Rasosomes originate from the Golgi to dispense Ras signals.

Ras proteins undergo an incompletely understood trafficking process in the cell. Rasosomes are protein nanoparticles of 80-100 nm diameter that carry lipidated Ras isoforms (H-Ras and N-Ras) as well as their effectors through the cytoplasm and near the plasma membrane (PM). In this study, we identified the subcellular origin of rasosomes and how they spread Ras proteins through the cell. We found no dependency of rasosome formation on galectins, or on the GDP-/GTP-bound state of Ras. We found that significantly more rasosomes are associated with forms of Ras that are localized to the Golgi, namely N-Ras or the singly palmitoylated H-Ras mutant (C181S). To explore the possibility that rasosome originate from the Golgi, we used photoactivatable (PA)-GFP-H-Ras mutants and showed that rasosomes bud from the Golgi in a two-step mechanism. Newly released rasosomes first move in an energy-dependent directed fashion and then convert to randomly diffusing rasosomes. Dual fluorescence time-lapse imaging revealed the appearance of dually labeled rasosomes, indicating a dynamic exchange of cytoplasmic and PM-associated Ras with rasosome-associated Ras. Finally, higher levels of rasosomes correlate with higher levels of ERK phosphorylation, a key marker of Ras downstream signaling. We suggest that H-Ras and N-Ras proteins exchange with rasosomes that can function as carriers of palmitoylated Ras and its signals.

Ras antagonist inhibits growth and chemosensitizes human epithelial ovarian cancer cells.

The objective of this article was to determine whether human ovarian carcinoma cells (OVCAR-3) express significant amounts of Ras oncogene and active Ras-guanosine triphosphate (GTP) and, if so, whether the Ras inhibitor farnesyl thiosalicylic acid (FTS) inhibits their growth and chemosensitizes them to cisplatin. We assayed Ras and Ras-GTP in OVCAR-3 cells before and after FTS treatment. The effect of FTS on OVCAR-3 cell growth was assessed in terms of cell number. Because the OVCAR-3 cell line was derived from a patient who was refractory to cisplatin, we examined whether FTS enables cisplatin to induce death of these cells. Significant amounts of Ras and active Ras-GTP were expressed by OVCAR-3 cells and were reduced by 40% by FTS. FTS inhibited OVCAR-3 cell growth in a dose-dependent manner. When combined with cisplatin, FTS reduced the number of OVCAR-3 cells by 80%, demonstrating synergism between FTS and cisplatin. FTS, at a concentration range that allows downregulation of Ras and Ras-GTP in OVCAR-3 cells, also chemosensitizes these cells and inhibits their growth. These results suggest that ovarian carcinomas might respond well to Ras inhibition, both alone and when combined with cisplatin. The combined treatment would allow the use of smaller doses of chemotherapy, resulting in decreased cytotoxicity.

Galectin-1 binds oncogenic H-Ras to mediate Ras membrane anchorage and cell transformation.

Ras genes, frequently mutated in human tumors, promote malignant transformation. Ras transformation requires membrane anchorage, which is promoted by Ras farnesylcysteine carboxymethylester and by a second signal. Previously we showed that the farnesylcysteine mimetic, farnesylthiosalicylic acid (FTS) disrupts Ras membrane anchorage. To understand how this disruption contributes to inhibition of cell transformation we searched for new Ras-interacting proteins and identified galectin-1, a lectin implicated in human tumors, as a selective binding partner of oncogenic H-Ras(12V). The observed size of H-Ras(12V)-galectin-1 complex, which is equal to the sum of the molecular weights of Ras and galectin-1 indicates a direct binding interaction between the two proteins. FTS disrupted H-Ras(12V)-galectin-1 interactions. Overexpression of galectin-1 increased membrane-associated Ras, Ras-GTP, and active ERK resulting in cell transformation, which was blocked by dominant negative Ras. Galectin-1 antisense RNA inhibited transformation by H-Ras(12V) and abolished membrane anchorage of green fluorescent protein (GFP)-H-Ras(12V) but not of GFP-H-Ras wild-type (wt), GFP-K-Ras(12V), or GFP-N-Ras(13V). H-Ras(12V)-galectin-1 interactions establish an essential link between two proteins associated with cell transformation and human malignancies that can be exploited to selectively target oncogenic Ras proteins.

