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A sensitive and specific method for determining three forms of methylarginine, i.e., NG-monomethylarginine, NG,NG-dimethylarginine, and NG,NG-dimethylarginine, in mammalian tissues was developed. After partial purification by ion-exchange chromatography, the methyl-arginines were derivatized to phenylthiocarbamyl compounds and quantitatively determined using HPLC with a reverse-phase C18 column. In rat organs, the highest concentrations of methylarginines were observed in the spleen. In rat brain, cerebellum and olfactory bulb contained large amounts of NG-monomethylarginine and NG,NG-dimethylarginine. A detailed study of the distribution of methylarginines in the bovine brain was also made, and the concentration of NG,NG-dimethylarginine was almost the same in all regions. The cerebellar gray matter, hippocampus, and hypothalamus contained large amounts of methylarginines. The distribution of methylarginines seems to parallel the distribution of nitric oxide synthase, which is known to be inhibited by NG-monomethylarginine. This may indicate that methylarginines play some role in controlling nitric oxide synthase activity.

The 3'-untranslated region (UTR) of the myelin basic protein (MBP) mRNA has been found previously to enhance the translational efficiency of the coding region by two-fold in cell-free translational systems. In this study, we transfected eukaryotic expression vectors containing the reporter cDNA for chloramphenicol acetyltransferase (CAT) with or without the mouse MBP cDNA 3'-UTR into cultured cells. CAT activity in the mouse oligodendrocyte cell line, N20.1, transfected with a CAT cDNA containing the MBP 3'-UTR [CAT-MBP 3'-UTR], was twice as high as that of the CAT cDNA without the 3'-UTR; CAT activities for the two constructs were the same in the mouse fibroblast cell line, NIH 3T3. Using reverse transcriptase PCR quantitative analysis, the expression of mRNA was determined. The level of the [CAT-MBP 3'-UTR] mRNA was about ten times higher than CAT mRNA in N20.1 cells but they were the same in NIH 3T3 cells. We conclude that the 3'-UTR of MBP gene increases gene expression at both the mRNA and protein levels in oligodrocyte cell lines, probably through a post-transcriptional mechanism such a message stabilization.

Prosaposin, the protein precursor of saposins A, B, C, and D which activate sphingolipid hydrolases, is abundant in several brain regions including the hippocampus. We infused prosaposin continuously for 7 days into the lateral ventricle of gerbils starting 3 hours before 3-min of forebrain ischemia. Using the step-down passive avoidance task, we demonstrated that ischemia-induced learning disability is prevented almost completely by prosaposin infusion. Subsequent light and electron microscopic examinations showed that pyramidal neurons in the CA1 field of the hippocampus as well as synapses within the strata moleculare, lacunosum/radiatum and oriens of the field were significantly more numerous in gerbils infused with prosaposin infusion than in those receiving saline infusion. These findings suggest that prosaposin possesses neurotrotrophic activity to protect hippocampal CA1 neurons from lethal ischemic damage.

パーキンソン病の治療の開発の歴史を概観した.その中でL-DOPA療法の開発の一部に筆者の一人が関与したので,発想や苦労話も加えて記述した.治療法の開発は他の病気のそれと同じく,病因がわかって,理論的に行われたものはない.偶然の思いつき(抗コリン薬),研究的関心(ドーパミン),臨床の偶然の経験(アマンタジン)などが発端になっている.薬物の密造の失敗(MPTP)も新たな研究への端緒になっている.

Methylarginines in free form were identified in bovine brain. Three compounds were isolated from the basic aliphatic amino acid fraction of bovine brain with several ion-exchange chromatographies. They showed the same Rf values in paper and thin-layer chromatographies as those of authentic NG-monomethylarginine, NG,NG-dimethylarginine, and NG,N'G-dimethylarginine. The migration distance of the isolated compounds in high-voltage paper electrophoresis and the retention times in ion-exchange HPLC were also identical to those of the above authentic methylarginines. We concluded that these three compounds are the methyl derivatives of arginine described above. The amount of these three compounds isolated from 1,090 g of bovine brain was 0.3 mumol of NG-monomethylarginine, 0.1 mumol of NG,NG-dimethylarginine, and 0.5 mumol of NG,N'G-dimethylarginine. The occurrence of these free methylarginines may have an important role in regulating the signal transduction through the nitric oxide system.

