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1.
Trachana V Christophorides E Kouzi-Koliakos K Koliakos G 《The international journal of biochemistry & cell biology》2005,37(2):478-492
Monocytes encounter basement membranes and interact with laminins while crossing the vascular barrier. It is known that these cells possess ecto-protein kinase activity on their surface. Several proteins of the extracellular matrix can be phosphorylated by ectokinases. Therefore, it has been hypothesized that monocyte ectokinases could phosphorylate laminins and influence their biological properties. In order to test the above hypothesis, we used intact human monocytes and adenosine triphosphate labeled with radioactive phosphate at the third phosphate ([gamma-32P]-ATP) to phosphorylate laminin-1. Autoradiography after sodium dodecyl sulphate polyacrylamyde gel electrophoresis (SDS-PAGE) electrophoresis indicated phosphorylation of laminin-1 on the beta and/or gamma chains. After phosphorylation, phosphoserine could be detected on Western blots by a specific monoclonal antibody. Phosphorylation was not detected when monocytes were pre-treated with trypsin and was inhibited by a specific ecto-protein kinase inhibitor (K252b). Laminin phosphorylation was also inhibited by heparin, a known inhibitor of casein kinase II and by pretreatment of monocytes by a monoclonal anti-casein kinase II antibody. Heparin binding, cell attachment and proliferation, and monocyte migration were enhanced on the phosphorylated laminin-1 as compared to the non-phosphorylated controls. These data indicate that laminin-1 can be phosphorylated by monocyte casein kinase II type ectokinase. This phosphorylation influences important functions of laminin and therefore could provide an additional means for the interaction of monocytes with basement membranes. 相似文献
2.
Phosphophoryns are the major non-collagenous proteins of the mineralized matrix of rat incisor dentin. Nearly half the phosphophoryn residues are serines, and 85-90% of these are phosphorylated. Since phosphorylation may be important for phosphophoryn function, it was of interest to identify the kinase(s) responsible for catalyzing their phosphophorylation. Rat osteosarcoma (ROS) 17/2.8 osteoblast-like cells were selected as the enzyme source. Native rat incisor phosphophoryns (RIPP-I, II, III) were not substrates for any of the ROS 17/2.8 messenger-dependent kinases but were phosphorylated by membrane-associated endogenous messenger-independent kinases. These were resolved chromatographically and identified as casein kinase (CK) I and II by elution properties and immunoblotting with a CKII antibody. The CKI preferentially used RIPP-III as substrate, while CKII preferred RIPP-I and II. Heparin at 100 and 500 ng/assay and NaCl at 0.25-0.4 M inhibited phosphorylation of the RIPP by CKI and CKII in parallel. At 10 mM spermine, phosphorylation of RIPP-I and II by CKII, and of RIPP-III by CKI were inhibited, but phosphorylation of RIPP-III by CKII was enhanced. Purified sea star oocyte CKII demonstrated the same substrate specificity and spermine concentration shift as the ROS 17/2.8 CKII. These data show that osteoblast-like cells are a rich source of membrane-bound CKI and CKII activity. The different patterns of phosphorylation of RIPP-I, II, and III further show that they are distinct synthetic products of the odontoblast. 相似文献
3.
Phosphorylation of the light-harvesting chlorophyll-protein regulates excitation energy distribution between photosystem II and photosystem I 总被引:8,自引:0,他引:8
The kinetics of thylakoid membrane protein phosphorylation in the presence of light and adenosine triphosphate is correlated to an incease in the 77 °K fluorescence emission at 735 nm (F735) relative to that at 685 nm (F685). Analysis of detergent-derived submembrane fractions indicate phosphorylation only of the polypeptides of Photosystem II, and the light-harvesting chlorophyll-protein complex serving Photosystem II (LHC-II). Although several polypeptides are phosphorylated, only the dephosphorylation kinetics of LHC-II follow the kinetics of the decrease of the fluorescence emission ratios. The relative quantum yield of Photosystem II was significantly lower in phosphorylated membranes compared to dephosphorylated membranes. Reversible LHC-II phosphorylation thus provides the physiological mechanism for the control of the distribution of absorbed excitation energy between the two photosystems. 相似文献
4.