Targeting of K-Ras 4B by S-trans,trans-farnesyl thiosalicylic acid.

Ras proteins regulate cell growth, differentiation and apoptosis. Their activities depend on their anchorage to the inner surface of the plasma membrane, which is promoted by their common carboxy-terminal S-farnesylcysteine and either a stretch of lysine residues (K-Ras 4B) or S-palmitoyl moieties (H-Ras, N-Ras and K-Ras 4A). We previously demonstrated dislodgment of H-Ras from EJ cell membranes by S-trans,trans-farnesylthiosalicylic acid (FTS), and proposed that FTS disrupts the interactions between the S-prenyl moiety of Ras and the membrane anchorage domains. In support of this hypothesis, we now show that FTS, which is not a farnesyltransferase inhibitor, inhibits growth of NIH3T3 cells transformed by the non-palmitoylated K-Ras 4B(12V) or by its farnesylated, but unmethylated, K-Ras 4B(12) CVYM mutant. The growth-inhibitory effects of FTS followed the dislodgment and accelerated degradation of K-Ras 4B(12V), leading in turn to a decrease in its amount in the cells and inhibition of MAPK activity. FTS did not affect the rate of degradation of the K-Ras 4B, SVIM mutant which is not modified post-translationally, suggesting that only farnesylated Ras isoforms are substrates for facilitated degradation. The putative Ras-recognition sites (within domains in the cell membrane) appear to tolerate both C(15) and C(20) S-prenyl moeities, since geranylgeranyl thiosalicylic acid mimicked the growth-inhibitory effects of FTS in K-Ras 4B(12V)-transformed cells and FTS inhibited the growth of cells transformed by the geranylgeranylated K-Ras 4B(12V) CVIL isoform. The results suggest that FTS acts as a domain-targeted compound that disrupts Ras-membrane interactions. The fact that FTS can target K-Ras 4B(12V), which is insensitive to inhibition by farnesyltransfarase inhibitors, suggests that FTS may target Ras (and other prenylated proteins important for transformed cell growth) in an efficient manner that speaks well for its potential as an anticancer therapeutic agent.

Stringent structural requirements for anti-Ras activity of S-prenyl analogues.

The carboxy terminal S-farnesylcysteine of Ras oncoproteins is required for their membrane anchorage and transforming activities. We showed previously that S-farnesylthiosalicylic acid (FTS) affects the membrane anchorage of activated H-Ras in EJ cells and inhibits their growth. We report here on structural elements in S-prenyl derivatives that specifically inhibit the growth of EJ cells, but not of untransformed Rat-1 cells. Inhibition of the Ras-dependent extracellular signal-regulated protein kinase (ERK), of DNA synthesis and of EJ cell growth were apparent after treatment with FTS or its 5-fluoro, 5-chloro and 4-fluoro derivatives or with the C20 S-geranylgeranyl derivative of thiosalicylic acid. The 4-Cl-FTS analogue was a weak inhibitor of EJ cell growth. The 3-Cl-FTS analogue and the FTS carboxyl methyl ester were inactive, as were the C10 S-geranyl derivative of thiosalicylic acid, farnesoic acid, N-acetyl-S-farnesyl-L-cysteine and S-farne-sylthiopropionic acid. The structural requirements for anti-Ras activity of S-prenyl analogues thus appear to be rather stringent. With regard to chain length, the C15 farnesyl group linked to a rigid backbone seems to be necessary and sufficient. A free carboxyl group in an appropriately rigid orientation, as in thiosalicylic acid, is also required. Halogenic substitutents on the benzene ring of the thiosalicylic acid are tolerated only at position 5 or 4. This information may facilitate the design of potent Ras antagonists and deepen our understanding of the mode of association of Ras with the plasma membrane.

