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31.
Expression vectors for cDNA of the κ and λ1 chains of a monoclonal antibody directed against creatine kinase were introduced into murine myeloma cells. κ and γ1 cDNA were either under the control of the SV40 early promoter or of the cognate promoters and enhancers of the light- and heavy-chain genes. Secretion of immuno-reactive κ and γ1 chains into the culture medium was demonstrated with the SV40 promoter as well as with the cognate promoters. Expression of y 1 cDNA with the SV40 early promoter was about twice as high as with the heavy-chain promoter and enhancer. Expression of κ cDNA under the control of the S V40 early promoter was about 17 times higher than with the light-chain promoter and enhancer. These expression levels were compared to those of a genomic immunoglobulin (Ig) κ determinant, including introns. Such an entire κ gene led to expression of the light chain at levels double those with the κ cDNA construction using the SV40 promoter and about 35 times as high when using κ cDNA and the cognate promoter and enhancer. This result might indicate that, besides the cognate promoter and enhancer elements, other intragenic elements are involved in the regulation of Ig expression. However, the SV40 early promoter seems to be able to compensate for the absence of these postulated regulatory elements probably located in the introns. 相似文献
32.
Florence Levy-Favatier Lydie Tichnonicky Jacques Kruh Marc Delpech 《Biochimie》1989,71(11-12):1157-1161
We have previously described in rat liver two protein kinases tightly bound to DNA, one is serine-specific, the other arginine-specific. In this work we show that both enzymes are present in various rat tissues and in liver from various species. Both kinase specific activities are strongly decreased in methyl-DBA-induced hepatomas and in HTC cells but not in regenerating liver after hepatectomy. This decrease is then not related to cell proliferation. 相似文献
33.
Jackie R. Vandenheede Sigrid Staquet Wilfried Merlevede 《Molecular and cellular biochemistry》1989,87(1):31-39
Summary Fractionation of rabbit skeletal muscle cytosol on Aminohexyl-Sepharose has resulted in the identification of a latent ATP, Mg-dependent protein phosphatase whose catalytic subunit is in the active conformation, but is inhibited by the presence of more than one modulator unit. The partially purified enzyme is converted to an inactive, kinase FA-dependent form upon incubation at 30°C unless modulator-specific polyclonal antibodies are added to the preparation. The immunoglobulins also relieve the inhibition which is responsible for the low basal phosphatase activity of the enzyme, and they counteract all of the heat-stable inhibitor activity present in the preparation. Addition of free catalytic subunit abolishes the inhibition of the latent enzyme in a dose-dependent way, but cannot prevent the inactivation process. The inactivated phosphatase and the original latent enzyme exhibit the same apparent M
r in sucrose density-gradient centrifugation (70 000) and in gel filtration (110 000).Abbreviations PMSF
Phenylmethanesulphonyl Fluoride
- TLCK
L-l-chloro-3-(4-tosylamido)-7-amino2-heptanone-hydrochloride
- TPCK
L-l-chloro-3-(4-tosvlamido)-4-phenyl-2-butanone 相似文献
34.
Cholesterylphosphoryldimethylethanolamine is a zwitterionic compound which is a good bilayer stabilizer. As has been found with many other compounds having these properties, cholesterylphosphoryldimethylethanolamine is found to be a potent inhibitor of protein kinase C in both vesicle and micelle assay systems. The kinetics of the inhibition in Triton X-100 micelles was non-competitive with respect to ATP, histone, diolein, phorbol ester and Ca2+. It has a Ki of about 30 m. The inhibition kinetics as a function of phosphatidylserine concentration is more complex but suggestive of competitive inhibition. Cholesterylphosphoryldimethylethanolamine does not prevent the partitioning of protein kinase C into the membrane. This inhibitor lowers the Ca2+-phosphatidylserine-independent phosphorylation of protamine sulfate by protein kinase C and directly affects the catalytic segment of the enzyme generated by tryptic hydrolysis. Thus, this zwitterionic bilayer stabilizing inhibitor of protein kinase C both competes with the binding of phosphatidylserine as well as affects the active site of protein kinase C.Abbreviation CPD
cholesterylphosphoryldimethylethanolamine 相似文献
35.
Rat ovarian membrane LH/hCG receptor was solubilized in various detergents and reconstituted into proteoliposomes. Upon removal of sodium cholate by active absorption on Bio-Beads SM-2, the functional interaction between receptor and adenylate cyclase was restored. Adenylate cyclase was stimulated by hCG, HCG+GTP or GppNHp and NaF. Reconstituted proteoliposomes bound more 125I-hCG (528 fmol/mg protein) than membrane-bound receptors (384 fmol/mg protein). There was no difference, however, in the relative affinity of reconstituted receptor preparations for hCG. 相似文献
36.
Helmut Krüger Werner Schr?der Klaus Buchner Ferdinand Hucho 《Journal of Protein Chemistry》1990,9(4):467-473
Inhibition of protein kinase C (PKC) by calmodulin is investigated and we describe the localization of inhibitory sequences within the calmodulin molecule. We present evidence that calmodulin inhibits PKC through an inhibition of the activation of PKC associated with lipid membranes: Binding of PKC to lipid vesicles is not affected, but activation is abolished. The potent calmodulin antagonist R24571 (calmidazol) did not affect the inhibition of PKC by calmodulin at concentrations up to 10–5 M. Two tryptic fragments of calmodulin were isolated which inhibited PKC. They were only slightly less potent than intact calmodulin with an IC50 of 6 µ M compared to 1 µ M of intact calmodulin. They were identified as Ser38-Arg74 and His107-Lys148. Each of the inhibiting fragments contains an intact Ca2+-binding domain with complete helix-loop-helix structure (EF hand). Other calmodulin peptides showed only weak inhibitory activity. Both fragments did not stimulate cAMP phosphodiesterase even at concentrations 100-fold higher than the calmodulin concentration needed for maximal stimulation. None of the fragments acted as a calmodulin antagonist. 相似文献
37.
