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排序方式: 共有173条查询结果,搜索用时 363 毫秒
1.
Cytosolic protein phosphotyrosine phosphatases from rabbit kidney. Purification of two distinct enzymes that bind to Zn2+-iminodiacetate agarose 总被引:14,自引:0,他引:14
Cytosolic protein phosphotyrosine (PPT) phosphatase was measured using a new substrate, Tyr(32P)-labeled bovine serum albumin. Kidney was found as a particularly rich tissue source of PPT-phosphatase activity, containing twice as much as liver and over 10-fold more than brain, heart, lung, or skeletal muscle. An affinity column of Zn2+-iminodiacetate agarose adsorbed up to 60% of the PPT-phosphatase present in kidney extracts. Subsequent chromatography on DEAE-Sepharose separated the phosphatase into two peaks, labeled I and II, that had Mr = 34,000 and 37,000, respectively, upon gel filtration with Sephadex G-75 Superfine. Overall purification of 850- and 1100-fold was achieved with a net 4% yield. Both phosphatases hydrolyzed p-nitrophenylphosphate as well as the protein substrate in the presence of EDTA. Peak I phosphatase activity displayed a neutral pH optimum, had an absolute requirement for sulfhydryl compounds, and was sensitive to trypsin, whereas Peak II activity had an acidic pH optimum and was active without mercaptans. The two proteins also gave different fragmentation patterns by gel electrophoresis after digestion with S. aureus V8 protease. The results show that multiple forms of PPT phosphatase specifically interact with Zn2+ and provide a basis for further structural and functional comparisons among different members of the phosphoprotein phosphatase family. 相似文献
2.
P A Gruppuso C L Shriner D L Brautigan 《Biochemical and biophysical research communications》1987,148(3):1174-1181
We have examined the characteristics of partially purified forms of rabbit skeletal muscle type-1 protein phosphatase (PP-1). Over 90% of PP-1 in unfractionated extracts and in glycogen particles was inactive, but could be activated by the divalent cations, Mn2+ or Co2+ (Me2+) plus trypsin. Gel filtration of muscle extracts revealed two inactive forms of PP-1; one activated by Me2+ alone or Me2+ plus trypsin, and a second containing inhibitor-2 and activated only by Me2+ plus trypsin. The kinetics of Me2+ plus trypsin activation revealed that after DEAE-chromatography, PP-1 was altered from the forms in crude extracts, gel filtered extracts or glycogen particles. The results indicate that the purified form of PP-1 catalytic subunit has properties which distinguish it from the forms of the enzyme in muscle extracts. 相似文献
3.
Calyculin A and okadaic acid: inhibitors of protein phosphatase activity 总被引:44,自引:0,他引:44
H Ishihara B L Martin D L Brautigan H Karaki H Ozaki Y Kato N Fusetani S Watabe K Hashimoto D Uemura 《Biochemical and biophysical research communications》1989,159(3):871-877
Calyculin A and okadaic acid induce contraction in smooth muscle fibers. Okadaic acid is an inhibitor of phosphatase activity and the aims of this study were to determine if calyculin A also inhibits phosphatase and to screen effects of both compounds on various phosphatases. Neither compound inhibited acid or alkaline phosphatases, nor the phosphotyrosine protein phosphatase. Both compounds were potent inhibitors of the catalytic subunit of type-2A phosphatase, with IC50 values of 0.5 to 1 nM. With the catalytic subunit of protein phosphatase type-1, calyculin A was a more effective inhibitor than okadaic acid, IC50 values for calyculin A were about 2 nM and for okadaic acid between 60 and 500 nM. The endogenous phosphatase of smooth muscle myosin B was inhibited by both compounds with IC50 values of 0.3 to 0.7 nM and 15 to 70 nM, for calyculin A and okadaic acid, respectively. The partially purified catalytic subunit from myosin B had IC50 values of 0.7 and 200 nM for calyculin A and okadaic acid, respectively. The pattern of inhibition for the phosphatase in myosin B therefore is similar to that of the type-1 enzyme. 相似文献
4.