Dislodgment and accelerated degradation of Ras.

Membrane anchorage of Ras oncoproteins, required for transforming activity, depends on their carboxy-terminal farnesylcysteine. We previously showed that S-trans,trans-farnesylthiosalicylic acid (FTS), a synthetic farnesylcysteine mimetic, inhibits growth of ErbB2- and Ras-transformed cells, but not of v-Raf-transformed cells, suggesting that FTS interferes specifically with Ras functions. Here we demonstrate that FTS dislodges Ras from membranes of H-Ras-transformed (EJ) cells, facilitating its degradation and decreasing total cellular Ras. The dislodged Ras that was transiently present in the cytosol was degraded relatively rapidly, causing a decrease of up to 80% in total cellular Ras. The half-life of Ras was 10 +/- 4 h in FTS-treated EJ cells and 27 +/- 4 h in controls. The dislodgment of membrane Ras and decrease in total cellular Ras were dose-dependent: 50% of the effects occurred at 10-15 microM, comparable to concentrations (7-10 microM) required for 50% growth inhibition in EJ cells. Higher concentrations of FTS (25-50 microM) were required to dislodge Ras from Rat-1 cell membranes expressing normal Ras, suggesting some selectivity of FTS toward oncogenic Ras. Membrane localization of the prenylated G beta gamma of heterotrimeric G proteins was not affected by FTS in EJ cells. An FTS-related compound, N-acetyl-S-farnesyl-L-cysteine, which does not inhibit EJ cell growth, did not affect Ras. FTS did not inhibit growth of Rat-1 cells transformed by N-myristylated H-Ras and did not reduce the total amount of this Ras isoform. The results suggest that FTS affects docking of Ras in the cell membrane in a rather specific manner, rendering the protein susceptible to proteolytic degradation.

The Ras antagonist S-farnesylthiosalicylic acid induces inhibition of MAPK activation.

Inhibition of Ras-dependent signaling and of oncogenic Ras function by farnesyl transferase inhibitors that block Ras membrane anchorage is limited due to alternative prenylation of Ras. Here we demonstrate that inhibition of the Ras-dependent Raf-1-MAPK (mitogen activated protein kinase) cascade is achieved by S-farnesylthiosalicylic acid (FTS) which affects Ras membrane association but not Ras farnesylation. FTS interferes with the activation of Raf-1 and MAPK and inhibits DNA synthesis in Ras-transformed EJ cells at concentrations similar to those at which it inhibits EJ cell growth (5-25 microM). FTS also inhibits MAPK activity and DNA synthesis stimulated by serum, EGF or thrombin in serum-starved untransformed Rat-1 cells, demonstrating the generality of its effects on Ras-dependent signaling. The effects of FTS on MAPK activity developed relatively rapidly (within 2-6 h) consistent with its rapid effect on Ras membrane anchorage. FTS represents a new class of Ras antagonists that may be useful for the inhibition of various types of oncogenic Ras isoforms independently of their prenylation.

Selective inhibition of Ras-dependent cell growth by farnesylthiosalisylic acid.

