Voprosy biokhimii mozga最新文献

Acid and neutral proteinases were isolated with the purpose of investigating their participation in the breakdown of hypothalamic peptides and proteins. The acid proteinase was purified about 1000-fold from hypothalamus by precipitation with acetone, chromatography on SP-Sephadex G-50, gel filtration through column of G-100 and chromatography on DEAE-Sephadex A-50. The molecular weight of the enzyme was approximately 50.000. Maximal activity against hemoglobin was obtained at pH 3,2--3,5: serum albumin was split much more slowly. Hypothalamus acid proteinase was partially inhibited by beta-phenyl pyruvate, benzothonium cloride, and was completely inhibited by low concentrations of pepstatin. This proteinase splits somatostatin, Substance P and some C-fragments of Substance P. The probable sites of enzyme action on these peptides were determined by the end group dansyl technique. Neutral proteinase was isolated from the supernatant fraction(100.000 g) of a 0,3 M sucrose homogenate of bovine hypothalamus by chromatography on DEAE Sephadex A-50, gel filtration through Sephadex G-100 and rechromatography on DEAE sephadex A-50 using luliberin as substrate. The rates of breakdown of luliberin and denaturated hemoglobin were measured by fluorometric estimation of acid-soluble peptides wieht o-phthaldialdehyde. The purifed enzyme preparations have a pH optimum of activity at 7--7,5. The enzymes molecular weight was approximatelyy 30--40.000. Enzyme activity was inhibited by L-1-tosylamide-2-phenylethylchloromethyl ketone, p-chloromercuribenzoate and divalent ions Co2+, Zn2+ and was significantly enhanced by dithiothreitol. The Km values for the reaction of hydrolysis of luliberin and hemoglobin were 1,33.10(-5) and 5,2.10(-5) M respectively. The neutral proteinase from the hypothalamus cleaves luliberin, somatostatin and Substance P. Sites of action of the enzyme upon those peptides were determined by means of the dansyl technique. The acid proteinase, most likely cathepsin D, and neutral proteinase from hypothalamus, may play an important role in the formation and breakdown of peptide hormones in the hypothalamus.

It has been studied the role of intracysternal administration of neuropeptides--five analogous of 8-lysinvasopressin (LVP) [Des-9-Gly-cine-amide-LVP (DGLVP), Tri-Gly-LVP, deamino-LVP (1--beta-mercaptopropionic acid-LVP), Leu4-LVP, Asn1,6-LVP] and two cardiotrop hexapeptides (acetylated and non) separated from the hypothalamus in the regulation of various types of RNA (GC and AU types n-RNA, r-RNA, t-RNA) biosynthesis in the brain. Using the combined method of phenol extraction, differential ultracentrifugation, gel-filtration and ultraviolet spectroscory of various types of RNA has been shown the increase in the content of r-RNA and t-RNA under the influence of DGLVP, Leu4-LVP and cardiotrop hexapeptide. Deamino-LVP, Tri-Gly-LVP and acetylated hexapeptide stimulate the rise in the content of r-RNA and AU type n-RNA (precursor of m-RNA). Ans1,6-LVP shows a nonspecific influence on the biosynthesis of the brain RNA. It has been suggested a hypothesis about a participation of the neuropeptides in the regulation of various types of the brain RNA biosynthesis, as a primary messanger (starting signals) during the stimulation of the initiation of cycloadenylate- and cycloguanylate-sensitive transcription, via phosphorylation of diverse RNA-polymerases. It was given a scheme of interaction between the messanger conception of neuropeptides and cyclic-3', 5'-monophosphate nucleotide-dependent transcription by which the cycloadenylate-dependent transcription appears to play a great role in the r-RNA and t-RNA biosynthesis and the cycloguanylate-dependent transcription -- in the biosynthesis of m-RNA.

