of the (protein) histone VIII-S binding site. The possible role of the enzyme in morphological control and its potential as a chemotherapeutic target are discussed.     

Restriction and modification in Bacillus subtilis: two DNA methyltransferases with BsuRI specificity. II. Catalytic properties, substrate specificity, and mode of action

The properties of two DNA methyltransferases, termed M. BsuRIa and M. BsuRIb, whose isolation was described in the preceding paper (Günthert, U., Freund, M., and Trautner, T. A. (1981) J. Biol. Chem. 256, 9340-9345) were compared. Both enzymes recognize the same target sequence in double-stranded DNA, leading to methylation of the internal cytosine: 5'GGCC. The enzymes have identical reaction constants with their substrates, DNA (km = 2.7 nM for the 5' GGCC sequence), and S-adenosyl-L-methionine (km = 0.7 microM). Initial rates of methyl group transfer were proportional to enzyme concentration over a range of 50-fold, indicating absence of aggregation. The enzymes are different in their ionic strength requirements using Tris-HCl, pH 8.4. M. BsuRIa is most active at 100 mM, M. BsuRIb at 440 mM. As measured by incorporation kinetics and heat inactivation, M. BsuRIa is the more stable enzyme of the two. Equilibrium dialysis was used to study the mode of methyl group transfer to the DNA with either enzyme. The data indicate that initially S-adenosyl-L-methionine binds to methyltransferase. This complex attaches to either modified or nonmodified DNA. The methyl group will then be transferred to a nonmodified target sequence, leading to the dissociation of enzyme and S-adenosyl-L-homocysteine from the DNA.     

Partial purification and properties of adenosine nucleosidase from leaves of spinach beet (Beta vulgaris L.)

Summary Adenosine nucleosidase (EC 3.2.2.7), which catalyses the irreversible hydrolysis of adenosine to adenine and ribose, has been isolated and purified about 40-fold from leaves of spinach beet (Beta vulgaris L.). The enzyme appeared to be specific for adenosine only among the naturally-occurring nucleosides, but comparable activity was also found with adenosine N-oxide. Adenosine hydrolysis, which had an optimum at pH 4.5, did not require phosphate ions nor was it stimulated by their presence. The Michaelis constant for this substrate was 11 M. Whereas the rate of adenosine hydrolysis was unaffected by DL-homocysteine, L-methionine and ribose, it was sensitive to the presence of adenine, S-adenosyl-L-methionine, S-adenosyl-L-homocysteine, AMP and deoxyadenosine. The role of this enzyme in plant metabolism is discussed.Abbreviations BSA bovine serum albumin - SAH S-adenosyl-L-homocysteine - SAM S-adenosyl-L-methionine     

An endogenous proteinacious inhibitor in porcine liver for S-adenosyl-L-methionine dependent methylation reactions: identification as oligosaccharide-linked acyl carrier protein

A proteinacious inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent transmethylation reactions was purified to homogeneity from porcine liver by size exclusion chromatography and FPLC. The molecular weight of the inhibitor was 12,222 Da. A 7400 Da polypeptide fragment of the purified inhibitor was sequenced by matrix-associated laser desorption ionization; time-of-flight MS, and was found to be identical with the known sequence of spinach acyl carrier protein (ACP). Although the remainder of the molecule was not clearly defined, 1H and H-H correlation of spectroscopy (COSY) NMR analysis revealed the presence of an oligosaccharide with alpha-glycosidic linkage. The purified oligosaccharide-linked ACP inhibited several AdoMet-dependent transmethylation reactions such as protein methylase I and II. S-farnesylcysteine O-methyltransferase, DNA methyltransferase and phospholipid methyltransferase. Protein methylase II was inhibited with a Ki value of 2.4 x 10(-3) M in a mixed inhibition pattern, whereas a well-known competitive product inhibitor S-adenosyl-L-homocysteine (AdoHcy) had Ki value of 6.3 x 10(-6) M. Commercially available active ACP fragments (65-74) and ACP from Escherichia coli had less inhibitory activity toward S-farnesylcysteine O-methyltransferase than the purified inhibitor. The biological significance of this oligosaccharide-linked ACP which has two seemingly unrelated functions (inhibitor for transmethylation and fatty acid biosynthesis) remains to be elucidated.     

Yeast mRNA cap methyltransferase is a 50-kilodalton protein encoded by an essential gene.

RNA (guanine-7-)methyltransferase, the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA, was isolated from extracts of Saccharomyces cerevisiae. The yeast enzyme catalyzed methyl group transfer from S-adenosyl-L-methionine to the guanosine base of capped, unmethylated poly(A). Cap methylation was stimulated by low concentrations of salt and was inhibited by S-adenosyl-L-homocysteine, a presumptive product of the reaction, but not by S-adenosyl-D-homocysteine. The methyltransferase sedimented in a glycerol gradient as a single discrete component of 3.2S. A likely candidate for the gene encoding yeast cap methyltransferase was singled out on phylogenetic grounds. The ABD1 gene, located on yeast chromosome II, encodes a 436-amino-acid (50-kDa) polypeptide that displays regional similarity to the catalytic domain of the vaccinia virus cap methyltransferase. That the ABD1 gene product is indeed RNA (guanine-7-)methyltransferase was established by expressing the ABD1 protein in bacteria, purifying the protein to homogeneity, and characterizing the cap methyltransferase activity intrinsic to recombinant ABD1. The physical and biochemical properties of recombinant ABD1 methyltransferase were indistinguishable from those of the cap methyltransferase isolated and partially purified from whole-cell yeast extracts. Our finding that the ABD1 gene is required for yeast growth provides the first genetic evidence that a cap methyltransferase (and, by inference, the cap methyl group) plays an essential role in cellular function in vivo.     