S-trans,trans-Farnesylthiosalicylic acid (FTS) is a novel farnesylated rigid carboxylic acid derivative. In cell-free systems, it acts as a potent competitive inhibitor (Ki = 2.6 microM) of the enzyme prenylated protein methyltransferase (PPMTase), which methylates the carboxyl-terminal S-prenylcysteine in a large number of prenylated proteins including Ras. In such systems, FTS inhibits Ras methylation but not Ras farnesylation. Inhibition of the PPMTase by FTS in homogenates or membranes of a variety of tissues and cell lines is inferred from a block in the methylation of exogenously added substrates such as N-acetyl-S-trans,trans-farnesyl-L-cysteine and of endogenous substrates including small GTP-binding proteins. FTS can also inhibit methylation of these proteins in intact cells (e.g. in Rat-1 fibroblasts, Ras-transformed Rat-1, and B16 melanoma cells). Unlike in cell-free systems, however, relatively high concentrations of FTS (50-100 microM) are required for partial blocking (10-40%) of protein methylation in the intact cells. Thus, FTS is a weak inhibitor of methylation in intact cells. Because methylation is the last step in the processing of Ras and related proteins, FTS is not likely to affect steps that precede it, e.g. protein prenylation. This may explain why the growth and gross morphology of a variety of cultured cell types (including Chinese hamster ovary, NIH3T3, Rat1, B16 melanoma, and PC12) is not affected by up to 25 microM FTS and is consistent with the observed lack of FTS-induced cytotoxicity. Nevertheless, FTS reduces the levels of Ras in cell membranes and can inhibit Ras-dependent cell growth in vitro, independently of methylation. It inhibits the growth of human Ha-ras-transformed cells (EJ cells) and reverses their transformed morphology in a dose-dependent manner (0.1-10 microM). The drug does not interfere with the growth of cells transformed by v-Raf or T-antigen but inhibits the growth of ErbB2-transformed cells and blocks the mitogenic effects of epidermal and basic fibroblast growth factors, thus implying its selectivity toward Ras growth signaling, possibly via modulation of Ras-Raf communication. Taken together, the results raise the possibility that FTS may specifically interfere with the interaction of Ras with a farnesylcysteine recognition domain in the cell membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Farnesyl derivatives of rigid carboxylic acids-inhibitors of ras-dependent cell growth.

Inhibitors of the enzyme that methylates ras proteins, the prenylated protein methyltransferase (PPMTase), are described. They are farnesyl derivatives of rigid carboxylic acids that recognize the farnesylcysteine recognition domain of the enzyme but do not serve as substrates. They also inhibit ras-dependent cell growth by a mechanism that is probably unrelated to inhibition of ras methylation, even though their potencies as PPMTase inhibitors and cell-growth inhibitors correlate well. The most potent inhibitor is S-trans,trans-farnesylthiosalicylic acid (FTS) (2). FTS (2) selectively inhibits the growth of human Ha-ras-transformed Rat1 cells in vitro (EC50 = 7.5 microM).

Prenylated protein methyltransferase of rat cerebellum is developmentally co-expressed with its substrates.

High levels of prenylated protein methyltransferase are expressed in the developing rat cerebellum and are responsible for methylation of endogenous G-proteins and 50-52 kDa synaptosomal proteins. Enzyme activity in cerebellar synaptosomes of 3 week postnatal rats is 2-fold higher than that found in adult rat cerebellum. A 10-fold rise in activity occurs at the end of the second and during the third postnatal weeks, followed by a subsequent decline. Expression of the enzymes' substrates follows the same pattern. The high methyltransferase activity in 3-week-old cerebellum coincides with the period of granule cell migration and synaptogenesis, suggesting a regulatory role for the enzyme and its substrates in cerebellar ontogenesis.

The uniquely distributed isoprenylated protein methyltransferase activity in the rat brain is highly expressed in the cerebellum.

Isoprenylated protein methyltransferase, the enzyme which catalyzes the reversible methylation of signal transducing G-proteins was studied in nine brain regions of the rat brain using S-farnesyl cysteine analogs as substrates. Enzyme activity, as determined with N-acetyl-S-farnesyl-L-cysteine (AFC) was found in the nuclear, synaptosomal and microsomal fractions of all brain regions but not in the cytosol. The enzyme is a unique methyltransferase with respect to its brain distribution. The rank order of activity of the enzyme is cerebellum >> midbrain > medulla > forebrain regions, where activities in cerebellar synaptosomal and nuclear fractions (28-32 pmol AFC [methyl-3H]ester formed/min/mg prot) are 20 to 30 times higher than those of the corresponding fraction of the forebrain regions. This distribution is reminiscent of that of neurotransmitter receptors and signal transduction molecules and suggests a regulatory role for the enzyme, particularly in the cerebellum.

