Protein Carboxyl Methylase Activity Research Articles

Changes in germ cell adenylate cyclase and protein carboxyl methylase activities in rat testicular tissue during bilateral cryptorchidism and after orchidopexy.

Soluble Mn2+-dependent adenylate cyclase and protein carboxyl methylase are two enzymes that are primarily localized in haploid germ cels of rat testicular tissue, and both enzymes exhibit an increase in activity in association with sexual maturation. Experimental cryptorchidism (surgery at 17 days of age) in immature rats prevented the age-dependent increase in the activity of these two testicular enzymes. After orchidopexy at 34 days of age the activities of these two enzymes increased to normal control values in association with testicular growth. These observations show that biochemical markers such as soluble Mn2+-dependent adenylate cyclase and protein carboxyl methylase can be used to follow germ cell differentiation.

The relation between protein carboxyl methylating and demethylating enzyme activities and noradrenaline efflux in platelets.

To test the hypothesis that the raised noradrenaline efflux rate constant in platelets observed in hypertensive individuals and some normotensive relatives to hypertensives could be ascribed to disturbances in the protein carboxyl methylation, protein carboxyl methylase activity and noradrenaline efflux rate constant were measured in platelets from 33 young healthy males. Sixteen had inheritance of hypertension. Protein methyl esterase activity and the noradrenaline efflux rate constant were measured in 43 men, 22 of whom had inheritance of hypertension. No correlation was found between unstimulated noradrenaline efflux from platelets and enzyme activity.

The relationship between glycolysis, mitochondrial respiration, protein-carboxyl methylation and motility in hamster epididymal spermatozoa.

Endogenous protein-carboxyl methylase activity can be measured in intact motile spermatozoa by using [3H]methionine as a precursor of the methyl donor, S-adenosylmethionine (SAM). Since the conversion of methionine to SAM requires ATP, the relationship between the energy metabolism of spermatozoa and methylation was investigated using inhibitors of glycolysis and mitochondrial respiration. When hamster spermatozoa from cauda epididymides were incubated in 12.2 mM glucose, glycolysis was progressively inhibited as concentrations of 2-deoxyglucose (2-DOG) increased. On the other hand, endogenous protein-carboxyl methylation showed a biphasic response being stimulated at low concentrations of 2-DOG and inhibited at higher concentrations. Sperm movement was also altered by 2-Dog. Increasing concentrations of 2-DOG in the incubation medium resulted in an increase in beat amplitude and a corresponding decrease in beat frequency. When the glucose concentration of the medium was reduced to 5 mM, protein methylation was inhibited at all concentrations of 2-DOG. The biphasic effect of 2-DOG on methylation at 12.2 mM and its monophasic effect at 5 mM suggested that this reaction was related to the effective glucose concentration. To investigate this possibility, endogenous protein-carboxyl methylation was measured after incubation of sperm in glucose concentrations ranging from 0-48.8 mM. Low glucose concentrations stimulated protein methylation (up to 6.1 mM for maximal effect), but further increases in the glucose concentration (10-48.8 mM) suppressed the rate of protein methylation to that observed in the absence of glucose. 2-Deoxyglucose decreased conversion of pyruvate to CO2 consistent with diminished mitochondrial respiration. The inhibition of mitochondrial respiration by antimycin A also inhibited endogenous methylation and motility. The results of these studies suggest that maximal endogenous protein-carboxyl methylation in intact spermatozoa from hamster epididymides depends upon active glycolysis as well as mitochondrial respiration.

Protein carboxyl methylation increases in parallel with differentiation of neuroblastoma cells.

Cells of mouse neuroblastoma clone N1E-115 in the confluent phase of growth can catalyze the formation of endogenous protein carboxyl methyl esters, using a protein carboxyl methylase and membrane-bound methyl acceptor proteins. The enzyme is localized predominantly in the cytosol of the cells and has a molecular weight of about 20,000 daltons. Treatment of the cells with dimethylsulfoxide (DMSO) or hexamethylene-bisacetamide (HMBA), agents that induce morphological and electrophysiological differentiation, results in a marked increase in protein carboxyl methylase activity. Maximal levels are reached 6-7 days after exposure to the agents, a time course that closely parallels the development of electrical excitability mechanisms in these cells. Serum deprivation also causes neurite outgrowth but does not enhance electrical excitability or enzyme activity. The capacity of membrane-bound neuroblastoma protein(s) to be carboxyl methylated is increased by the differentiation procedures that have been examined. However, the increase in methyl acceptor proteins induced by DMSO or HMBA is the largest, and its time course parallels electrophysiological differentiation. In contrast, serum deprivation induced a small increase that reached maximal levels within 24 h. The data suggest that increased protein carboxyl methylation is a developmentally regulated property of neuroblastoma cells and that at least two groups of methyl acceptor proteins are induced during differentiation: a minor group related to morphological differentiation, and a major group that may be related to ionic permeability mechanisms of the excitable membrane.