Prosaposin is the precursor protein for saposins, which are small lysosomal proteins required for the hydrolysis of sphingolipids by specific lysosomal hydrolases. Prosaposin, in addition to generating the saposins in the lysosomes, also exists as an unprocessed approximately 70-kDa protein in many tissues and secretory fluids. In this study, we isolated prosaposin from human milk. Milk was fractioned by ammonium sulfate precipitation, then chromatographed with DEAE-Sephacel and G-3000 SW gel permeation-HPLC. A fraction containing prosaposin was finally purified with the anti-saposin C IgG attached affinity column. The protein staining of the purified preparation on SDS-PAGE and the Western blotting showed a single band. The sequence of the initial 10 amino acids from N-terminus of the purified protein was identical to the sequence of prosaposin deduced from cDNA. Although prosaposin itself showed beta-glucosidase activator activity at a slight degree, the activity increased much after trypsin treatment. Western blotting of the trypsin-treated sample confirmed the formation of small saposin-like bands from prosaposin by the action of trypsin.

Sphingolipid hydrolases are activated by activator proteins or saposins. The precursor protein has been expected from the studies on the cDNA for saposins. Here we demonstrate that prosaposin occurs in various kinds of human secretory fluids such as cerebrospinal fluid, semen, milk, pancreatic juice, and bile. However, mature type saposins were not detected in these fluids. In human milk the amount of prosaposin changed during the lactating period; it became high in concentration within a few days after delivery, decreased during the transitional milk lactating stage, and then increased again toward the mature milk lactating stage. Prosaposin was released from human platelets in response to stimulation by thrombin, but mature saposins were not. From the time course of the release of prosaposin induced by thrombin and from the fact that weak platelet agonists, ADP, epinephrine, and collagen, did not cause the release of prosaposin, prosaposin secretion from platelets seemed to be from lysosome like granules. We postulate that some prosaposin works as a precursor for saposins in the lysosomes and the other serves as an extracellular protein with other specific roles.

A high concentration of a basic unidentified amino compound was found in the blood of rats. It was isolated and identified as N ϵ , N ϵ , N ϵ -trimethyllsine 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 trimethyllsine 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.

Neuroendocrine abnormality of the hypothalamic-pituitary-adrenal axis was investigated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated dogs as a model of Parkinson's disease. Blood samples were collected every 4 h for 3–4 days before MPTP treatment, around 2 and 4 weeks after the treatment of MPTP (2.5 mg/kg). Adrenocorticotrophin (ACTH) and cortisol concentrations in plasma were determined by radioimmunoassay. The concentrations of plasma ACTH and cortisol increased by 40% and 60% after MPTP treatment, respectively. Circadian rhythms of neither plasma ACTH nor cortisol concentrations were observed in both MPTP-treated and control dogs. This high hypothalamic-pituitary-adrenal function in MPTP-treated dogs suggest that MPTP may influence the activity of the hypothalamic neurons of the dog.

Studies on the circadian rhythm of urine excretion in healthy men have demonstrated that the maximal urine flow occurs in the early afternoon and the minimal around midnight. In this study, an abnormality in the variation of urine volume was found in parkinsonian patients. Urine samples were collected during daytime (9:00-21:00) and nighttime (21:00-9:00). Fifteen healthy control subjects were examined and found to excrete 60% during the daytime and 40% during the nighttime of the total urine volume. Sixteen parkinsonian patients excreted 43% during the daytime and 57% during the nighttime. In contrast to the control subjects, the parkinsonian patients excreted a smaller volume of their urine during the daytime than during the nighttime. This finding might be related to the degeneration of dopaminergic and/or nondopaminergic neurons in the brain which control urinary excretion.