Purification, properties and kinetic mechanism of flavonol 8-O-methyltransferase from Lotus corniculatus L

A novel O-methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to the 8-hydroxyl group of flavonols was purified about 1200-fold from Lotus flower buds, by precipitation with ammonium sulfate and successive chromatography on columns of Sephadex G-100, S-adenosyl-L-homocysteine--Agarose, hydroxyapatite and Polybuffer ion exchanger. The enzyme exhibited strict specificity for position 8 of 8-hydroxyquercetin and 8-hydroxykaempferol, a pH optimum at 7.9, a pI value of 5.5, an Mr of 55 X 10(3) and required Mg2+ and SH groups for activity. The Km values for 8-hydroxykaempferol and S-adenosyl-L-methionine were 1.3 microM and 53 microM, respectively. The data obtained from substrate interaction and product inhibition studies are expected for a steady-state ordered bi-bi mechanism, with 8-hydroxyflavonol binding before S-adenosyl-L-methionine followed by the release of S-adenosyl-L-homocysteine and 8-methoxyflavonol. An alternative mechanism that may also fit the data is the mono-iso Theorell-Chance with the inverse binding sequence and an isomerization step of the free enzyme.     

A kinetic study of human protein arginine N-methyltransferase 6 reveals a distributive mechanism

Human protein arginine N-methyltransferase 6 (PRMT6) transfers methyl groups from the co-substrate S-adenosyl-L-methionine to arginine residues within proteins, forming S-adenosyl-L-homocysteine as well as omega-N(G)-monomethylarginine (MMA) and asymmetric dimethylarginine (aDMA) residues in the process. We have characterized the kinetic mechanism of recombinant His-tagged PRMT6 using a mass spectrometry method for monitoring the methylation of a series of peptides bearing a single arginine, MMA, or aDMA residue. We find that PRMT6 follows an ordered sequential mechanism in which S-adenosyl-L-methionine binds to the enzyme first and the methylated product is the first to dissociate. Furthermore, we find that the enzyme displays a preference for the monomethylated peptide substrate, exhibiting both lower K(m) and higher V(max) values than what are observed for the unmethylated peptide. This difference in substrate K(m) and V(max), as well as the lack of detectable aDMA-containing product from the unmethylated substrate, suggest a distributive rather than processive mechanism for multiple methylations of a single arginine residue. In addition, we speculate that the increased catalytic efficiency of PRMT6 for methylated substrates combined with lower K(m) values for native protein methyl acceptors may obscure this distributive mechanism to produce an apparently processive mechanism.     

Partial purification, characterization, and kinetic analysis of isoflavone 5-O-methyltransferase from yellow lupin roots

An isoflavone 5-O-methyltransferase was partially purified from the roots of yellow lupin (Lupinus luteus) by fractional precipitation with ammonium sulfate, followed by gel filtration and ion-exchange chromatography using a fast-protein liquid chromatography system. This enzyme, which was purified 810-fold, catalyzed position-specific methylation of the 5-hydroxyl group of a number of substituted isoflavones. The methyltransferase had a pH optimum of 7 in phosphate buffer, an apparent pI of 5.2, a molecular weight of 55,000, no requirement for Mg2+, and was inhibited by various SH-group reagents. Substrate interaction kinetics of the isoflavonoid substrate and S-adenosyl-L-methionine gave converging lines which were consistent with a sequential bireactant binding mechanism. Furthermore, product inhibition studies showed competitive inhibition between S-adenosyl-L-methionine and S-adenosyl-L-homocysteine and noncompetitive inhibition between the isoflavone and either S-adenosyl-L-homocysteine or the 5-O-methylisoflavone. The kinetic patterns obtained were consistent with an ordered bi bi mechanism, where S-adenosyl-L-methionine is the first substrate to bind to the enzyme and S-adenosyl-L-homocysteine is the final product released. The physiological role of this enzyme is discussed in relation to the biosynthesis of 5-O-methylisoflavones of this tissue.     

Protein-O-carboxylmethyltransferase from cytosol and membranes of chicken erythrocytes

Cytosolic protein-O-carboxylmethyltransferase was purified more than 4,000-fold in specific activity and membrane-associated protein-O-carboxylmethyltransferase carboxymethylase about 900-fold from chicken erythrocytes by use of a combination of affinity chromatography on immobilized S-adenosyl-L-homocysteine and gel filtration on Sephacryl S-200 (Pharmacia), together with 3-((3-cholamidopropyl)-dimethylammonio)-1-propane-sulfonate as a detergent to solubilize the membrane-associated enzyme. The two enzymes were characterized by examining the dependence of their activity on pH and on concentration of S-adenosyl-L-methionine using fetuin as an exogenous methyl-acceptor substrate, and were found to differ somewhat. The cytosolic enzyme had a pH optimum of 6.0 with an apparent Km value of 2.1 microM for S-adenosyl-L-methionine, whereas corresponding values for the membrane-associated enzyme were 6.5 and 0.71 microM. This report deals with the biochemical differences between purified cytosolic and membrane-associated protein carboxymethylase from the same cell source.