Regulation of various metabolic processes occurs by the phosphorylation/dephosphorylation of enzymes. Both the protein kinases that catalyze the phosphorylations and the protein phosphatases that catalyze the dephosphorylations display relatively broad specificity, reacting with a number of distinct sites in target enzymes. In this way changes in the activity of a particular kinase or phosphatase can cause coordinated and pleiotropic responses. However, the kinases and phosphatases do not exhibit a one-to-one correspondence in their reactions. Residues at different positions may be phosphorylated by a single kinase, yet dephosphorylated by different individual phosphatases. Conversely, sites which are substrates for different individual kinases may be dephosphorylated by a single phosphatase. In exploring the molecular basis for these differences this article shows that whereas kinases react with specific primary structures that often times appear as beta bends, the phosphatases recognize higher order structure, less strictly ruled by amino acid sequence surrounding the phosphorylated site. The differences, seen in the ability of these enzymes to utilize synthetic peptide substrates, might be rationalized in terms of function. Kinases need protruding segments of structure that can be enwrapped to exclude water, thereby minimizing ATP hydrolysis and enhancing phosphotransferase activity. On the other hand phosphatases are hydrolytic enzymes that may operate especially well on protein interfaces. Hydrolytic action often measured with p-nitrophenylphosphate is not necessarily indicative of a protein phosphatase and consideration of the mechanism reveals why this substrate can be misleading.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
5.
Phosphorylase phosphatase catalytic subunit. Evidence that the Mr = 33,000 enzyme fragment is derived from a native protein of Mr = 70,000 总被引:6,自引:0,他引:6
An active form of phosphorylase phosphatase of Mr = 33,000, referred to as the catalytic subunit for over a decade, was purified to near-homogeneity from rabbit skeletal muscle. Repeated immunization of a sheep produced immunoglobulins that blocked the activity of the phosphatase. These immunoglobulins were affinity-purified on columns of immobilized phosphorylase phosphatase and used as macromolecular probes in a "Western" immunoblotting procedure with peroxidase-conjugated rabbit anti-sheep immunoglobulins. Only one protein, of Mr = 33,000, was stained in samples of the immunogen, attesting to the specificity of the probes. However, the Mr = 33,000 phosphatase protein was not detected in muscle extracts or in partially purified preparations. Instead, a single protein of Mr = 70,000 was detected. Limited proteolysis, in particular by Staphylococcus aureus V8 protease and thermolysin, converted the immunoreactive protein from Mr = 70,000 to Mr = 33,000. Coagulation of the phosphatase preparation with 80% ethanol at room temperature rendered the Mr = 70,000 protein insoluble, but allowed extraction of the Mr = 33,000 protein from the precipitate. Thus, we conclude that the immunoreactive protein of Mr = 70,000 is the "catalytic subunit" of phosphorylase phosphatase with a catalytic domain of Mr = 33,000. Previous purification schemes have yielded only the fragment of Mr = 33,000 due to its relative resistance to proteolysis and coagulation. Gel filtration chromatography of the "native" form of phosphorylase phosphatase showed Mr approximately 230,000. Both the Mr = 70,000 catalytic subunit and a Mr = 60,000 protein related to inhibitor-2 were detected by immunoblotting in the same fractions that exhibited activity after treatment with Co2+ and trypsin. Only the Mr = 60,000 protein was degraded during this activation process. We propose that the native phosphorylase phosphatase is an elongated structure with two-fold symmetry, containing one catalytic subunit of Mr = 70,000 and one regulatory subunit of Mr = 60,000. 相似文献
6.