Santillo M Migliaro A Mondola P Laezza C Damiano S Stingo S Fiorentino L Andreozzi A Vitale M Bifulco M 《FEBS letters》1999,455(1-2):83-86
Ser-473 is solely phosphorylated in vivo in the tail region of neurofilament L (NF-L). With peptides including the native phosphorylation site, it was not possible to locate responsible kinases. We therefore adopted full-length dephosphorylated NF-L as the substrate, and employed MALDI/TOF (matrix-assisted laser desorption and ionization/time of flight) mass spectrometry and a site-specific phosphorylation-dependent antibody recognizing Ser-473 phosphorylation. The antibody showed that casein kinase I (CK I) as well as casein kinase II (CK II) phosphorylated Ser-473 in vitro, while neither GSK-3beta nor calcium/calmodulin-dependent protein kinase II did so. However, the mass spectra of the tail fragments of the phosphorylated NF-L indicated that CK II was the kinase mediating Ser-473 phosphorylation in vitro as opposed to CK I, because CK I phosphorylated another site as well as Ser-473 in vitro. The antibody also demonstrated that NF-L phosphorylated at Ser-473 was abundant in the neuronal perikarya of the rat cortex, indicating that phosphorylation of Ser-473 may take place there. This result may support the suggestion that CK II is the kinase responsible for Ser-473 phosphorylation. Despite many reports showing that CK I mediates phosphorylation of neurofilaments, CK II may phosphorylate NF-L in vivo. 相似文献
5.
Uptake of the four ribonucleosides normally present in RNA increases nearly 50-fold shortly after fertilization in eggs of the sea-urchin, Strongylocentrotus purpuratus. Uridine, adenosine and cytidine are phosphorylated (greater than 95%) to their mono-, di- and triphosphates immediately after transport into the fertilized egg. Although guanosine is transported to an extent equal to the other three ribonucleosides, less than 12% of its phosphorylated after transport. In vitro nucleoside and nucleotide kinase assays of unfertilized egg homogenates indicate that the uridine, adenosine and cytidine kinases as well as the uridylate, adenylate, cytidylate and guanylate kinases are present in the egg prior to fertilization. Substrate competition measurements indicate that adenosine phosphorylation is catalyzed by a monospecific enzyme, while uridine and cytidine phosphorylations are catalyzed by a common kinase. Guanosine kinase activity was not detectable in unfertilized egg homogenates. Between 3 h and 5 h after fertilization the phosphorylation of transported guanosine begins to increase as it enters the embryo. By 7 h after fertilization, more than 95% of the guanosine entering the embryo is phosphorylated to the mono-, di- and triphosphates. More than 80% is phosphorylated to guanosine triphosphate. The timing of increased guanosine phosphorylation correlates with a decrease in the acid-soluble GTP pools in the embryo, suggesting that increased guanosine kinase activity is a response to increased GTP demand. These results, in view of the importance of GTP in many cellular processes, imply a crucial role for guanosine kinase activation in GTP pool maintenance and cellular metabolism during early sea-urchin development. 相似文献
6.
Fodrin, an actin and calmodulin binding and spectrin-like protein present in many nonerythrocyte tissues, could be phosphorylated up to more than 1.5 mol of phosphate/mol of protein by a highly purified non-receptor-associated protein tyrosine kinase from bovine spleen. The protein phosphorylation was not affected by Ca2+/calmodulin or by F-actin. Km and Vmax values of the reaction were 91 nM and 0.35 nmol of P2 min-1 (mg of kinase)-1, respectively. Both subunits A and B of fodrin were phosphorylated, with the rate of subunit A phosphorylation much greater than that of subunit B phosphorylation. Tryptic phosphopeptide mapping of the phosphorylated subunits suggested that there were three major phosphorylation sites in subunit A and one in subunit B. Phosphotyrosylfodrin could be dephosphorylated by the calmodulin-stimulated phosphatase (calcineurin) in the presence of activating metal ions; Ni2+ was a much more effective activator than Mn2+ for this reaction. Fodrin phosphorylation by the spleen protein tyrosine kinase did not appear to alter the actin and calmodulin binding properties of the protein. On the other hand, the calmodulin-dependent stimulation of smooth muscle actomyosin Mg2+-ATPase by fodrin was enhanced by 101% +/- 3% (n = 3) upon fodrin phosphorylation. Ni2+-calcineurin, which was shown to effectively dephosphorylate the phosphotyrosyl residues on fodrin, could reverse the phosphorylation-enhanced Mg2+-ATPase stimulatory activity of fodrin. 相似文献
7.