Anticipation refers to the progressively earlier onset and increase in disease severity in successive generations. We studied four families with hereditary dentatorubral-pallidoluysian atrophy (DRPLA), a neurodegenerative disease, and anticipation was present in the mode of inheritance. In subsequent generations DRPLA shows an earlier onset and more severe as well as additional symptoms. Older onset patients suffer from cerebellar ataxia with or without dementia, whereas younger onset patients present as progressive myoclonus epilepsy syndrome, which consists of mental retardation, dementia, and cerebellar ataxia as well as epilepsy and myoclonus. Anticipation with paternal transmission was significantly greater than with maternal transmission.

Isoaspartyl protein carboxyl methyltransferase (PIMT) is widely distributed in mammalian tissues. Using a polymerase chain reaction-generated 124-bp DNA fragment from brain cDNA as a probe, four different sizes (approximately 4.0, 2.5, 1.7, and 1.1 kb) of transcripts were detected with northern blot analysis. They were expressed predominantly in rat brain and testis. The major transcripts were 2.5 and 1.7 kb in the brain and 2.5 and 1.1 kb in the testis. One of the major transcripts specific to the testis (1.1 kb) was determined to study the structural difference of major transcripts in the two tissues. This testicular cDNA had neither the 5' (94 nucleotides) nor the 3' (594 nucleotides) end of previously reported brain cDNA corresponding to 1.7 kb. The mRNA levels and enzyme activities of different regions and developmental changes were examined in the brain. The mRNA levels and enzyme activities were concomitantly high in cerebral cortex and hippocampus. Although they increased rapidly approximately 30 days after birth in the testis and decreased in aged rats, they increased gradually after birth and remained high during the aging of the brain. Both structural and developmental studies show that the expression of the PIMT gene in brain and testis is regulated in a tissue-specific manner.

Prosaposin is the precursor for saposins A, B, C, and D, which are small lysosomal proteins required for the hydrolysis of sphingolipids by specific lysosomal hydrolases. With a monospecific anti-saposin C antibody, which cross-reacts with prosaposin but not with saposin A, B, or D, the present immunoblot experiments showed that the rat brain expresses an unprocessed approximately 72 kDa protein (possibly prosaposin) and little saposin C. Regional analysis demonstrated that prosaposin is abundant in the brainstem, hypothalamus, cerebellum, striatum, and hippocampus, and less abundant in the cerebral cortex. Consistent with this finding, prosaposin-like immunoreactive neurons and fibers as revealed by immunohistochemistry were observed frequently in subcortical regions. The medial septum, diagonal bands, basal nucleus of Meynert, ventral striatum, medial habenular nucleus, and motor nuclei of cranial nerve had significant numbers of immunoreactive neurons. There were also nerve fibers with prosaposin-like immunoreactivity in several projection fields of the above nuclei. Other brain areas that contained prosaposin-like immunoreactive neurons and/or processes were: several brain nuclei (nucleus caudate putamen, globus pallidus, substantia nigra, red nucleus) constituting the so-called extrapyramidal system, reticular thalamic nucleus, entopeduncular nucleus, mammillary nuclei, auditory relay nuclei, cerebellum, sensory cranial nerve nuclei, and the reticular formation. The distribution pattern of prosaposin is apparently different from that of other neuroactive substances so far examined, and thus prosaposin may be involved in novel central events.

A new polymorphic 40-nucleotide repeat in the human dopamine transporter gene is described. It may underlie individual differences in susceptibility to several neuropsychiatric diseases.

Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to dogs produces clinical, pathological and neurological features in dog resembling human Parkinson's disease. Using this animal model, we studied the changes in diurnal rhythms of urine volume, creatinine in urine, and vasopressin, aldosterone and renin activity in plasma. Before MPTP treatment, urine volume showed a peak between 17.00 and 1.00 and plasma vasopressin concentration also showed a clear circadian rhythm with a peak at 13.00 and a minimum level at 5.00. Two weeks after MPTP treatment (2.5 mg/kg i.v.), the rhythm of urine volume disappeared and that of vasopressin became less clear. Plasma renin activity increased 2 and 4 weeks after MPTP treatment. The increase was, however, not enough to change the concentration of plasma aldosterone. We examined the effect of L-3,4-dihydroxyphenylalanine (levodopa), on the circadian pattern of urine volume and vasopressin attenuated by MPTP. Levodopa (4 mg/kg/day) was administered orally every day from the first week after MPTP treatment. The circadian rhythms of urine volume and vasopressin reappeared within one week after the start of levodopa administration.

