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101.
Summary Mitochondrial creatine kinase (CK) purified from canine myocardium showed a single protein band on SDS-PAGE and was free of
MMCK. Its amino acid composition was different than MMCK or BBCK and did not react to antiserum to MMCK or BBCK. Using purified
mitochondrial, MM and BBCK, the velocity of reaction (V) was estimated for creatine phosphate (CP), creatine (C), adenosine
triphosphate (ATP) and adenosine diphosphate (ADP) over a wide range of concentrations including those at Vmax. The values for Km (mM/L) derived from Lineweaver-Burke plots are shown:
The affinity of mitochondrial CK for C is much greater than MMCK which is compatible with the energy shuttle hypothesis, namely
ATP is converted by mitochondrial CK to CP, and then diffuses to the myofibril for conversion to ATP for utilization. 相似文献
102.
To determine which of the major isoenzymes of pyruvate kinase pancreatic islet pyruvate kinase most resembled, it was compared to pyruvate kinase from other tissues in kinetic and immunologic studies. The pattern of activation by fructose bisphosphate and the patterns of inhibition by alanine and phenylalanine were most similar to those of the M2 isoenzyme from kidney and were dissimilar to those of the isoenzymes from skeletal muscle (type M1) and liver (type L). The islet pyruvate kinase was inhibited by anti-M1 pyruvate kinase serum (which crossreacts with the M2 isoenzyme), but not by anti-L pyruvate kinase. These results are most consistent with islets possessing predominantly, if not exclusively, the M2 isoenzyme of pyruvate kinase. We previously showed that rat pancreatic islet cytosol contains protein kinases that can catalyze a calcium-activated phosphorylation of an endogenous peptide that has properties, such as subunit molecular weight and isoelectric pH, that are identical to those of the M2 and M, isoenzymes of pyruvate kinase, and that islet cytosol can catalyze phosphorylation of muscle pyruvate kinase. In the present study it was shown that incubating islet cytosol with ATP under conditions known to permit phosphorylation and inhibition of liver pyruvate kinase did not affect the islet pyruvate kinase activity. It is concluded that phosphorylation of the islet pyruvate kinase has no immediate effect on enzyme activity.Abbreviations EGTA
ethylene glycos his (-aminoethyl ether)-N,N,NN-tetraacetic acid
- Hepes
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 相似文献
103.
Abstract cAMP-dependent phosphoprotein changes were determined using 1-dimensional SDS-gel electrophoresis in a cAMP-requiring yeast mutant ( Saccharomyces cerevisiae AM18). During cAMP starvation, the yeast cells accumulated 3 32 P-labeled bands with M r / 72000, 54000, and 37000. The M r / 72000 protein was the most prominent phosphorylated protein. After the readdition of cAMP, these phosphoproteins lost their 32 P-label while phosphoproteins with M r / 76000, 65000, 56000 and 30000 were accumulated. Similar phosphoprotein changes were also detected in cdc35 at the nonpermissive temperature, but not in wildtype (A363A) or cdc7 strains of S. cerevisiae . 相似文献
104.
Bernhard Knig Patricia A. DiNitto Peter M. Blumberg 《Journal of cellular biochemistry》1985,29(1):37-44
The major phorbol ester receptor is the Ca++-activated, phospholipid-dependent protein kinase C. Diacylglycerol stimulates protein kinase C in a fashion similar to the phorbol esters. Likewise, it inhibits phorbol ester binding competitively. Both results suggest that diacylglycerol is the/an endogenous phorbol ester analogue. Alternatively, the diacylglycerol might simply be acting to modify the phospholipid environment of the protein. If diacylglycerol were indeed functioning as an analogue, it should interact with the receptor stoichiometrically. This interaction can be quantitated by measuring the perturbation in apparent diacylglycerol binding affinity as a function of the ratio of diacylglycerol to receptor. We report here that 1,2-dioleoylglycerol interacts with the receptor with the predicted stoichiometry. 相似文献
105.