GABA, its derivative -- gamma-hydroxybuturic acid and metabolite --succinic acid have a pronounced dilatatory activity on cerebral circulation in various brain parts. GABA increases cerebral circulation by 25.3%, gamma-hydroxybutyric acid by 35.9% and sucinic acid by 20.4%. In ischaemia of the brain a relationship has been established between cerebral circulation, changes in the GABA system in brain and in the walls of cerebral arteries. The content of GABA increases following enhancement of GAD activity and inhibition of GABA-T. The increase of endogenous GABA level in brain during hypoxia of the brain brings to an improvement of blood circulation through increasing collateral vessels. Experiments with GABA-T inhibition by aminooxyacetic acid give direct evidence about the role of the GABA system in cerebral blood circulation. This mechanism is evaluated by us as an example of an autoregulatory system that is realized by a feed-back mechanism providing the adaptability and compensatory function of cerebral haemodynamics to changing conditions.

Calculated on a protein basis the yield of neuronal and glial cell-enriched fractions obtained from the brain of aged and adult rats was found to be equal. In aged animals the high affinity uptake of 14C-GABA by neuronal fractions is reduced by 27.34% while that of glial fractions by 11.87%. At 0 degrees both age groups studied the uptake of 14C-GABA is greatly inhibited--by 66% in neurones and 92% in glia. The conclusion is drawn that glial cells are changed much less with age than neurones and thus their role as modulators of neuronal function increases with age.

Our previous studies have shown that the two coronarodilatatory hypothalamic proteins isolated by us are not only carriers for neurohormones "K" and "C" but also procursors of coronarodilatory substances. In the present study we have looked at some physico-chemical properties of these proteins through isoelectric focusing and gel electrophoresis as well as the possibility of obtaining coronaroactive fragments by proteolytic enzymes. The results obtained have shown that the isoelectric points of the coronaroactive proteins are between pH 6.2 and 6.4. Polyacrylamide gel-electrophoresis has shown that one of the coronaroactive proteins, the carrier of neurohormone "C", moves towards the anode and is homogenous while carrier of neurohormone "K" is made up of two protein fractions. Under the action of certain enzymes (trypsin and pepsin) two coronaroactive fragments are obtained from the "neutral" protein carrier. Such an effect is not observed following the action of pronase and chymotrypsin. The physico-chemical properties of these fragments require further studies.

In glial tumors of human brain the content of homocarnosine has been found to be lower than in brain tissue. In experimental animals cranial injury induces an increase of homocarnosine in brain tissue. Stimulation of the nervous system of traumatized animals by phenamin brings the metabolism of homocarnosine back to normal. The results of these studies make us suppose that homocarnosine is a substance involved in brain activity.

A comparative study of respiration of mitochondrial and synaptosomal fractions of adult rat brain cerebral cortex has shown that glutamic acid is oxidized in both fractions and that its oxidation is potentiated by ADP. Addition of ADP has, however, a different effect on the endogenous respiration of these two fractions; while it has no effect on the endogenous respiration of the synaptosomal fraction, it increases considerably the weak endogenous respiration of the mitochondrial fraction.

The changes in the rates of citrate biosynthesis and utilization in rat brain, liver, kidney and heart, produced by hypoxia, action of 2,4-DNP and thyreotoxicosis, were compared with changes of some regulatory parameters under the same conditions. The comparison of citrate-synthase activities, citrate levels in tissues and 14C-incorporation from different precursors into citric acid permitted us to establish that the biosynthesis of citrate in brain was more intensive than in other tissues studied. The main source of acetyl-CoA for citrate-synthase reaction in brain is the oxidation of pyruvate. The ratio of adenine nucleotides plays an important role in the control of citrate-synthase activity in brain, where the oxaloacetate control is not as significant as in liver. NAD-specific isocitrate dehydrogenase reaction was found to be the dominant pathway for citrate oxidation in brain: more than 60 percent of brain citrate were oxidized by NAD-ICDH, while less than 10 percent of citric acid were utilized by this enzyme in other tissues studied. The existance of an adenine nucleotide control of NAD-ICDH activity in brain may be an additional mechanism for the regulation of the first steps of energy metabolism in brain.