Rat sperm enzymes during epididymal transit.

The activities of cAMP and cGMP phosphodiesterases (EC 3.1.4.1), adenylate cyclase (EC 4.6.1.1) and protein carboxyl-methylase (EC 2.1.1.24) were measured in the particulate and soluble (105 000 g supernatant) fractions of washed spermatozoa isolated from five segments of the adult rat epididymis. The activities of both phosphodiesterases decreased during epididymal transit, whereas adenylate cyclase and protein carboxyl-methylase underwent a progressive increase, the latter showing the most marked alteration. Both cAMP and cGMP phosphodiesterases as well as the adenylate cyclase were all associated primarily with the particulate fraction, and the extent to which these enzymes were associated with the membranes increased as the spermatozoa passed through the epididymis. Sperm protein carboxyl-methylase activity was, on the other hand, predominantly soluble in all segments of the epididymis. Adenylate cyclase, cAMP phosphodiesterase and protein carboxyl-methylase activities were found predominantly in the sperm tails, whereas cGMP phosphodiesterase was equally distributed between heads and tails. These observations imply that the acknowledged increase in intracellular cAMP levels which occurs in spermatozoa during epididymal transit may be a consequence of both increased synthesis (adenylate cyclase) and reduced hydrolysis (phosphodiesterase).

Deficiency of protein-carboxyl methylase in immotile spermatozoa of infertile men.

We studied protein-carboxyl methylase, an enzyme involved in the regulation of cellular locomotion in both bacteria and leukocytes, in semen from 22 normal fertile men, 10 vasectomized volunteers, and nine infertile patients with nonmotile spermatozoa. In normally motile spermatozoa protein-carboxyl methylase activity was 68.8 +/- 5.5 pmol per milligram of protein (mean +/- S.E.). On the other hand, the enzyme activity in nonmotile spermatozoa from the infertile patients was low (17.4 +/- 3.4 pmol per milligram of protein) and similar to that in the cellular debris from the vasectomized volunteers (10.4 +/- 1.3 pmol per milligram of protein). The low enzyme activity in the infertile patients was not caused by the presence of dead spermatozoa or spermatozoa with leaky plasma membranes, since mitochondrial protein synthesis and lactate dehydrogenase activity were normal in these patients. The deficiency of protein-carboxyl methylase activity in nonmotile sperm is probably not due to a primary genetic defect, since the enzyme activity is normal in the red cells of these patients and spontaneous recovery of motility is associated with the return of enzyme activity.

Erythro-9-[3-(2-Hydroxynonyl)]adenine is an inhibitor of sperm motility that blocks dynein ATPase and protein carboxylmethylase activities.

Protein carboxylmethylase (S-adenosyl-L-methionine:protein O-methyltransferase, EC 2.1.1.24.) is believed to be involved in the regulation of sperm motility. To test this hypothesis, we investigated the effects of erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) which, in combination with adenosine and homocysteine thiolactone, inhibits protein carboxylmethylase activity in monocytes. This group of compounds inhibited sea urchin sperm motility. Unexpectedly, EHNA alone inhibited the motility., This observation was confirmed in intact spermatozoa from rats, rabbits, and humans. EHNA also inhibited the motility of demembranated, reactivated sea urchin and rat spermatozoa from which protein carboxylmethylase had been extracted. In these preparations, motility was restored by ATP. These observations suggested that EHNA arrests sperm motility by inhibiting the axonemal dynein ATPase on which motility depends. Kinetic analysis demonstrated that EHNA produced mixed inhibition of both the axonemal ATPase and the partially purified dynein 1 from sea urchin sperm tails, as well as the axonemal ATPase of rat sperm tails. These observations also provide evidence for the similarity of the active site of the dynein ATPase in sea urchin and rat spermatozoa.

Characterization of protein carboxyl methylase activities in rat testis: presence of testis specific charge isomers.

The physicochemical and molecular properties of testicular Protein Carboxyl Methylase (PCM) have been studied. The testicular enzyme was stable at high ionic strength, independent of metal ions and resistant to reducing or alkylating agents, and displayed a narrow pH optimum of pH 5.8-6.0. The enzyme had a molecular weight of approximately 25,000 daltons and a Km for S-adenosyl-L-methionine (SAM) of 1.3 microM. Varying concentrations of gelatine (exogenous methyl acceptor protein) did not change the affinity of the enzyme for SAM. The molecular properties of the enzyme were characterized using gel filtration, hydroxyapatite and DEAE-cellulose chromatography, and isoelectric focusing. Three charge isomers exhibiting PCM activity were isolated with pI values of 6.1, 6.7, and 7.35 while comparative data from the pituitary gland revealed primarily the presence of the most acid isozyme (pI 6.1). These results suggest that the different testicular charge isozymes may be involved in the selective methylation of specific testicular proteins.