Location of sites in human lipocortin I that are phosphorylated by protein tyrosine kinases and protein kinases A and C 总被引:3,自引:0,他引:3
L Varticovski S B Chahwala M Whitman L Cantley D Schindler E P Chow L K Sinclair R B Pepinsky 《Biochemistry》1988,27(10):3682-3690
Lipocortin I is a 39-kilodalton membrane-associated protein that in A431 cells is phosphorylated on tyrosine in response to epidermal growth factor (EGF). We have used recombinant human lipocortin I as a substrate for several protein kinases and identified phosphorylated residues by a combination of peptide mapping and sequence analysis. Lipocortin I was phosphorylated near the amino terminus at Tyr-21 by recombinant pp60c-src. The same tyrosine residue was phosphorylated by polyoma middle T/pp60c-src complex, by recombinant pp50v-abl, and with A431 cell membranes by the EGF receptor/kinase. The primary site of phosphorylation by protein kinase C was also near the amino terminus at Ser-27. The major site of phosphorylation by adenosine cyclic 3',5'-phosphate dependent protein kinase was on the carboxy-terminal half of the molecule at Thr-216. These sites are compared to the phosphorylation sites previously located in the structurally related protein lipocortin II. 相似文献
8.
The incubation of spinach chloroplasts with 1 mM N-ethylmaleimide in light for 60 to 90 s results in a partial, irreversible inhibition of photophosphorylation. The inhibition was not overcome at infinite light intensity or at infinite concentrations of the phosphorylation substrates. Although the inhibition diminished with decreasing concentrations of adenosine diphosphate in the assay of phosphorylation, the inhibition of guanosine diphosphate phosphorylation was independent of the concentration of this nucleotide. Although adenosine di- or triphosphate (10 to 30 muM) alone partially prevented the development of the N-ethylmaleimide inhibition of phosphorylation, these nucleotides were more effective when either 1 mM inorganic phosphate or arsenate was also present. The light-dependent incorporation of N-ethylmaleimide into chloroplast-bound coupling factor 1 was affected by adenosine triphosphate and inorganic phosphate in a manner similar to the onset of N-ethylmaleimide inhibition. Since guanosine diphosphate did not protect phosphorylation from N-ethylmaleimide inhibition but is phosphorylated at rapid rates, it is apparent that coupling factor 1 in chloroplasts has multiple nucleotide recognition sites. 相似文献
9.
Adenosine kinase was partially purified from wheat germ. This enzyme preparation, which was devoid of adenine phosphoribosyltransferase and nearly free of adenosine deaminase but contained adenylate kinase, rapidly phosphorylated adenosine and a cytokinin, N6-(δ2-isopentenyl)adenosine. Electrophoretic analysis indicated that only N6-(δ2-isopentenyl)adenosine-monophosphate was formed from the cytokinin while about 55% AMP, 45% ADP, and a trace of ATP were formed from adenosine. The biosynthesized nucleoside monophosphates were quantitatively hydrolyzed to the corresponding nucleosides by 5′-nucleotidase and the isopentenyl side chain of the phosphorylated cytokinin was not cleaved. The enzyme did not catalyze phosphorylation of inosine. 相似文献
10.
K Yamauchi K Hashizume K Ichikawa H Ohtsuka N Ohara T Miyamoto M Kobayashi T Yamada 《Endocrinologia japonica》1991,38(2):175-182
The effect of phosphorylation of insulin receptor with adenosine 3',5'-cyclic monophosphate-dependent protein kinase (A kinase) on its insulin binding activity was investigated by using insulin receptors prepared from rat liver in vitro. A 95 KDa protein was phosphorylated by stimulation of insulin receptor kinase. This protein was also phosphorylated by A kinase. Analysis of phosphoamino acid showed that tyrosine residue(s) was phosphorylated by activation of insulin receptor kinase, whereas phosphoserine and phosphothreonine were dominantly generated by activation of A kinase. [125I] Iodoinsulin binding activity was decreased by prior phosphorylation of the receptor with A kinase. Scatchard analysis showed that the affinity for insulin was decreased by the phosphorylation with A kinase. Although the maximal activity of insulin receptor kinase was not affected by phosphorylation with A kinase, the insulin concentration which induced half maximal activity (ED50) of the receptor kinase was increased by the phosphorylation with A kinase. These results suggested that counter regulatory hormones whose actions are mediated by the generation of adenosine 3',5'-cyclic monophosphate regulate the insulin binding to the alpha subunit through phosphorylation of the beta subunit of insulin receptor. 相似文献
11.