p38MAPK是丝裂原活化蛋白激酶(mitogen activated protein kinases,MAPK)家族的一个亚类,在高等脊椎动物免疫应答的信号转导过程中扮演着非常重要的角色。在日本七鳃鳗(Lampetra japonica)中发现,p38MAPK以两种异构体的形式存在。通过克隆它们的开放阅读框并进行同源序列比对和系统发育分析,鉴定它们分别为p38α(Lja-mapk14)和p38β(Lja-mapk11)。用混合菌刺激七鳃鳗,利用免疫印迹方法,检测Lja-mapk14在外周血类淋巴细胞、鳃组织和髓样小体中,分别在加强免疫36 h、24 h和24 h后,表达量达到峰值,分别为对照组的2.9、2.1和2.6倍;而Lja-mapk11在以上组织中,都在加强免疫36 h后达到表达量峰值,分别为对照组的2.2、2.5和6.3倍。实时荧光定量PCR检测发现,Lja-mapk14的mRNA表达水平在混合菌加强免疫36 h后,分别在类淋巴细胞、鳃组织和髓样小体中,上调2.3、1.5和3.4倍;而Lja-mapk11的则分别在类淋巴细胞、鳃组织和心肌中,上调1.3、2.6和1.6倍。以上结果在mRNA和蛋白质水平证明,Lja-mapk14和Lja-mapk11均参与七鳃鳗的免疫应答反应。采用B细胞和T细胞丝裂原LPS和PHA分别对七鳃鳗进行刺激,免疫印迹结果显示,Lja-mapk14和Lja-mapk11蛋白质表达量经LPS加强免疫36 h后,在类淋巴细胞、鳃组织和髓样小体中,上调表达1.3 ~ 4.1倍;而经PHA加强免疫36 h后,Lja-mapk14和Lja-mapk11在上述组织中表达量均不存在显著变化。以上结果说明,Lja-mapk14和Lja-mapk11可能参与了B细胞丝裂原LPS介导的VLRB类淋巴细胞亚群的免疫应答反应。 相似文献
106.
C. Lal Kapoor Gerald J. Chader 《Biochemical and biophysical research communications》1984,122(3):1397-1403
A calcium phospholipid-dependent protein kinase (C-kinase) activity was detected in the soluble fraction of rod outer segments (ROS) of the bovine retina. The enzyme required calcium, phosphatidylserine (PS) and diacylglycerol for maximal activity. In the presence of calcium and PS, C-kinase endogenously phosphorylated proteins with molecular weights of 95,000, 91,000, 31,000, 21,000, 19,000, 18,000, 16,000, 14,000 and 11,000. Addition of diolein in the reaction mixture further enhanced the endogenous phosphorylation of these proteins. Retinal was found to inhibit the phosphorylation of endogenous proteins by C-kinase in a concentration dependent manner. Half-maximal inhibition of enzyme activity was obtained at a retinal concentration of about 12μM. These results suggest that calcium, phospholipids and the C-kinase enzyme may play an important role in the functional regulation of rod photoreceptors and, with retinal, perhaps in the visual process as well. 相似文献
107.
Changes during growth in the activity of several enzymes involved in purine "salvage", adenine phosphoribosyltransferase (EC 2.4.2.7), guanine phosphoribosyl-transferase (EC 2.4.2.8), hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and adenosine kinase (EC 2.7.1.20), the enzymes which catalyze the conversion of nucleoside monophosphate to triphosphate, nucleoside monophosphate kinase (EC 2.7.4.4) and nucleoside diphosphate kinase (EC 2.7.4.6), and several degradation enzymes, deoxyribonucleae(s), ribonuclease(s). phosphatase(s), nucleosidase (EC 3.2.2.1), 3'-nucleotidase (EC 3.1.3.6) and 5'-nucleotidase (EC 3.1.3.5) were examined in cells of Catharanthus roseus (L.) G. Don cultured in suspension. In addition, the incorporation of [8-14 C] adenine, [8-14 C] adenine, [8-14 C]hypoxanthine. [8-14 C] adenosine and [8-14 C]inosine into nucleotides and nucleic acids was also determined using intact cells.
The activities of all purine "salvage" enzymes examined and those of nucleoside monophosphate and diphosphate kinases increased rapidly during the lag phase and decreased during the following cell division and cell expansion phases. The rate of incorporation of adenine, guanine, hypoxanthine, and adenosine into nucleotides and nucleic acids was higher in the lag phase cells than during the following three phases. The highest rate of [8-14 C]inosine incorporation was observed in the stationary phase cells. The activity of all degradation enzymes examined decreased when the stationary phase cells were transferred to a new medium.
