共查询到20条相似文献,搜索用时 31 毫秒
1.
V.K. Dombradi S.R. Silberman E.Y.C. Lee A.H. Caswell N.R. Brandt 《Archives of biochemistry and biophysics》1984,230(2):615-630
Evidence is presented for the association of a phosphorylase kinase activity with transverse tubules as well as terminal cisternae in triads isolated from rabbit skeletal muscle. This activity remained associated with T-tubules throughout the purification of triad junctions by one cycle of dissociation and reassociation. The possibility that the presence of phosphorylase kinase in these highly purified membrane vesicle preparations was due to its association with glycogen was eliminated by digestion of the latter with α-amylase. The phosphorylase kinase activity associated with the T-tubule membranes was similar to that reported for other membrane-bound phosphorylase kinases. The enzyme had a high activity ratio (0.4 – 0.7) and a high level of Ca2+ independent activity . The kinase activated and phosphorylated exogenous phosphorylase b with identical time courses. When mechanically disrupted triads were centrifuged on continuous sucrose gradients, the distribution of phosphorylase kinase activity was correlated with the distribution of a Mr 128,000 polypeptide in the gradients. This polypeptide and a Mr 143,000 polypeptide were labeled with 32P by endogenous and exogenous protein kinases. These findings suggest that the membrane-associated phosphorylase kinase may be similar to the cytosolic enzyme. Markers employed for the isolated organelles included a Mr 102,000 membrane polypeptide which followed the distribution of Ca2+-stimulated 3-O-methylfluorescein phosphatase activity, which is specific for the sarcoplasmic reticulum. A Mr 72,000 polypeptide was confirmed to be a T-tubule-specific protein. Several proteins of the triad component organelle were phosphorylated by the endogenous kinase in a Ca2+/calmodulin-stimulated manner, including a Mr ca. 72,000 polypeptide found only in the transverse tubule. 相似文献
2.
Georg Krohne Marie-Christine Dabauvalle Werner W. Franke 《Journal of molecular biology》1981,151(1):121-141
Nuclear envelopes from oocytes of Xenopus laevis are rich in pore complexes and contain a major polypeptide of apparent molecular weight (Mr) 68,000. A rapid extraction procedure using buffer containing 1% () Triton X-100 and 1.0 m-KCl allows the preparation of insoluble nuclear envelope skeletons showing only residual pore complex structures, with some interconnecting filament material, and one major polypeptide; i.e. that of Mr 68,000. This skeletal protein, which is not found in nuclear contents, reveals, on two-dimensional gel electrophoresis, a series of distinct isoelectric variants focusing in the pH range from 6.4 to 6.6. In living oocytes, this protein is continuously synthesized, as demonstrated by incorporation of labelled amino acids, and phosphorylated, A similar prominent skeletal protein has been found in nuclear envelopes of oocytes of other amphibia; however, slight but significant differences in electrophoretic mobility can be noted between different amphibian species.For comparison, nucleocortical lamina structures containing few pore complexes have been isolated, using similar extraction procedures, from various somatic cells of X. laevis, including erythrocytes. Laminae from these cells contain two major polypeptides, one (LI) of Mr 72,000 focusing at approximately pH 5.35 and another (LII) of Mr 69,000 focusing in several variants between pH 6.20 and 6.35. Similarly extracted “pore complex-lamina” fractions from rat liver contain a polypeptide of similar size and electrical charge as protein LI from Xenopus and, in addition, two other polypeptides (Mr values: 74,000 and 62,000) both focusing between pH 6.6 and 6.9.It is concluded that the pore complex-lamina structure of the oocyte nucleus is assembled by only one major protein of Mr 68,000. The results also show that the protein composition of this insoluble nucleocortical structure can be different in different cells of the same organism. The compositional differences of these nuclear envelope skeletons are discussed in relation to the relative proportions of pore complex and interporous (lamina) material in the nuclear envelopes of the specific cells. It is suggested that the Mr 68,000 protein predominant in oocyte nuclear envelopes contributes, as an architectural component, to the formation of the highly organized nuclear pore complex. 相似文献
3.
Human ceruloplasmin, which is usually cleaved by limited proteolysis into three major fragments during preparation (, 50,000, and 70,000) was isolated in good yield as an undegraded single-chain protein (). The cryosupernatant from fresh frozen plasma (100 liters) was fractionated with polyethylene glycol (PEG 4000) at + 5°C yielding a ceruloplasmin-enriched fraction in the 20% PEG supernatant. Three steps of chromatography on DEAE-Sephacel, hydroxyapatite, and Sephadex G-200 produced a homogeneous protein with maximal enzymatic activity and the ratio of 0.046 corresponding to 98–100% purity. Two forms of ceruloplasmin having this absorbance ratio were obtained; Form I was predominant and was studied further. The procedure separated both forms from apoceruloplasmin and degraded ceruloplasmin. The single-chain ceruloplasmin (Form I) had an NH2-terminal sequence of Lys-Glu-Lys-His-Tyr-Tyr-Ile-, the same as for the 70,000 fragment, and is suitable for structural study by sequence analysis and physicochemical methods. 相似文献
4.