The presence of protein kinase activity and its phosphorylated products has been demonstrated on the outer surface of the plasma membrane of endothelial cells. Extracellular phosphorylation was detected by incubation of primary endothelial cells (HUVEC's) and endothelial cell line EA.hy 926 with [gamma-32P]ATP. The reaction products were subjected to SDS/PAGE, autoradiography and scanning densitometry. Under the experimental conditions, five proteins with apparent molecular masses of 19, 23, 55, 88, and 110 kDa were prominently phosphorylated in both types of cells. Phosphorylation of the 19 kDa protein was the most rapid reaching maximum after 60 s and then the protein became dephosphorylated. Ecto-protein kinases responsible for the surface labeling of membrane proteins were characterized by using (a) protein kinase C inhibitors: K-252b, chelerythrine chloride, and [Ala113] myelin basic protein (104-118), (b) protein kinase A inhibitor Kemptide 8334, and (c) casein kinase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB). Stimulation of endothelial cells with tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) is associated with 20-80% reduction of extracellular phosphorylation of all membrane proteins. IFN gamma bound to membrane receptors becomes rapidly phosphorylated. Only in the case of IFN gamma it was associated with the appearance of a strongly phosphorylated band of 17 kDa corresponding to IFN gamma itself. Phosphorylation of this 17 kDa exogenous substrate was prevented by an ecto-kinase inhibitor K-252b. The existence of ecto-phosphoprotein phosphatase activity in endothelial cells was evidenced by testing the effect of microcystin LR--a membrane impermeable reagent that inhibits both PP-1 and PP-2a phosphoprotein phosphatases. The extent of phosphorylation of 19 kDa and 110 kDa phosphoproteins significantly increased in the presence of microcystin. Our results suggest the presence of at least two ecto-kinase activities on endothelial cells that may play a significant role(s) in the regulation of cytokines function. 相似文献
12.
A solid-phase assay for the phosphorylation of proteins blotted on nitrocellulose membrane filters 总被引:1,自引:0,他引:1
A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters, and the blotted polypeptides are phosphorylated with the catalytic subunit of cyclic AMP (adenosine 3':5'-monophosphate)-dependent protein kinase. The method was developed for the assay of dephosphosynapsin I, but it has also proven suitable for the phosphorylation of other proteins. The patterns of phosphorylation of tissue samples phosphorylated using the new method are similar to those obtained using the conventional test tube assay. Once phosphorylated, the adsorbed proteins can be digested with proteases and subjected to phosphopeptide mapping. The phosphorylated blotted proteins can also be analyzed by overlay techniques for the immunological detection of polypeptides. 相似文献
13.
Four ubiquitin-peptide extensions prepared as cloned products in E. coli were tested as casein kinase II substrates. Two extensions containing the sequence Ser-Glu-Glu-Glu-Glu-Glu were readily phosphorylated by partially purified rabbit reticulocyte casein kinase II. The other two fusion proteins, which lack a consensus phosphorylation site for casein kinase II, did not serve as substrates under identical reaction conditions. Native ubiquitin was not phosphorylated by reticulocyte casein kinase II, nor have we observed its phosphorylation in crude extracts from HeLa cells, mouse liver, or Xenopus eggs. Ubiquitin's apparent lack of phosphorylatable residues coupled with its remarkable heat stability and rapid migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels make the protein an attractive carrier for carboxyl-terminal peptides containing specific phosphorylation sites. Such ubiquitin extension proteins should prove valuable as protein kinase substrates. 相似文献
14.