Robert M. Levin Penelope A. Longhurst Sheila S. Levin Niels Haugaard Alan J. Wein 《Molecular and cellular biochemistry》1990,97(2):153-159
The urinary bladder depends on intracellular ATP for the support of a number of essential intracellular processes including contraction. The concentration of ATP is maintained constant primarily via the rapid transfer of a phosphate from creatine phosphate (CP) to ADP catalyzed by the enzyme creatine kinase (CK). Since muscular pathologies associated with diabetes are in part related to intracellular alterations in metabolism, we have characterized the CK activity in both skeletal muscle and urinary bladder from control and streptozotocin-diabetic rats.The following is a summary of the results: 1) Bladder tissue from control rats showed linear kinetics with a Vmax = 390 nmoles/mg protein/min, and a Km = 275 µM. 2) Urinary bladder tissue isolated from diabetic rats displayed biphasic kinetics with Vmax = 65 and 324 nmoles/mg protein/min, and Km's = 10 µM and 190 µM respectively. 3) Skeletal muscle isolated from control rats showed linear kinetics with an approximate Vmax of 800 nmoles/mg protein/min and a Km of 280 µM CP. 4) Homogenates of skeletal muscle from diabetic rats showed complex kinetics not separable into distict component forms. 5) The Km for ADP for both skeletal muscle and bladder was approximately 10 µM.These studies demonstrate that whereas bladders isolated from both control and diabetic rats possess a low-affinity isomer(s) of CK with similar maximum enzymatic activity, there is a high affinity isomer present within the urinary bladder muscle of diabetic rats that is not present in bladder tissue isolated from control rats. Skeletal muscle isolated from both diabetic and control rats exhibited a maximal activity 2 to 3 times higher than that of the bladder. 相似文献
38.
In the present communication, we report the identification of a new gene family which encodes the protein subunits of the proteasome. The proteasome is a high-Mr complex possessing proteolytic activity. Screening a Drosophila λgt11 cDNA expression library with the proteasome-specific antibody N19-28 we isolated a clone encoding the 28-kDa No. 1 proteasome protein subunit. In accordance with the nomenclature of proteasome subunits in Drosophila, the corresponding gene is designated PROS-28.1, and it encodes an mRNA of 1.1 kb with an open reading frame of 249 amino acids (aa). Genomic Southern-blot hybridization shows PROS-28.1 to be a member of a family of related genes. Analysis of the predicted aa sequence reveals a potential nuclear targeting signal, a potential site for tyrosine kinase and a potential cAMP/cGMP-dependent phosphorylation site. The aa sequence comparison of the products of PROS-28.1 and PROS-35 with the C2 proteasome subunit of rat shows a strong sequence similarity between the different proteasome subunits. The data suggest that at least a subset of the proteasome-encoding genes belongs to a family of related genes (PROS gene family) which may have evolved from a common ancestral PROS gene. 相似文献
39.
Protein phosphorylation in Streptomyces albus 总被引:1,自引:0,他引:1
Z. Dobrová M. Jirsová T. Petík P. Ryavý J. Náprstek J. Janeek 《FEMS microbiology letters》1990,71(1-2):145-148
The phosphorylated proteins of Streptomyces albus, radioactively labeled with [32P]orthophosphate have been analyzed by gel electrophoresis and autoradiography. More than 10 protein species were found to be phosphorylated. With [32P]ATP as substrate cell free extracts phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. From cell extract which exhibited active phosphorylated in vitro, a protein kinase has been partially purified. The kinase activity was identified in fractions corresponding to a 90 kDa protein. 相似文献
40.
Preston Gadson Judy McCoy Ann Charlotte Wikstrm Jan-Ake Gustafsson 《Journal of cellular biochemistry》1990,43(2):185-198
Exposure of fibroblasts derived from keloid tissues, desmoid and dermal tissue from individuals with Gardner's syndrome (GS) to dexamethasone resulted in the suppression of protein kinase C (PKC) activity and [3H]thymidine incorporation into DNA, and a 20-fold induction of glutamine synthetase activity. Treatment of GS and keloid fibroblasts with 0.1 microM dexamethasone for 36 h increased glucocorticoid receptor (GR) synthesis, as determined by [35S]methionine labeling and immunoprecipitation with a monoclonal antibody to the human GR. The suppression of PKC activity by dexamethasone was shown to result from a loss of protein mass as determined by immunoblotting using an antibody to PKC type III. In contrast to these results, exposure of fibroblasts isolated from normal tissues to dexamethasone did not result in the suppression PKC and [3H]thymidine incorporation, there was only a sixfold induction of glutamine synthetase, and a decrease of GR synthesis. As no primary receptor binding defect could be detected, the altered response of tumor cells to steroid-occupied receptor indicates a partial post-receptor binding defect in GS and keloid cells. 相似文献