These results indicated that the increased activity of purine "salvage" enzymes observed in the lag phase cells may contribute to an active purine "salvage" which is required to initiate a subsequent cell division. 相似文献
The activities of all purine "salvage" enzymes examined and those of nucleoside monophosphate and diphosphate kinases increased rapidly during the lag phase and decreased during the following cell division and cell expansion phases. The rate of incorporation of adenine, guanine, hypoxanthine, and adenosine into nucleotides and nucleic acids was higher in the lag phase cells than during the following three phases. The highest rate of [8-
These results indicated that the increased activity of purine "salvage" enzymes observed in the lag phase cells may contribute to an active purine "salvage" which is required to initiate a subsequent cell division. 相似文献
108.
The levels of adenylate nucleotides were examined in 4th-instar Trichoplusia ni larvae 3 days after parasitization by the insect parasite Hyposoter exiguae. In general, parasitization caused a decrease in the level of ATP and increased ADP and AMP levels. These changes resulted in alteration of the adenylate kinase mass-action ratio. The overall energy status of parasitized larvae, however, as indicated by energy ratios, including the “energy charge,” was affected only slightly. The result demonstrates that the host maintained an active and viable metabolic state despite extensive alterations in physiology which occur at this stage of the parasite-host association. 相似文献
109.
Characterization of Cyclic AMP-Dependent Phosphorylation of Neuronal Membrane Proteins 总被引:4,自引:4,他引:0
We examined the patterns of cyclic AMP-dependent protein phosphorylation in membranes prepared from rat cortical synaptosomes following gel electrophoresis and autoradiography. We determined the optimum pH (6.2), time (20 s), Mg2+ concentration (10 mM) and cyclic AMP concentration (5 microM) for the reaction. We also found that the detergents Triton X-100 and gramicidin S enhanced cyclic AMP-dependent protein phosphorylation. Inhibitors of the Na+, K+ ATPase (ouabain, NaF, vanadate) enhanced protein phosphorylation. This effect occurred in the presence but not in the absence of detergent. The addition of purified bovine brain cyclic AMP-dependent protein kinase catalytic subunit enhanced membrane protein phosphorylation. The addition of homogeneous neural (bovine brain) and non-neural (bovine skeletal muscle) cyclic AMP-dependent protein kinase type II regulatory subunit partially inhibited protein phosphorylation. Both neural and non-neural regulatory subunits behaved similarly. In addition to cyclic AMP-dependent phosphorylation, the alpha-subunit of pyruvate dehydrogenase (Mr = 41,000) is phosphorylated in a cyclic AMP-independent fashion. We also examined the phosphorylation pattern of membranes prepared from rat heart and found that the number of acceptor substrates was much less than that from the nervous system. 相似文献
110.
Phospholipid-Sensitive Ca2+ -Dependent Protein Kinase Preferentially Phosphorylates Serine-115 of Bovine Myelin Basic Protein 总被引:1,自引:0,他引:1
R. Scott Turner C.-H. Jen Chou† Gonzalo J. Mazzei Philip Dembure ‡ J. F. Kuo 《Journal of neurochemistry》1984,43(5):1257-1264
Phospholipid-sensitive Ca2+ -dependent protein kinase (PL-Ca-PK) and cyclic AMP-dependent protein kinase (A-PK) both preferentially phosphorylated serine residues of bovine myelin basic protein (MBP). Tryptic peptide maps of MBP phosphorylated by PL-Ca-PK or A-PK, however, revealed different phosphopeptides, suggesting a difference in the intramolecular substrate specificity for the two enzymes. Serine-115 of MBP, in the sequence (-Arg-Phe-Ser(115)-Trp-), was found to be a preferred and probably major phosphorylation site for PL-Ca-PK. Because serine-115 of bovine MBP corresponds to serine-113 of rabbit MBP, an in vivo phosphorylation site reported by Martenson et al. (1983), and PL-Ca-PK is present at a very high level in brain and myelin, it is suggested that the enzyme may be responsible for the in vivo phosphorylation of this and other sites in MBP. 相似文献