Saposin-C, a small acidic glycoprotein that can activate glucosylceramide-s-glucosidase, has been isolated from bovine spleen. The complete amino acid sequence of bovine saposin-C was determined by Edman degradation of the purified protein and its fragmented peptides. It contains 80 amino acids, one carbohydrate chain attached to a single aspargine residue and six cysteine residues in oxidized form. The sequence of bovine saposin-C is 76 and 65% identical with the sequences of saposin-C from human spleen and guinea pig liver, respectively. Hydropathy profiles of the sequence of saposin-C from three species were similar despite the significant residue substitutions. Bovine saposin-C had a stronger effect in stimulating bovine s-glucosidase compared to human saposin-C. However, the effect of human saposin-C in stimulating human enzyme was stronger than that of bovine saposin-C. The region around residue 35, which is next to the extremely hydrophilic region, seems to be important to produce an interaction with the enzyme.

A unique dipeptide was isolated from bovine brain using five steps of ion-exchange chromatography. Its acid hydrolysate contained equimolar amounts of β-alanine and hypusine. The structure of the peptide was elucidated as α-(β-alanyl)hypusine using dansylation technique. About 1 μmol of the compound was isolated from 1090 g of bovine brain.

D -3-Aminoisobutyrate-pyruvate aminotransferase (EC 2.6.1.40, D -BAIB aminotransferase) participates in the metabolism of thymine. Recently we purified this enzyme from rat liver. We have studied D -BAIB aminotransferase further to clarify its physiological function. Among our findings were the following. (1) The enzyme activity was widely distributed in the organs of guinea pigs and rats. The kidney, liver, and lung showed high specific activities. (2) Using the livers of six vertebrates, difference between species were studies. Activity was detected in all species, the human liver showing the lowest activity among them. (3) Developmental study using rat liver showed that the activity was low at birth, increased sharply thereafter for 10 days, and then subsequently declined to the adult level. (4) Intraperitoneal injection of BAIB and β-alanine in rats was performed to determine whether they induce activity of this aminotransferase. Only BAIB increased the activity of the aminotransferase in the liver significantly. (5) Subcellular distribution study of this aminotransferase in rat liver revealed that it is a mitochondrial enzyme.

D-3-Aminoisobutyrate-pyruvate aminotransferase (EC 2.6.1.40) was purified 1900-fold from rat liver extract. The purified enzyme showed a molecular mass of 180 kDa by gel-permeation HPLC analysis using a TSK gel G3000SW column. Reductive polyacrylamide gel electrophoresis in sodium dodecyl sulfate resulted in identification of a single band of approx. 50 kDa, indicating that the native enzyme is probably a tetrametric protein. The specific activity of the purified enzyme was 1.14 mumol/min per mg protein. D-3-Aminoisobutyrate and beta-alanine were good amino donors. The Km value for L-3-aminoisobutyrate was 100-times larger than that for the D-isomer. The apparent Km values for D-3-aminoisobutyrate and beta-alanine were 35 and 282 microM, respectively. Pyruvate, glyoxylate, oxalacetate, 2-oxo-n-valerate, and 2-oxo-n-butyrate were good amino acceptors. The apparent Km values for pyruvate and glyoxylate were 32 and 44 microM, respectively.

Activator proteins for sphingolipid hydrolases (saposins) are small acidic, heat-stable glycoproteins that stimulate the hydrolysis of sphingolipids by lysosomal enzymes. The molecular mass of each stimulator is about 10 kDa, but glycosylated forms of higher mass exist too. The distribution and developmental changes in two saposins and their precursor proteins were studied with the aid of monospecific antibodies against saposin-B and saposin-C. They show a wide distribution in rat organs and forms intermediate between saposin and prosaposin (the precursor protein containing four different saposin units) could be seen. The amount of saposin and the degree of processing from prosaposin are quite different in different tissues. The saposins are the dominant forms in spleen, lung, liver, and kidney, while skeletal muscle, heart, and brain contain mainly precursor forms. In human blood, leukocytes contain mainly saposin, while plasma contains mainly precursor forms and platelets show many forms. Their subcellular distribution was studied using rat liver. The saposins of approximately 20 kDa are dominant in the light mitochondrial, mitochondrial, and microsomal fractions, following the distribution of the activity of a lysosomal marker enzyme. The nuclear fraction exhibits bands corresponding to non-glycosylated saposin. The soluble fraction contained much precursor forms. A developmental study of rat brain showed that the concentration of saposin precursors increased with age.

