牛脑Ca~(2+)/CaM依赖性蛋白激酶Ⅱ的纯化和鉴定

通过一系列层析法,首次从牛脑纯化得到胶凝电泳匀一的Ca~(2+)/CaM PKⅡ。凝胶过滤法测定全酶分子量为550kD,SDS-PAGE法测定亚基分子量为55kD,推测牛脑Ca~(2+)/CaM PK Ⅱ由十个相同的亚基组成。该酶活性绝对依赖于Ca~(2+)和CaM,以63kD PDE同工酶为底物,其AC_(50)分别为0.85μmol/L和0.18μmol/L;以酪蛋白为底物,其AC_(50)分别为0.22μmol/L和0.06μmol/L。牛脑Ca~(2+)/CaM PK Ⅱ旣能催化63kD PDE同工酶等多种蛋白或酶磷酸化,又能进行自身磷酸化。该酶催化63kD PDE同工酶最大磷酸参入量为1mol/mol亚基。磷酸化型63kD PDE同工酶的Ca~(2+)的AC_(50)高于非磷酸化型。     

心肌肌浆网膜钙泵调节蛋白——受磷蛋白的结构与功能

受磷蛋白(phospholamban,PHL)是心肌肌浆网膜钙泵的调节蛋白。它是由五个相同亚基组成的分子量为25000Da的寡聚体膜蛋白。它对心肌钙泵的调节是通过磷酸化和脱磷酸化作用来实现的。磷酸化作用使它在SDS-PAGE上表观分子量的变化显示出其亚分子结构的复杂性。本文着重介绍了在心肌兴奋-收缩偶联过程中,受磷蛋白的调节作用以及它的结构特征。     

REGULATION OF THE LEVEL OF ENDOGENOUS PHOSPHORYLATION OF SPECIFIC BRAIN PROTEINS BY DIPHENYLHYDANTOIN1

Diphenylhydantoin (DPH) in therapeutic concentrations caused a decrease in the net level of endogenous phosphorylation of two specific proteins from rat brain cerebri, while not significantly affecting the phosphorylation of other protein substrates. The apparent molecular weights of the DPH- specific substrate proteins were 60-63,000 and 49-52,000, and were designated proteins DPH-L and DPH-M, respectively. DPH decreased both the initial rate and the net level of [³²P] phosphate incorporation from [γ-³²P] ATP into proteins DPH-L and DPH-M. The concentrations of DPH required to produce a half maximal decrease in the levels of phosphorylation of proteins DPH-L and DPH-M was 3 × 10^-4 and 8 × 10 ^-4 M, respectively. The effects of DPH on the incorporation of [³²P] phosphate into these specific brain proteins were independent of the concentration of ATP over a wide range of ATP concentrations. The DPH-specific proteins were demonstrated to be present in synaptosomal preparations. The results are compatible with the hypothesis that some of the stabilizing actions of DPH on neuronal tissue and seizure discharge may be mediated by the effect of this anticonvulsant on the phosphorylation of specific brain protein substrates.     

吗啡及第二信使对鼠脑细胞膜蛋白质磷酸化的调节

 本文研究了几种蛋白激酶活化剂及吗啡对脑细胞膜蛋白质磷酸化的调节。cAMP刺激了一种68KDa蛋白质和几种60KDa相关的蛋白质的磷酸化作用,Ca~(++)刺激68KDa和50KDa蛋白质的磷酸化。μ吗啡受体的特异性兴奋剂D-脑啡肽(DAGO)增加68KDa蛋白质的磷酸化,而吗啡K受体的特异性兴奋剂,Bremazocyne抑制这一蛋白质的磷酸化。蛋白激酶c的特异性活化剂——磷脂酰丝氨酸(PS)和甘油二油酸酯(DO)不促进这一磷酸化。相反,却抑制cAMP、Ca~(++)、和DAGO所刺激的68KDa蛋白质的磷酸化。结果表明,在鼠脑细胞膜存在一种68KDa专一的蛋白激酶,其活性受吗啡及几种细胞内信使分子,如cAMP、Ca~(++)和DO的调节。     

溴氰菊酯对鸡脑突触膜蛋白磷酸化和Ca-ATP酶活力的影响

研究了神经毒性杀虫剂———溴氰菊酯对体内源性蛋白质磷酸化作用的影响。结果表明,浓度为１０－５ｍｏｌ／Ｌ溴氰菊酯明显抑制正常鸡和经三甲基苯基磷酸酯处理的鸡脑突触膜中５５ｋＤ和６０ｋＤ两种蛋白的磷酸化。而０２５ｍｍｏｌ／ＬＣａ２＋加０２５ｍｍｏｌ／Ｌ的钙调蛋白则明显地促进这两种蛋白质的磷酸化,但较低浓度（１０－６ｍｏｌ／Ｌ）时,溴氰菊酯明显抑制４８ｋＤ蛋白的磷酸化。而００３ｍｍｏｌ／ＬＣａ２＋加００３ｍｍｏｌ／Ｌ的钙调蛋白则明显地增强４８ｋＤ和４５ｋＤ两种蛋白的磷酸化。此外,还发现溴氰菊酯可抑制鸡脑突触膜中ＣａＡＴＰ酶活力。     