Release and activation of phosphorylase phosphatase upon rupture of organelles from rat liver 总被引:6,自引:0,他引:6
Liver extracts (8000 × for 10 min) from fasted rats contain about 4 times more phosphorylase phosphatase activity when the liver was homogenized in a hypotonic medium or frozen before homogenization. This increase is caused by: (i) release of partially latent phosphatases (r=60 000 and 45 000 in sucrose gradient centrifugation) from ruptured organelles; (ii) rapid activation of phosphatase in the ruptured pellet by endogenous protease(s) which can be blocked by p-tosyl-L-lysine chloromethyl ketone. Only the r=60 000 enzyme, associated with the nuclei, can be activated proteolytically, with conversion to an r=45 000. 相似文献
5.
Chris Thoen Lauris Van Hove Etienne Piot Herman Slegers 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1984,783(2):105-113
The mRNP-associated protein kinase is purified to near homogeneity by ion-exchange chromatography on phosphocellulose and affinity chromatography on casein-Sepharose 4B and ATP-agarose. The cyclic nucleotide-independent enzyme phosphorylates casein using either ATP or GTP. The enzyme exists in two forms composed of subunits with Mr 36 500 (α) and 28 000 (β) and of subunits with Mr 36 500 (α), 33 000 (α′) and 28 000 (β). The undegraded enzyme has an Mr of 136 000 ± 7000. The enzyme is inhibited by heparin and hemin and stimulated by spermine. The mRNP-associated protein kinase may be classified as a casein kinase II. Main mRNP protein phosphate acceptors have Mr values of 112 000, 72 000, 65 000, 53 000, 38 000, 28 000, 23 500 and 21 000. Phosphorylation of the Mr 38 000 poly(A)-binding protein resulted in the generation of different acidic ionic species. From the observed inhibition of the translational activity after phosphorylation by the mRNP-associated protein kinase a function in the repression of mRNP is proposed. 相似文献
6.
Stuart L. Marcus Gregg Lipschik Generosa Trueba Cyrus J. Bacchi 《Biochemical and biophysical research communications》1980,93(4):1027-1035
A single peak of DNA polymerase activity from extracts of , obtained by DEAE-cellulose and phosphocellulose ion-exchange chromatography, was resolved into two peaks differing in KCl concentration necessary to elute them from a DNA-agarose column. Peak I (eluting at 0.2 M KCl) and Peak II (eluting at 0.4 M KCl), differed in response to increasing KCl concentrations, although both functioned optimally with Mg2+ as divalent cation when DNA synthesis was directed either by activated DNA or poly (dC)·(dG)12–18. Due to the potential significance of polyamines in the metabolism of , the effect of exogenous polyamine on rates of DNA synthesis by the peak I and II enzymes was compared with that of murine DNA polymerase alpha. Only the peak I enzyme was significantly stimulated (up to 4-fold) by the biologically active polyamines spermine and spermidine at physiological concentrations. The response of the peak I enzyme resembled that of the alpha polymerase. This result suggests a possible functional difference between peak I and II enzymes, as well as a potential target site for trypanocidal drug development. 相似文献
7.
In the presence of [γ32-P] ATP or [γ32-P] GTP 4 non ribosomal proteins (Mr 110,000; 105,000; 89,000 and 25,000) of the native 40S subunit became phosphorylated. The protein kinase responsible for this phosphorylation could be removed by treatment with 0.5 KCl. Sucrose density gradient analysis showed that the endogenous enzyme activity sedimented with approx. 7.5S. 相似文献
8.
A Rosner K M Jakob J Gressel D Sagher 《Biochemical and biophysical research communications》1975,67(1):383-391
A 0.5 × 106r RNA found in plastids of the aquatic angiosperm , is synthesized at a much higher rate than any other rapidly labeling RNA species about h after dark-grown plants are transferred to light. The pulse labeling kinetics of the 0.5 × 106r RNA after transfer to light, argue against its involvement in the biogenesis of plant rRNAs. Although poly(A) RNA is found in , poly(A) sequences are not detected in the 0.5 × 106r RNA; yet a sucrose gradient fraction which includes RNA of this r stimulates amino acid incorporation by an cell free extract more than other RNA fractions. The possible involvement of the 0.5 × 106r RNA as a chloroplast messenger is discussed. 相似文献
9.