The excretion of 3-methylhistidine increased in the urine of dystrophic mice C57BL/6J. The content of 3-methylhistidine residue decreased in the muscle proteins of dystrophic mice, but not in other organs. Methylated proteins in the skeletal muscle, actin and myosin, were partially purified from the dystrophic and control muscles. The amount of 3-methylhistidine residue in unit weight of the actin and myosin preparations was normal in dystrophic muscle. These three facts indicate that the turnover rates of actin and myosin are increased in the muscle of the dystrophic mice.

The developmental changes of N-acetylaspartic acid (NA-Asp), N-acetyl-alpha-aspartylglutamic acid (NA-Asp-Glu), and beta-citryl-L-glutamic acid (beta-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 beta-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 beta-CG content decreased to an adult level at birth.

Summary 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 γ-globulin. The Km of myelin basic protein (25 μM) 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.

—An unknown radioactive compound was detected in the basic fraction of the trichloroacetic acid extract of rat brain injected with radioactive putrescine. This compound was purified from bovine brain and identified as γ-glutamylputrescine by comparison of its behaviour with that of the synthesized glutamylamides. The amide seemed to be metabolized as rapidly as putrescine in rat brain.

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.

Schlussfolgerung Wir haben schon berichtet22, dass wir den Anhaltspunkt fur den Zusamnienhang zwischen Serotonin-Stoffwechsel und Schizophrenie nicht linden konnten, womit Woolley's Annahme, Serotonin-Defizite im Hirn hatten mit Schizophrenie zu tun, abgelehnt worden ist. Inzwischen hat M. Bleuler23 uber die psychiatrischen Irrtumer in der Serotoninforschung geschrieben. Immerhin ist es interessant, dass nach unseren Ergebnissen21,22. Chlorpromnzin die Desaminicrung des Serotonins hemmt. Zwar haben Chlorpromazin und Reserpin, die mindestens als Symptomatica gegen bestimmte psychische Storungen verwendet werden, mit dem Serotoninstoffwechsel zu tun. In der vorliegenden Arbeit haben wir gezeigt, dass die Mehrzahl der einzelnen korperlichen Symptome unter der Reserpinkur mit der Mehrzahl der Symptomc bei der Serotoninbelastung und dem malignen Karzinoid- patienten ubereinstimmen. Selbstverstandlich konnen wir auf einige Unstimmigkeiten in heiden Fallen z. B. Parkinsonistische Zustande bei der Reserpinkur und Herzveranderungen beim malignen Karzinoid, hinweisen. Der Unter-schied der Symptonibilder konnte auf einen wesentlichen Unterschied in der Entstehung der Serotoninstolfwechselstorung bei den beiden verschiedenen Fallen zuruckgefuhrt werden. Beim Karzinoidsyndrom wird Serotonin im Organismus immer vermehrt produziert und unter der Reserpinwirkung wird Serotonin aus Gewebe ins Hlut ausge-schwemmt. In veiden Fallcn zeigt sich eine erhebliche Scrotoninamie bei den Wallungsanfallen und im Anfang der Reserpinkur. Die Zusaninienhange zwischen Karzinoid-Syndrom und Reserpin-Syndrom ergibt sich aus dem folgenden Schema (Fig. 13). Zusammenfassung. 1. Wenn man die korperlichen Erscheinungen unter der Reser-pinkur analysiert, kann man ahnliche Symptome wie beim malignen Diinndarmkarzinoid feststellen. 2. Beim Menschen wird sowohl bei Serotoninbelastung als auch bei Reserpinverabreichung einc erheblich vermehrte Ausscheidung der 5-OIES im Urin beobachtet. 3. Die Vermehrung der 5-OIES im Urin und die deutliche Oligurie gehen in beiden Fallen deutlich parallel. 4. Die Verminderung der Chlorid im Urin geht auch deutlich parallel. 5. Die hamatologischen Befunde bei Wallungen der malignen Kar-zinoidpatienten, wie- Vermehrung der Blutplattchen, Verkurzung der Blutungszeit, Verminderung der eosinophilen Leukozyten usw. konnen durch Serotoninbelastung kunstlich erzeugt werden. Dieselben hamatologischen Veranderungen werden auch durch Reserpininjektion hervor-gerufen und die Schwankungen der Blutbefunde gehen mit dem 5-OIES-Spiegel im Urin parallel. 6. Peristaltiksteigerung des Verdauungssystems, die beim Karzi-noid und bei der Serotoninbelastung beobachtet wird, kann bei der Reserpinverabreichung ebenfalls beobachtet werden. Die “Spike-Patterns” bei der Serotoninbelastung und nach der Reserpininjektion sind im Enteromyogramme identisch. Wir verdanken der Firma Ciba fur freundliche Unterstutzung unserer Arbeit.

