Masaharu Miyake’s research while affiliated with Ehime University and other places

A high concentration of a basic unidentified amino compound was found in the blood of rats. It was isolated and identified as N epsilon,N epsilon,N epsilon-trimethyllysine by paper chromatography, thin-layer chromatography, high-performance liquid chromatography and amino acid analyzer. It was localized exclusively in red blood cells in the blood of rats. Free trimethyllysine was also determined in the liver, kidney, spleen, brain, muscle, heart and testis of rat. The concentration of free trimethyllysine in red blood cells was more than 10-times as high as that in the other tissues. This compound in red blood cells was found in different species of animals. The relationship between this free trimethyllysine and carnitine was discussed.

The effects of some neurotransmitters, adenosine (Ad), and homocysteine (Hcys) on protein carboxyl methylation in synaptic plasma membranes from rat cerebral cortex were examined. Neither any of the neurotransmitters nor Ad had a detectable effect. Incubation of membrane with DL-Hcys alone (5 X 10(-5) M), the combination of both Ad (5 X 10(-5)) and DL-Hcys (5 X 10(-5)), or S-adenosyl-L-homocysteine (SAH) (1 X 10(-6)) strongly decreased the methyl ester formation. The inhibitory effect of the combination of both compounds may be interpreted in terms of the increased SAH concentration due to the presence of SAH hydrolase in the membrane. The inhibitory effect of Hcys alone was blocked by preincubation with Ad deaminase or Neplanocin A, a potent inhibitor of SAH hydrolase, suggesting the presence of Ad-bound SAH hydrolase in the synaptic membrane. Ad-bound SAH hydrolase activity estimated by the inhibition of methylation in the presence of Hcys was located in the membrane fractions including synaptosomes, myelin, and microsomes (about 70%), but the SAH hydrolase activity estimated on the basis of the inhibitory effect of the combination of both Ad and Hcys was localized exclusively in the soluble fraction (about 90%). The distribution of the latter activity is coincident with that of SAH hydrolase reported to date. Incubation of the synaptic membrane with Hcys markedly increased the SAH concentration. The stimulatory effect of Hcys alone was blocked by Ad deaminase.

A S-adenosylmethionine:protein-lysine N-methyltransferase (EC 2.1.1.43) has been purified from rat brain cytosol 7,080-fold with a yield of 8%, using octopus calmodulin as a substrate. It contains a lysine residue that is not fully methylated. The enzyme was purified by ammonium sulfate fractionation, Sephacryl S-200 gel filtration, and phosphocellulose and octopus calmodulin-Sepharose affinity chromatographies. Among protein substrates, it was highly specific toward octupus calmodulin. The Km values for octopus calmodulin and S-adenosyl-L-methionine were found to be 2.2 X 10(-8) M and 0.8 X 10(-6) M, respectively. The molecular weight was estimated to be 57,000 by gel filtration and the pH optimum was between 7.5 and 8.5. The enzyme was stimulated in the presence of 10(-7) M Mn2+ and 10(-4) M Ca2+. HPLC of the acid hydrolysate of methyl-3H-labeled calmodulin showed the formation of epsilon-N-mono, epsilon-N-di, and epsilon-N-trimethyllysine. Reverse-phase HPLC of tryptic peptides of the methyl-3H-labeled calmodulin demonstrated that the labeled N-methyllysine lies in the 107-126 peptide. These findings suggest that this enzyme methylated a specific lysine residue of octopus calmodulin.

During a study to find natural substrate proteins of carboxymethylation, myelin basic protein was found to be a good substrate. The two protein carboxymethylases were purified partially using myelin basic protein as a substrate. These two enzymes may be identical with protein carboxymethylase I and II, which have been found to methylate gamma-globulin. The Km of myelin basic protein (25 microM) was very small compared with other substrates. The activities of the two carboxymethylases were high in the rat brain in comparison to the other rat organs. The activity increased during the period of myelination in the rat brain. These findings suggest that carboxymethylation of myelin basic protein may play an important role in myelination.