Treatment of BALB/c-3T3 mouse fibroblasts with 3′-led to a rapid accumulation of 3′-phosphates and the kinetics of this process has been determined. Concomitant with accumulation of these compounds, the adenine ribonucleotide pool was reduced. The kinetics of the two processes suggested that they were tightly coupled. The inhibitory effect of relatively high concentrations of coformycin indicated that IMP was an intermediate in the catabolic pathway. Similar experiments with Ehrlich ascites tumor cells were performed in Ringer-Hepes solution at pH 6.5 or 7.5 and with varying concentrations of orthophosphate. The experiments were performed with cells where ATP was [3H]-. This allowed the determination of the catabolism of adenine ribonucleotides to labeled nucleosides under conditions where added adenosine was phosphorylated. The results showed that at low phosphate concentration (5.8 mM) at pH 6.5 adenosine may be phosphorylated at a rate that was completely balanced to the concomitant catabolism of adenine ribonucleotides; that is, there was apparently a tight kinetic coupling between anabolism of adenosine and catabolism of adenine ribonucleotides. With 3′-a corresponding effect was obtained although the apparent coupling between phosphorylation of 3′-and catabolism of adenine ribonucleotides was not complete. When experiments were performed at the same pH but at high concentration of phosphate (45 mM) there was in contrast no coupling between the two processes; that is, ATP was present in constant amounts while 3′-phosphates accumulated at a high rate. In experiments with adenosine under these conditions there was still some although a relatively limited degree of apparent coupling between phosphorylation of adenosine and catabolism of adenine ribonucleotides. In both lines of cells used and with both adenosine and 3′-, the main products of the catabolism of adenine ribonucleotides were inosine and hypoxanthine. With 3′-there was in addition (about 20%) formation of xanthosine, suggesting that IMP dehydrogenase had also been activated. These results lead to the suggestion that adenosine (or 3′-) may be phosphorylated in two ways. 1) Phosphorylation may depend on an adenosine kinase unrelated to catabolism of adenine ribonucleotides. 2) Phosphorylation may be tightly coupled to catabolism of adenine ribonucleotides. A nucleoside phosphotransferase may catalyze the transfer of a phosphoryl group from IMP to adenosine (or 3′-) to form AMP (or 3′-) and inosine, a process that may be tightly coupled to an AMP deaminase reaction. The IMP formed in the latter reaction may not be released but transferred to the phosphotransferase. In contrast, the AMP formed in the phosphotransferase reaction should be in equilibrium with soluble AMP. It is assumed that a physical complex may exist, possibly in a membrane bound form, between AMP deaminase and the nucleoside phosphotransferase. © 1993 Wiley-Liss, Inc. 相似文献
15.
16.
Yamamoto H Yamauchi E Taniguchi H Ono T Miyamoto E 《Archives of biochemistry and biophysics》2002,408(2):255-262
The paired helical filaments (PHF) found in Alzheimer's disease (AD) brain are composed mainly of the hyperphosphorylated form of microtubule-associated protein tau (PHF-tau). It is well known that tau is a good in vitro substrate for Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). To establish the phosphorylation sites, the longest human tau (hTau40) was bacterially expressed and phosphorylated by CaM kinase II, followed by digestion with lysyl endoprotease. The digests were subjected to liquid chromatography/mass spectrometry. We found that 5 of 22 identified peptides were phosphorylated. From the tandem mass spectrometry, two phosphorylation sites (serines 262 and 356) were identified in the tubulin binding sites. When tau was phosphorylated by CaM kinase II, the binding of tau to taxol-stabilized microtubules was remarkably impaired. As both serines 262 and 356 are reportedly phosphorylated in PHF-tau, CaM kinase II may be involved in hyperphosphorylation of tau in AD brain. 相似文献
17.
《Biochemical medicine》1981,25(3):288-297
The apparent rate of phosphorylation of deoxyadenosine has been studied in crude extracts of human leukemic cells. Since detection and quantitation of phosphorylated products of deoxyadenosine are not possible in the presence of the competing enzyme, adenosine deaminase, an assay system has been devised in which deaminase activity is totally inhibited. Two inhibitors of adenosine deaminase, erythro-9-(2-hydroxy-3-nonyl)adenine and 2′-deoxycoformycin were tested. Complete inhibition of adenosine deaminase cannot be achieved with erythro-9-(2-hydroxy-3-nonyl)adenine, but can be achieved with 2′-deoxycoformycin at concentrations greater than 100 μ m. Use of deoxycoformycin allows an accurate assessment of phosphorylation of deoxyadenosine and its nucleoside analogs. Cellular extracts from patients with several types of leukemia contained a 30-fold difference in relative deoxyadenosine kinase activity. Adenine arabinoside is a competitive inhibitor of deoxyadenosine, but not adenosine phosphorylation with a Ki (app) of 3.2 mm. This inhibition pattern is consistent with a common pathway of phosphorylation for deoxyadenosine and adenine arabinoside in human leukemic cells. 相似文献
18.