P Plevani L Capucci G Badaracco D Breviario N Sacchi G Cattoretti E Ginelli 《Biochemical and biophysical research communications》1982,108(3):1196-1203
The polypeptide structure of terminal transferase purified from human lymphoblasts was examined with an immunoblot procedure using rabbit anti-calf thymus terminal transferase antibodies. Two doublets of bands of Mr 58-56,000 and Mr 44-42,000 are the major immunoreactive polypeptides. Only the Mr 44-42,000 polypeptides can be efficiently renatured after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Controlled degradation with trypsin produces fully active enzyme containing the α and β polypeptides typical of the low molecular weight terminal transferase, suggesting that the different forms of purified terminal transferase may arise by proteolysis of the Mr 58,000 polypeptide. 相似文献
10.
Larry J. Takemoto Jeff S. Hansen Bruce J. Nicholson Michael Hunkapiller Jean-Paul Revel Joseph Horwitz 《生物化学与生物物理学报:生物膜》1983,731(2):267-274
A protein of 26 000 has been shown to be the major component of eye-lens junctions, which are similar but not identical to the gap junctions of liver and other tissues. Cyanogen bromide cleavage of the 26 000 polypeptide from bovine lenses yields a major fragment of 15 000 (fragment 1). However, if the junctions are first treated with trypsin or carboxypeptidase Y, cyanogen bromide treatment yields a fragment of reduced molecular weight. Since protease treatment has been shown to cleave residues almost exclusively from the carboxy-terminal end of the 26 000 polypeptide, it follows that fragment 1 represents the carboxy-terminal half of this molecule, part of which is exposed to proteolytic attack outside the membrane. This latter result is corroborated by the fact that antisera which recognize both the 26 000 polypeptide and fragment 1 fail to do so after preadsorption with intact membranes. In addition, comparative amino acid and partial sequence analyses of the 26 000 polypeptide and fragment 1 indicate that fragment 1 is more hydrophilic in character, suggesting that much of the amino-terminal half of the 26 000 polypeptide is buried within the lipid bilayer. 相似文献
11.
A type 1 phosphoprotein phosphatase active with phosphorylated Mr = 68,000 initiation factor 2 kinase 总被引:7,自引:0,他引:7
R Szyszka W Kudlicki G Kramer B Hardesty J Galabru A Hovanessian 《The Journal of biological chemistry》1989,264(7):3827-3831
A type 1 protein phosphatase from reticulocytes is shown to efficiently dephosphorylate the Mr = 68,000 phosphopeptide of the double-stranded RNA-dependent kinase that phosphorylates the alpha subunit of eukaryotic peptide initiation factor 2, eIF-2. The kinase, activated in the presence of double-stranded RNA with concomitant phosphorylation of the Mr = 68,000 peptide, causes inhibition of peptide initiation and thereby effects translational control of protein synthesis. The Mn2+-dependent phosphatase is classified as a type 1 enzyme in that it is inhibited by inhibitor 2 in nanomolar concentrations and appears to have a Mr = 35,000 catalytic subunit. Dephosphorylation of the Mr = 68,000 peptide by the phosphatase is directly associated with a loss in kinase activity which can be restored by incubation with double-stranded RNA in the presence of ATP. The results demonstrate that the eIF-2 alpha kinase can undergo cyclic activation-inactivation that appears to be directly related to the phosphorylation state of the Mr = 68,000 peptide. They strongly support the previous conclusion that double-stranded RNA is required only for activation of the kinase and phosphorylation of the Mr = 68,000 peptide. 相似文献
12.
Regulation of Protein Synthesis in Plant Embryo by Protein Phosphorylation : I. Purification and Characterization of a cAMP-Independent Protein Kinase and Its Endogenous Substrate 总被引:1,自引:1,他引:0
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A cyclic AMP-independent protein kinase, which strongly inhibits in vitro protein synthesis, was purified to homogeneity from barley embryo by affinity and ion exchange chromatography. The Mr of the purified enzyme is 95,000 with two nonidentical subunits of Mr 58,000 and 39,000. The enzyme activity is not stimulated by cAMP, cGMP, or calmodulin. The endogenous phosphate acceptor of this kinase is a protein of Mr 52,000, was isolated by purified protein kinase immobilized Sepharose column. Using antibodies raised against this protein kinase, the levels of the enzyme during embryogenesis and germination are determined. An inverse relationship has been observed between protein kinase level and rate of protein synthesis. 相似文献
13.
About ScienceDirect 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,802(3):390-393
Photoaffinity labeling techniques have recently demonstrated that mammalian β1- and β2-adrenergic receptors reside on peptides of Mr 62 000–64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of Mr 30 000–55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the β2-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of Mr 40000–55 000 are fully functional with respect to their ability to bind β-adrenergic antagonist radioligands such as [3H]dihydroalprenolol and β-adrenergic antagonist photoaffinity reagents such as variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of β-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein. 相似文献
14.