A simple and sensitive method for determining hypusine in proteins was developed. A greater part of amino acids in the acid hydrolysate of proteins was separated from hypusine by treatment with an ion-exchange resin. The sample containing partially purified hypusine was then analyzed by high-performance liquid chromatography using the post-column derivatization method with o-phthalaldehyde. The recovery rate of hypusine through the overall procedure was more than 95%. Using this method, the distribution and developmental changes of hypusine in proteins were determined. The amino acid was found in proteins of all examined organs of rat. Its concentration was 5-40 nmol/g protein. The subcellular distribution in rat liver was also determined. About 60% of total amount of hypusine was present in the proteins of cytoplasmic and microsomal fractions and its relative concentration was high in the proteins of microsome and lysosome and low in mitochondria. In developing rat, the concentration of hypusine in the brain proteins was relatively high during the first 2 or 3 weeks of postnatal life and then decreased until adulthood. Its concentration in the liver proteins was highest at birth and then decreased continuously to the adult level.

An enzyme responsible for the deacylation of beta-citryl-L-glutamate to citrate and glutamate has been characterized in rat testis. The enzyme required manganese ion for full activity and was strongly inhibited by nucleotides such as ATP or GTP. The activity was localized in the particulate fractions. The enzyme favored N-formyl-L-glutamate greater than beta-citrly-L-glutamate greater than beta-citryl-L-glutamine in a decreasing order. The amidohydrolyase activity was highest in the testis and lung, a moderate activity was detected in heart, kidney and intestine, and low in brain, thymus, stomach, skeletal muscle, spleen and liver. These findings suggest that the amidohydrolase is different from any of amidohydrolases reported so far, amidohydrolase I (EC 3.5.1.14), II (EC 3.5.1.15), III, N-acetyl-lysine deacylase (EC 3.5.1.17) and N-acetyl-beta-alanine deacetylase (EC 3.5.1.21), and various peptidases.

beta-Citryl-L-glutamic acid, which is known to be highly concentrated in the brains of immature animals, is preferentially localized in the testes of various adult animals, including mammals, amphibians and fish, mainly in the germinal cells. In young rats, the citrylglutamate concentration increases with age and coincides with the development of late spermatocytes into early spermatids. Rats with seminiferous tubule failure induced by ductuli efferentes ligation and experimental cryptorchidism are infertile as a result of germ cell depletion, especially spermatocytes and early spermatids. In these animals, the testicular citrylglutamate content was much lower than in normal testes.

N-Acetyl-L-aspartic acid (NA-Asp), N-acetyl-alpha-L-aspartyl-L-glutamic acid (NA-Asp-Glu) and beta-citryl-L-glutamic acid (beta-CG), which are known to occur in the brain, have been isolated from human urine. Their identities were proved by comparing them with synthetic NA-Asp, NA-Asp-Glu and beta-CG using electrophoretic and chromatographic methods and by acid hydrolysis. A method was developed for the quantitation of NA-Asp, NA-Asp-Glu and beta-CG in human urine. It consists of ion-exchange chromatography followed by gas-chromatographic analysis. The amounts of urinary excretion of NA-Asp, NA-Asp-Glu and beta-CG were 41.2 +/- 10.1 (n = 27), 20.8 +/- 9.6 (n = 27) and 30.2 +/- 13.2 (n = 21) mumol/g creatinine in adult males, and 62.2 +/- 16.3 (n = 27), 24.0 +/- 8.2 (n = 27) and 40.5 +/- 21.1 (n = 24) mumol/g creatinine in adult females, respectively.

Abstract The developmental changes of N-acetylaspartic acid (NA-Asp), N-acetyl--aspartylglutamic acid (NA-Asp-Glu), and β-citryl-L-glutamic acid (β-CG) have been examined in the cerebrum, cerebellum, brain stem and spinal cord of both rat and guinea pig by the gas chromatographic method developed in our studies. A rapid increase in the concentration of NA-Asp was observed postnatally in every region of the rat brain. On the other hand, all regions of guinea pig brain showed the prenatal increases. NA-Asp-Glu showed a different developmental profile, depending on region of the brain, in the two species. The concentration of NA-Asp-Glu remained constantly low during brain maturation in the rostral regions. In the caudal portions it showed a marked increase during maturation and reached a high level in the adult brain. The concentration of β-CG was highest at birth in all regions of rat brain and rapidly decreased by 20 days after birth and remained low thereafter. The rapid decrease occurred in the guinea pig during the foetal period, and β-CG content decreased to an adult level at birth.