Identification of the phosphorylation sequence in the cytoplasmic tail of the varicella-zoster virus Fc receptor glycoprotein gpI. 总被引:15,自引:13,他引:2
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Varicella-zoster virus (VZV) glycoprotein gpI, the homolog of herpes simplex virus gE, functions as a receptor for the Fc portion of immunoglobulin G. Like other cell surface receptors, this viral receptor is highly phosphorylated in cell culture. To identify the precise location of the cellular kinase-mediated phosphorylation, we generated a tailless deletion mutant and several point mutants which had altered serine and threonine residues within the cytoplasmic domain of gpI. The mutated and wild-type genes of gpI were transfected and expressed within a vaccinia virus-T7 polymerase transfection system in order to determine what effect these mutations had on the phosphorylation state of the protein in vivo and in vitro. Truncation of the cytoplasmic domain of gpI diminished the phosphorylation of gpI in vivo. Examination of the point mutants established that the major phosphorylation sequence of gpI was located between amino acids 593 and 598, a site which included four phosphorylatable serine and threonine residues. Phosphorylation analyses of the mutant and wild-type glycoproteins confirmed that gpI was a substrate for casein kinase II, with threonines 596 and 598 being critical residues. Although the mutant glycoproteins were phosphorylated by casein kinase I, protease V8 partial digestion profiles suggested that casein kinase II exerted the major effect. Thus, these mutagenesis studies demonstrated that the gpI cytoplasmic sequence Ser-Glu-Ser-Thr-Asp-Thr was phosphorylated in mammalian cells in the absence of any other herpesvirus products. Since the region defined by transfection was consistent with results obtained with in vitro phosphorylation by casein kinase II, we propose that VZV gpI is a physiologic substrate for casein kinase II. Immunofluorescence and pulse-chase experiments demonstrated that the mutant glycoproteins were processed and transported to the outer cell membrane. 相似文献
19.
《BBA》1985,809(1):17-26
Treatment of spinach thylakoids with the adenosine affinity inhibitor 5′-p-fluorosulfonylbenzoyl adenosine (FSBA) resulted in at least 95% inhibition of phosphorylation of the light-harvesting protein complex of Photosystem II (LHC II), while the Mr 10 000 polypeptide showed a 35% decrease in phosphorylation. This residual kinase activity after FSBA treatment appears to have the same properties as the control, since phosphorylation of the Mr 10 000 polypeptide subsequent to FSBA treatment could be achieved with either light or reducing conditions in the dark. [14C]FSBA labelled several polypeptides, but only the Mr 50 000 band was protected against the label by prior addition of ADP or adenosine, making it a possible candidate for the LHC II kinase. FSBA had no effect on electron transport, and [14C]FSBA did not label LHC II or the Mr 10 000 polypeptide, indicating that the FSBA was not interfering with activation of the kinase or modifying the substrates, but rather acting at the level of the LHC II protein kinase. Inhibition of LHC II phosphorylation by FSBA resulted in the elimination of the slow ATP-induced decrease in variable fluorescence, a parameter believed to be associated with phosphorylation of the LHC II. The half-times and time-course for inhibition of LHC II phosphorylation and inhibition of the ATP-induced decrease of fluorescence yield were identical, consistent with the concept that LHC II phosphorylation plays a major role in this fluorescence change. 相似文献
20.
Topoisomerase I phosphorylation in vitro and in rapidly growing Novikoff hepatoma cells. 总被引:7,自引:1,他引:6
Changes in phosphorylation modulate the activity of topoisomerase I in vitro. Specifically, enzymatic activity is stimulated by phosphorylation with a purified protein kinase (casein kinase type II). The purpose of this study was to compare the sites that are phosphorylated in vitro by casein kinase type II with the site(s) phosphorylated in vivo in rapidly growing Novikoff hepatoma cells. Topoisomerase I labeled in vitro was characterized by three major tryptic phosphopeptides (I-III). Separation of these peptides by a C18-reverse phase h.p.l.c. column resulted in their elution at fractions 18 (I), 27 (II) and 44 (III) with 17%, 22.5% and 33% acetonitrile, respectively. In contrast, only one major phosphopeptide was identified by h.p.l.c. in topoisomerase I labeled in vivo. This phosphopeptide eluted at fraction 18 corresponding to the elution properties of phosphopeptide I labeled in vitro. It also co-migrated with tryptic phosphopeptide I when subjected to high-voltage electrophoresis on thin-layer cellulose plates. Preliminary experiments suggest that phosphorylation occurs at a serine residue six amino acids from the N-terminus of the peptide. These data indicate that topoisomerase I is phosphorylated in vivo and in vitro within the same tryptic peptide and suggest that topoisomerase I is phosphorylated in vivo by casein kinase II. 相似文献