Nerve growth factor induces a succession of increases in isoprenylated methylated small GTP-binding proteins of PC-12 pheochromocytoma cells.

Pheochromocytoma (PC-12) cells exposed to nerve growth factor (NGF) acquire a sympathetic neuron-like phenotype. This NGF-response is blocked by methylation inhibitors and can be mimicked by the farnesylated methylated small GTP-binding protein p21ras. The implicated involvement of prenylation, methylation and a small GTP-binding protein in the NGF-response has been studied by directly measuring 3H-mevalonic acid (MVA)-metabolites incorporated into proteins, protein carboxy [methyl-3H]ester formation and levels of [alpha-32P]GTP-binding proteins in NGF-induced PC-12 cells. We demonstrate that NGF induces a 2-3-fold increase in 21-24 kDa methylated membrane proteins that incorporate 3H-MVA-metabolites, and bind GTP. Levels of [alpha-32P]GTP-binding in these proteins were increased by 2-3-fold. Methylation and membrane association of the small GTP-binding proteins were blocked by lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which also enhanced their labeling by 3H-MVA-metabolites. Cycloheximide reduced the levels of [methyl-3H] labeled 21-24 kDa proteins and of the overlapping [alpha-32P]GTP binding-proteins. About 70% of the [methyl-3H]-groups found in these proteins were recovered from two dimensional gel blots in nine distinct spots of [alpha-32P]GTP-binding proteins. Taken together these results strongly suggest that in PC-12 cells, NGF induces an increase in the synthesis of prenylated methylated small GTP-binding proteins. The efficacy of lovastatin blockage of protein methylation and enhancement of 3H-MVA-metabolites incorporation into GTP-binding proteins was lower in NGF-induced cells than in controls. This suggests that NGF also induces an increase in HMG-CoA reductase activity. At the early phase of the NGF response in PC-12 cells (15 min-1 h), the levels of two small GTP-binding proteins (molecular mass of 21-22 kDa and 23-24 kDa) were increased. Thus, at least two proteins, of which one but not the other may be p21ras, appear to be involved in the early response. After a lag period of 24 h with NGF, a second more robust phase of increase in methylated small GTP-binding proteins was apparent. This relatively late response, which was almost completed within 24 h, may reflect involvement of small GTP-binding proteins in neurite-outgrowth and in the functional activity of the differentiated cells. Many small GTP-binding proteins were increased during the second phase, precluding electrophoretic separation of all of them. 3 proteins, however, were well separated (one 23-24 kDa protein and two 21-22 kDa proteins).(ABSTRACT TRUNCATED AT 400 WORDS)

Isoprenylation and carboxylmethylation in small GTP-binding proteins of pheochromocytoma (PC-12) cells.