THE ACTIVITY OF PARTIAL REACTIONS IN THE CHAIN ELONGATION OF PALMITOYL-CoA AND STEAROYL-CoA BY MOUSE BRAIN MICROSOMES

Partial reactions in the overall chain elongation of palmitoyl-CoA and stearoyl-CoA by mouse brain microsomes have been analyzed. The rate of the initial condensation reaction between palmitoyl-CoA and malonyl-CoA was more than 5 times greater than the rate obtained with stearoyl-CoA, and in both cases good agreement between condensation and overall chain elongation rates was observed. By contrast, both β-hydroxyoctadecanoyl-CoA and β-hydroxyeicosanoyl-CoA were quite rapidly dehydrated by brain microsomes at similar rates. Similar results were obtained with 2-trans-octadecenoyl-CoA and 2-trans-eicosenoyl-CoA in which both substrates were rapidly reduced at nearly the same rate in the presence of NADPH. In all cases, intermediate reactions subsequent to condensation were much more rapid than overall chain elongation. These results suggest that the mechanism of malonyl-CoA-dependent fatty acid chain elongation in brain microsomes is similar to that observed in other tissues, and are consistent with an overall regulation of chain elongation mediated primarily by the initial condensation reaction.     

TRANSIENT PHOSPHORYLATION OF DEOXYRIBOSIDES AND REGULATION OF DEOXYRIBONUCLEIC ACID SYNTHESIS

Microspores isolated from Lilium longiflorum and Trillium erectum were studied with respect to their capacities for phosphorylating deoxyribosides in vitro. It was found that such capacities are manifest only during brief intervals of time adjacent to periods of DNA synthesis, and that none of the neighboring cells in the anther acquire them. The observed patterns of behavior are interpreted in terms of enzyme induction as a device for regulating DNA synthesis.     

ACTIVITY OF CYCLIC AMP-DEPENDENT MICROSOMAL PROTEIN KINASE AND PHOSPHORYLATION OF RIBOSOMAL PROTEIN IN RAT BRAIN DURING POSTNATAL DEVELOPMENT

Abstract— Microsomes from rat brain exhibited protein kinase activity which was stimulated by cyclic AMP when assayed in the presence of exogenous protein substrate, such as thymus histone. In the absence of exogenous substrate some phosphorylation of microsomal protein occurred, but no stimulation by cyclic AMP could be discerned, probably because of limitations of substrate. The maximal activity of microsomal protein kinase observed in the presence of saturating concentrations of histone and the optimal concentration (5 μ m ) of cyclic AMP remained essentially unchanged from birth to early adulthood, but the magnitude of the stimulation by cyclic AMP was significantly higher at birth than at 30 days of age. Brain ribosomal proteins could be phosphorylated by the cyclic AMP-dependent brain protein kinase. Their total capacity for acceptance of phosphate by means of this phosphorylation reaction remained unchanged throughout the postnatal development of the brain. Our results are consistent with the possibility that phosphorylation of ribosomal protein mediated by cyclic AMP-dependent protein kinase may play a a role in the postnatal regulation of cerebral protein synthesis, as a result of the changes in the levels of cyclic AMP known to occur in brain during postnatal maturation.     

DIFFERENCES IN THE REGULATION OF TYROSINE AMINOTRANSFERASE IN BRAIN AND LIVER

Abstract— l -Tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5) activity in rat brain is not regulated in the same way as in rat liver. No diurnal rhythm in the activity of the cerebral enzyme was found in rats fed ad lib. although there was a marked diurnal variation in the activity of the hepatic enzyme. In adrenalectomized rats, hydrocortisone and glucagon induced the enzyme in liver but had no effect on the enzyme in brain. In normal rats, treatment with reserpine or exposure to cold elevated the activity of the hepatic enzyme without affecting the enzyme in brain. Thus, the tyrosine aminotransferase of brain differed from the enzyme in liver since it did not exhibit diurnal variations of activity and was not affected by hormones, drugs, or stress.     

ISOLATION AND CHARACTERIZATION OF BOVINE BRAIN CATHEPSIN D

Bovine brain cathepsin D was purified 1774-fold with a 19% recovery by affinity chromatography on immobilized pepstatin. Approximately 2 mg of enzyme protein were isolated from 150 g (wet weight) of bovine brain. The enzyme eluted from gel filtration as a single peak with a molecular weight of 40,000–42,000. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the predominant band migrated with a molecular weight of 48,000: however, less distinct bands were also present in the molecular weight ranges of 31,000 and 13,000. The isolated enzyme had isoelectric points over a range of pH 5–7 with 3 major peaks occurring at pH 5.6, 6.1, and 6.6. The amino acid composition of brain cathepsin D showed substantial differences from that reported for cathepsin D isolated from bovine spleen. Amino-terminal sequence analysis revealed an Asp-Val-lle sequence by Edman degradation. With hemoglobin as the substrate the enzyme had an apparent K, of 60mM.