An enzyme that hydrolyzes Na-(γ-aminobutyryl)lysine, a constituent of mammalian brain, was purified 62-fold from the hog kidney. Substrate specificity was investigated for 52 peptides and their derivatives. Thie enzyme seems to be a new peptidase, which hydrolyzes dipeptides having lysine, arginine, or ornithine as C-terminal. Heat stability, substrate specificity and its distribution in organs indicate that this enzyme is different from carboxypeptidase B. The pH optimum was between 6.1 and 6.8, and the Km for Na-(γ-aminobutyryl)lysine was 9.8 mM. Cadmium acetate, CuSO4, ZnSO4, HgCl2, o-phenanthroline and basic aliphatic amino acids, products of the reaction, inhibited its hydrolytic activity. The enzymic activity was widely distributed in various organs of the rat and rabbit with high activity in the skeletal muscle and kidney.

β-Aminoisobutyrate-α-ketoglutarate transaminase described by Kupiecki and Coon was further purified from hog kidney. The enzyme catalyzed the transamination with its l isomer while the d isomer, the natural form, was practically inactive as the substrate. The activity of the enzyme in the kidney of an hereditary high excretor of β-aminoisobutyric acid was not different from the activities of the low excretors. These findings indicate that the enzyme is not the principle enzyme catalyzing the degradation of the amino acid in vivo, and suggest the occurrence of another enzyme reaction by which d -β-aminoisobutyric acid is metabolized.

A basic amino acid, putreanine, was found in mammalian brain but not in other organs. It was purified from bovine brain by ion exchange chromatography and was crystallized as the sulfate. From 82 kg of brain, 535 mg of the sulfate were obtained. Elementary analysis gave its empirical formula as C7H16N2O2. Its structure was subsequently determined as N-(4-aminobutyl)-3-aminopropionic acid by Shiba and Kaneko (see "Appendix"). The concentration of putreanine was determined by preliminary purification of the basic amino acid fraction from a trichloracetic acid extract of mammalian tissues followed by analysis in an automatic amino acid analyzer. It was only detected in the central nervous system of mammalian and avian organisms. Its concentration was highest in caudal regions of the brain, and the white matter of the cerebral and cerebellar cortices and the spinal cord contained more than grey matter of the corresponding areas. Putreanine appears in the brain of rats 2 weeks after birth and subsequently its concentration increases for several months.

— α-(γ-Aminobutyryi)-lysine was identified in rabbit brain. This compound was detected exclusively in the brain of mammals, but not in other tissues. It is not concentrated in any particular region of the rabbit brain.