1. A group of 21 to 24-kDa proteins of pheochromocytoma (PC-12) cells was found in blot overlay assays to bind specifically [alpha-32P]GTP. Binding was inhibited by GTP analogues but not by ATP. Such small GTP-binding proteins were found in the cytosolic and in the particulate fraction of the cells, but they were unevenly distributed: about 75% of the small GTP-binding proteins were localized within the particulate fraction of the cells. Separation of these proteins by two-dimensional gel electrophoresis revealed the existence of seven distinct [alpha-32P]GTP-binding proteins. 2. Targeting of the small GTP-binding proteins to the particulate fraction of PC-12 cells requires modification by isoprenoids, since depleting the cells of the isoprenoid precursor mevalonic acid (MVA) by the use of lovastatin resulted in a 50% decrease in membrane-bound small GTP-binding proteins, with a proportionate increase in the cytosolic form. This blocking effect of lovastatin was reversed by exogenously added MVA. 3. In addition, metabolic labeling of PC-12 cells with [3H]MVA revealed incorporation of [3H]MVA metabolites into the cluster of 21 to 24-kDa proteins in a form typical of isoprenoids; the label was not removed from the proteins by hydroxylamine, and labeling was enhanced in cells incubated with lovastatin. The latter effect reflects a decrease in the isotopic dilution of the exogenously added [3H]MVA, as the addition of exogenous MVA reversed the effect of lovastatin on [3H]MVA-metabolite incorporation into the 21 to 24-kDa proteins. 4. Additional experiments demonstrated that isoprenylation is required not only for membrane association of small GTP-binding proteins, but also for their further modification by a methylation enzyme. This was evident in experiments in which the cells were metabolically labeled with [methyl-3H]methionine, a methylation precursor. The group of 21 to 24-kDa proteins was labeled with a methyl-3H group in a form typical of C-terminal-cysteinyl carboxylmethyl esters. Their methylation was blocked by the methylation inhibitors methylthioadenosine (MTA), 3-deazadenosine and homocysteine thiolactone as well as by lovastatin. MVA reversed the lovastatin block of methylation. 5. Two-dimensional gel analysis of the [3H]methylated proteins detected seven methylated small GTP-binding proteins that correspond to the isoprenylated proteins. Levels of the small GTP-binding proteins as well as isoprenylation and methylation were reduced by cycloheximide. 6. Distribution of the methylated proteins between particulate and cytosolic fractions was found to be similar to that of the small GTP-binding proteins (i.e., a 4:1 ratio).(ABSTRACT TRUNCATED AT 400 WORDS)

Relationship among methylation, isoprenylation, and GTP binding in 21- to 23-kDa proteins of neuroblastoma.

1. Dimethylsulfoxide-induced differentiated neuroblastoma express high levels of membrane 21 to 23-kDa carboxyl methylated proteins. Relationships among methylation, isoprenylation, and GTP binding in these proteins were investigated. Protein carboxyl methylation, protein isoprenylation, and [alpha-32P]GTP binding were determined in the electrophoretically separated proteins of cells labeled with the methylation precursor [methyl-3H]methionine or with an isoprenoid precursor [3H]mevalonate. 2. A broad band of GTP-binding proteins, which overlaps with the methylated 21 to 23-kDa proteins, was detected in [alpha-32P]GTP blot overlay assays. This band of proteins was separated in two-dimensional gels into nine methylated proteins, of which four bound GTP. 3. The carboxyl-methylated 21 to 23-kDa proteins incorporated [3H]mevalonate metabolites with characteristics of protein isoprenylation. The label was not removed by organic solvents or destroyed by hydroxylamine. Incorporation of radioactivity from [3H]mevalonate was enhanced when endogenous levels of mevalonate were reduced by lovastatin, an inhibitor of mevalonate synthesis. Lovastatin blocked methylation of the 21 to 23-kDa proteins as well (greater than 70%). 4. Methylthioadenosine, a methylation inhibitor, inhibited methylation of these proteins (greater than 80%) but did not affect their labeling by [3H]mevalonate. The results suggest that methylation of the 21 to 23-kDa proteins depends on, and is subsequent to, isoprenylation. The sequence of events may be similar to that known in ras proteins, i.e., carboxyl methylation of a C-terminal cysteine that is isoprenylated. 5. Lovastatin reduced the level of small GTP-binding proteins in the membranes and increased GTP binding in the cytosol. Methylthioadensoine blocked methylation without affecting GTP binding. 6. Thus, isoprenylation appears to precede methylation and to be important for membrane association, while methylation is not required for GTP binding or membrane association. The role of methylation remains to be determined but might be related to specific interactions of the small GTP-binding proteins with other proteins.

Methylation of 21-23 kD membrane proteins by a membrane-associated protein carboxyl methyltransferase in neuroblastoma cells. Increased methylation in differentiated cells.