Identification of two cDNA clones coding for androgen-dependent polypeptides in rat ventral prostate
Y.H. Xu O.D. Mpanias T.Y. Wang 《Biochemical and biophysical research communications》1983,111(2):624-629
cDNA clones were obtained by transformation of x1776 with pBR322 containing insert of ds cDNA synthesized from total rat prostate poly(A) RNA. Two prostate-specific cDNA clones were isolated by colony hybridization and identified by message selection/translation as encoding polypeptides of Mr: 13,500 and 9,300. Hybridization of poly(A) RNA from normal and castrated rat prostates to the cloned cDNAs indicated that the levels of mRNAs coding for Mr: 13,500 and 9,300 polypeptides are regulated by testosterone. 相似文献
15.
Cyclic AMP-dependent protein kinase of Neurospora crassa 总被引:3,自引:0,他引:3
was surveyed for cyclic AMP-dependent protein kinase activity. Two peaks (I and II) of protein kinase activity were demonstrated by DEAE-cellulose chromatography of wild type extracts. Peak I was stimulated by cyclic AMP, eluted below 60 mM NaCl and had high activity using histone H2B as substrate. Peak II eluted at 200–250 mM NaCl; its activity was not cyclic AMP stimulated and was highest with dephosphorylated casein as a substrate. Cyclic AMP binding to a protein associated with the protein kinase is specifically inhibited by certain cyclic AMP analogs. 相似文献
16.
Rat ventral prostate spermine synthetase was inhibited by 5′-methylthioadenosine and by S-adenosylhomocysteine at concentrations which did not inhibit spermidine synthetase from the same tissue. S-Adenosylethionine inhibited both enzymes to an equal extent. These aminopropyltransferases were also inhibited by diamines not normally present in mammalian cells. All the α,ω-diamines with 3 to 12 C atoms had inhibitory activity, but 1,3-diaminopropane and 1,5-diaminopentane were most active. Spermine synthetase was more sensitive than spermidine synthetase to the effects of these diamines. These results suggest that the relative rates of spermidine and spermine formation , might be affected by the intracellular concentration of nucleosides such as S-adenosylhomocysteine. They also raise the possibility that these rates of synthesis could be selectively affected by administration of one or the other of these inhibitors. 相似文献
17.
Marian B. Meyers June L. Biedler 《Biochemical and biophysical research communications》1981,99(1):228-235
A 19,000-dalton peptide (pI = 5.7) that is synthesized in increased amounts in vincristine-resistant Chinese hamster cells () has been identified by two-dimensional gel electrophoresis. Reduced amounts of the protein were present in a revertant line of , and only trace amounts were detected in control DC-3F cells. A similar protein (Mr = 19,000; pI = 5.7) was also found in a vincristine-resistant mouse line. Two vincristine-resistant human neuroblastoma cell lines likewise contained elevated levels of a low molecular weight acidic protein. Increased biosynthesis of the 19,000-dalton polypeptide in cells coincides with the presence of a homogeneously staining region, HSR, on a metaphase chromosome. 相似文献
18.
19.
《Insect Biochemistry》1985,15(1):35-44
Isolation of glutathione from the New Zealand grass grub, is complicated by the marked loss of activity from crude homogenates. This loss may be due to proteolysis or to modification by endogenous chemicals. The effect may be minimized by immediate fractionation with ammonium sulphate and by inclusion of 5mM glutathione in homogenates.Two enzymes species, isoelectric at pH 8.7 and 5.9 respectively, could be isolated by ammonium sulphate fractionation, affinity chromatography, anion exchange chromatography and chromatography on hydroxyl apatite. They had different substrate specificities and had differing subunit structure. The pI 8.7 enzyme appeared to be a homodimer of subunits of 23,700 and the pI 5.9 enzyme one of subunit 22,500.A third major enzyme species, isoelectric at pH 4.3 differed from the other two enzymes in having low affinity for the affinity matrix. This preparation was heterogeneous. The enzymically active species in this preparation had the same molecular weight as that of the pI 8.7 enzyme, had a very similar substrate specificity to the basic enzyme species and was characterized by kinetic parameters almost identical to those of the pI 8.7 enzyme. 相似文献
20.
Spermidine and spermine are found in unfertilized eggs of the sea urchin, . Putrescine becomes detectable and concentrations of spermidine and spermine increase in the eggs upon fertilization. Then, concentrations of these polyamines decrease after respective peaks in polyamine concentrations. The peaks in the concentrations are found at 15 minutes post fertilization for putrescien, at 30 minutes for spermidine and at 30–40 minutes for spermine respectively. Levels of polyamines elevate again and reduce after the 2nd concentration peaks of respective compounds, and then the first cleavage of the eggs takes place. Cyclic change in each polyamine concentration is also observed after the first cleavage, and egg cleavage occurs at decreasing phase of polyamine concentrations. 相似文献