— Transfer of the methyl group of S-adenosyl [Me-14C]methionine into cerebral proteins, an encephalitogenic protein and histones was studied using extracts of bovine and rat brains. The brain extract contains multiple substrate proteins and their lysine and arginine residues were methylated to form Ne-mono-, -di- and -trimethyl-lysine and NG-mono-, NG, NG- and NG,NG-dimethylarginine residues respectively, at different rates. The enzyme which catalyses the methylation of arginine residues was differentiated by ammonium sulphate fractionation from that methylating lysine residues. Methylation of arginine and lysine residues of proteins was stepwise in general, from mono- to dimethyl-arginine and from mono- to di- and trimethyl-lysines. Two different enzymes which methylate histone and the encephalitogenic basic protein respectively were obtained from the cytoplasmic fraction of rat brain and their enzymic properties were examined.

1. Eight different substances were isolated in crystalline forms by ion exchange chromatography of the aliphatic basic amino acid fraction of human urine. 2. Structures of guanidino-N,N-dimethylarginine and guanidino-N,N'-dimethylarginine were assigned for two substances by analyses of the degradation products, elemental compositions, and nuclear magnetic resonance spectra. The identities of the compounds were confirmed by synthesis followed by comparison of infrared and nuclear magnetic resonance spectra and of chromatographic properties with those of the isolated compounds. The concentrations of these two compounds in human urine are higher than that of arginine. Their natural occurrence has not been reported previously. 3. Three compounds, Ne-methyl-, Ne,Ne-dimethyl-, and Ne,Ne,Ne-trimethyllysine, were identified by elementary analyses and syntheses. Occurrence of the last two compounds in free state in nature has not been reported previously. 4. Two compounds, glucosylgalactosyl- and galactosyl-δ-hydroxylysine, whose occurrence in human urine has been reported previously, were isolated as crystals for the first time. 5. The concentrations of these N-methyl derivatives of lysine and arginine in the urine were not changed by oral loading of either lysine or arginine. Protein-free diet did not affect the amounts of these compounds in urine. The possibility that these compounds are derived from tissue proteins in which lysine and arginine residues are methylated was discussed.

An enzyme which catalyzes a reversible transamination between d-β-aminoisobutyrate and pyruvate was found in the liver of rat, guinea pig, and hog, and the enzyme in an extract of hog liver acetone powder was purified about 200-fold. The enzyme was localized almost exclusively in the liver among the tissues examined in the guinea pig. It was most active against d-β-aminoisobutyrate among the ω-amino acids examined, and had little effect on the l isomer; this specificity is markedly different from that of β-aminoisobutyrate:2-oxoglutarate aminotransferase, for which the d isomer is not a substrate. Since the β-aminoisobutyrate in human urine and liver has the d configuration and a genetically determined high excretor cannot destroy the amino acid, it was postulated that the enzyme activity might be absent from the liver of genetic high excretors of β-aminoisobutyrate. The activity of the enzyme in liver and the concentration of β-aminoisobutyrate in urine were measured in material obtained at autopsy of 15 bodies. The enzyme activity was always found in the liver specimens from eight low excretors and not found in five of seven high excretors. Thus the genetically determined high excretion of β-aminoisobutyrate occurs most probably because of the absence of the aminotransferase from the liver of excretors.

The distribution of alpha-(gamma-aminobutyryl)-hypusine was examined in several organs of the rabbit and in the brain of the rat, rabbit, dog, ox, and monkey. The peptide occurred only in the brains, but appeared to be absent from dog brain. Concentrations were higher in the cerebral hemispheres than in other portions of the brain. No significant difference between white and gray matter was observed.

– γ-Glutamyl amides of histamine, serotonin and dopamine were formed from these amines by the transfer of the γ-glutamyl moiety from γ-glutamyl peptides in the presence of γ-glutamyl transpeptidase. [I4C]Histamine was injected intraventricular into rats, and the formation of γ-glutamyl-[14C] histamine in the brain was confirmed by purification and identification with the authentic compound. The radioactivity was highest 30 min after the injection. The possible significance of γ-glutamyl amides in nerve transmission is discussed.

γ-Glutamylhistidine was purified from 7.5 kg of bovine brain. ldentification was based on the comparison with the synthetized compound. γ-Glutamyllysine and γ-glutamylarginine were not detected. The present study added another γ-glutamyl amino acid 10 the list of the previously known 10 γ-glutamyl compounds.