Membranes of neuroblastoma N1E-115 cells contain a specific protein carboxyl methyltransferase that methylates a 70 kD protein and a group of 21-23 kD proteins which are tightly bound to the membranes. The enzyme catalyzes the transfer of [methyl-3H] groups from [methyl-3H]S-adenosyl-L-methionine (Km = 0.22 microM) to these proteins to form base-labile carboxymethylesters. These protein methylesters are relatively stable compared to other protein methylesters, as shown by the ability of the 21-23 kD methylated proteins to retain their [methyl-3H] groups at pH values of 7 to 8.5 for at least 12 hr at room temperature. The extent of methylation of the 21-23 kD proteins, but not that of the 70 kD protein, was increased in membranes of cells induced to differentiate by 2% dimethyl sulfoxide (from a basal level of 0.1-0.2 to 0.9-1.2 pmol [methyl-3H] groups incorporated per mg membrane protein). This increase appeared after a lag period of 3 days of growth in the presence of the dimethyl sulfoxide and developed in parallel with the appearance of neurite-like processes in the cells. Kinetic experiments suggest that the amounts of 21-23 kD proteins available for methylation in the membranes of the undifferentiated and of the differentiated cells are limited. This and the previously observed low turnover of methylated 21-23 kD proteins in the intact cells suggest that the differentiated cells express and methylate more 21-23 kD proteins than the undifferentiated cells. These methylated proteins may be involved in differentiation or other functions of the differentiated cell membranes.

Carboxyl methylation of 21-23 kDa membrane proteins in intact neuroblastoma cells is increased with differentiation.

Evidence is presented for specific enzymatic methylation of 21-23 kDa membrane proteins in intact neuroblastoma N1E 115 cells, which is increased in dimethylsulfoxide-induced differentiated cells. Methylation of these proteins has characteristics typical of enzymatic reactions in which base labile volatile methyl groups are incorporated into proteins, consistent with the formation of protein carboxyl methylesters. However, these methylesters of the 21-23 kDa proteins are relatively stable compared to other protein carboxyl methylesters. The 3-fold increase in methylated 21-23 kDa proteins in the differentiated cells suggest biological significance in differentiation of the cell membranes.

Purification and characterization of protein carboxyl methyltransferase from Torpedo ocellata electric organ.

Posttranslational modification of proteins by the enzyme protein carboxyl methyltransferase (PCM) has been associated with a variety of cellular functions. A prerequisite for the understanding of cellular mechanisms associated with PCM is the characterization of purified PCMs from different tissues. We describe here the purification and characterization of PCM from the electric organ of Torpedo ocellata. The enzyme was purified to homogeneity by ion-exchange chromatography and ammonium sulfate precipitation, followed by chromatography on Sephadex G-100 and hydroxylapatite columns. When visualized by silver staining, the 700-fold-purified PCM exhibited a single band on sodium dodecyl sulfate-polyacrylamide gels, corresponding to a polypeptide of Mr 29,000. The molecular weight of the nondenatured enzyme (as determined by rechromatography on Sephadex G-100 column) was also 29,000, suggesting that the enzyme is a monomer. Two isoelectric forms of PCM (pI = 6.1 and pI = 6.4) were detected in the purified enzyme preparation. The enzyme methylates various exogenous and endogenous proteins, including the acetylcholine receptor. Of the four different polypeptides of the acetylcholine receptor, the gamma and beta polypeptides were selectively methylated by the purified PCM. Purified Torpedo PCM is highly sensitive to sulfhydryl reagents. The competitive inhibitor of PCM S-adenosyl-L-homocysteine (AdoHcy) protected the enzyme from inactivation by sulfhydryl reagents, suggesting the existence of a cysteine residue at the active site of the enzyme. The purified PCM has a low affinity toward DEAE-cellulose and toward AdoHcy-agarose. This property, as well as the relatively high molecular weight and the marked sensitivity to sulfhydryl reagents, distinguishes between the electric organ PCM and analogous enzymes of mammalian tissues.