L-(Methyl- 14 C)-methionine was administered i.p. to mice, and the incorporation of radioactive methionine into proteins and methyllysine and methylarginine residues formed by the transfer of the methyl- 14 C group of methionine were measured. Tissue protein was actively methylated in organs having a high activity of protein synthesis, and the in vivo methylating activity in organs was not correlated with the protein methylating activity of the organs determined in vitro. Puromycin inhibited both protein synthesis and protein methylation in mouse organs to a similar degree. Neither the formation of S-adenosyl-(methyl- 14 C)-methionine nor protein methylase was inhibited by puromycin. The data suggests that proteins are methylated immediately after protein synthesis, that is, newly synthesized proteins are the substrates of protein methylation. Radioactive methionine and the [C 14 ] methyl groups of methyllysine and methylarginine residues of tissue proteins are degraded in parallel over a period of 3 wk, suggesting that protein methylation is an irreversible type of protein modification.

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.

Ab unknwon compound containing glutamic acid residue was found in newborn rat brain. The compound occurred predominantly in brain. Its concentration was approx. 1 μmol/g tissue at birth and decreased to one-tenth 24 days after birth. The compound was isolated from newborn rat brains, and subjected to elementary analysis and to infrared and mass spectrometric analysis. Glutamic acid and citric acid were formed from the compound on acid hydrolysis. The compound was presumed to be a citryglutamic acid. Two isomers, α- and β-citrylglutamic acid, were sunthesized. The unknown compound was identified as β-citryl- L -glutamic acid. The occurrence of this compound has not been reported in nature.

The concentration of D-beta-aminoisobutyric acid (D-BAIB) in the liver and kidney was twice as high and dropped more slowly in the female mouse than in the male after an intraperitoneal injection of thymine. The concentration of beta-alanine, formed from uracil by the same enzyme system catalyzing formation of D-BAIB from thymine, was not different in the liver and kidney of both sexes after an intraperitoneal injection of uracil. After the intraperitoneal injection of D-BAIB, the concentration of BAIB in male liver decreased faster than that in female liver. Inhibition of D-BAIB: pyruvate aminotransferase caused by injection of D-cycloserine resulted in a significant increase in the concentration of BAIB in liver of both sexes after injection of thymine, but the concentration dropped more rapidly in the male. The activity of D-BAIB: pyruvate aminotransferase was not different in the livers of male and female mice. Under the action of probenecid, an inhibitor of active transport systems, the sex difference in accumulation and disappearance of the amino acid in the liver was not observed. This suggested that the excretion of BAIB is more active in the renal tubules of the male mouse than in those of the female. However, the amount of BAIB excreted in the urine after injection of thymine was larger in the female mice than in the male mice. There may be another probenecid-sensitive enzyme for the disposal of BAIB in male mice.

A simple and sensitive gas-chromatographic method for the determination of N-acetyl-L-aspartic acid (NA-Asp), N-acetyl-alpha-aspartylglutamic acid (NA-Asp-Glu) and beta-citryl-L-glutamic acid (beta-CG) was developed. The organ, regional and phylogenetic distributions of these compounds were studied. NA-Asp and NA-Asp-Glu were highly concentrated in nervous tissue, and less than 1% of the amounts in the nervous tissues were found in non-nervous organs. These two compounds showed a reciprocal relationship in their regional distribution in mature brains, but such a relationship was not evident or was even reversed in immature brains. The two compounds also showed different developmental changes in different regions of the brain. Fish brain contained a relatively high concentration of NA-Asp, but only a trace amount of NA-Asp-Glu. By contrast, a 10 times higher concentration of NA-Asp-Glu than NA-Asp was found in frog brain. Reptilian brain contained similar amounts of each compound. Avian and mammalian brain had NA-Asp at a roughly 10 times higher concentration than NA-Asp-Glu. beta-CG occurred at the highest concentration in the immature brain of rat and guinea pig, but disappeared in the mature brains. The adult frog brain, however, contained a large amount of beta-CG. In the adult rat, testis contained the highest concentration of beta-CG.