The major intracellular protein tyrosine phosphatase (PTP1B) is a 50kDa protein, localized to the endoplasmic reticulum. This PTP is recovered in the particulate fraction of mamalian cells and can be solubilized as a complex of 150 kDa by extraction with non-ionic detergents. Previous work from this laboratory implicated phosphorylation of serine/threonine residues in the regulation of this PTP. Activity was several-fold higher in cells treated with activators of cAMP-dependent or Ca2+/phospholipid-dependent protein kinases or inhibitors of protein phosphatase 2A. Here we show that these treatments result in more than an 8-fold increase in the phosphorylation of the 50kDa PTP catalytic subunit within the 150kDa form of the phosphatase in HeLa cells. The phosphorylation occurred exclusively on serine residues, and the same tryptic and cyanogen bromide,32P-phosphopeptides were recovered in the PTP from control and stimulated cells. Either multiple kinases phosphorylate a common site in the PTP1B, or a single kinase is activated downstream of cAMP- and Ca2+/phospholipid-dependent kinases. The results indicate that phosphorylation of a serine residue in the segment 283–364, probably serine 352 in the sequence Lys-Gly-Ser-Pro-Leu, occurs in response to cell stimulation. Phosphorylation in this region of PTP1B, between the N-terminal catalytic domain and the C-terminal membrane localization segment, is proposed to regulate phosphatase activity. 相似文献
7.
DNA sequences were determined for three to five alleles of the bride-of-
sevenless (boss) gene in each of four species of Drosophila. The product of
boss is a transmembrane receptor for a ligand coded by the sevenless gene
that triggers differentiation of the R7 photoreceptor cell in the compound
eye. Population parameters affecting the rate and pattern of molecular
evolution of boss were estimated from the multinomial configurations of
nucleotide polymorphisms of synonymous codons. The time of divergence
between D. melanogaster and D. simulans was estimated as approximately 1
Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and
that between the two pairs of sibling species as approximately 2 Myr. (The
boss genes themselves have estimated divergence times approximately 50%
greater than the species divergence times.) The effective size of the
species was estimated as approximately 5 x 10(6), and the average mutation
rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of
amino acid polymorphisms within species to fixed differences between
species suggests that approximately 25% of all possible single-step amino
acid replacements in the boss gene product may be selectively neutral or
nearly neutral. The data also imply that random genetic drift has been
responsible for virtually all of the observed differences in the portion of
the boss gene analyzed among the four species.
相似文献
8.
John P. Williams Hanjoong Jo Ruthann E. Hunnicutt David L. Brautigan Jay M. McDonald Dr. 《Journal of cellular biochemistry》1995,57(3):415-422
Inhibitor 2 is a heat-stable protein that complexes with the catalytic subunit of type-1 protein phosphatase. The reversible phosphorylation of Thr 72 of the inhibitor in this complex has been shown to regulate phosphatase activity. Here we show that inhibitor 2 can also be phosphorylated on tyrosine residues. Inhibitor 2 was 32P-labeled by the insulin receptor kinase in vitro, in the presence of polylysine. Phosphorylation of inhibitor 2 was accompanied by decreased electrophoretic mobility. Dephosphorylation of inhibitor 2 by tyrosine phosphatase 1B, restored normal electrophoretic mobility. Phosphotyrosine in inhibitor 2 was detected by immunoblotting with antiphosphotyrosine antibodies and phosphoamino acid analysis. In addition, following tryptic digestion, one predominant phosphopeptide was recovered at the anode. The ability of inhibitor 2 to inhibit type-1 phosphatase activity was diminished with increasing phosphorylation up to a stoichiometry of 1 mole phosphate incorporated/mole of inhibitor 2, where inhibitory activity was completely lost. These data demonstrate that inhibitor 2 can be phosphorylated on tyrosine residues by the insulin receptor kinase, resulting in a molecule with decreased ability to inhibit type-1 phosphatase activity. 相似文献
9.
The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum 总被引:29,自引:22,他引:7 下载免费PDF全文
Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature. 相似文献
10.
Partial purification and characterization of phosphotyrosyl-protein phosphatase from Ehrlich ascites tumor cells 总被引:4,自引:0,他引:4
We have previously described a phosphotyrosylprotein phosphatase in membrane vesicles from human epidermoid carcinoma A431 cells which is inhibited by micromolar concentration of Zn2+ and is insensitive to ethylenediaminetetraacetic acid (EDTA) and NaF [Brautigan, D. L., Bornstein, P., & Gallis, B. (1981) J. Biol. Chem. 256, 6519-6522]. Here we present the identification and partial purification of a similar enzyme from lysates of Ehrlich ascites tumor cells. the enzyme was purified by using diethylaminoethyl-Sephadex, Zn2+ affinity, and Sephadex G-75 chromatography. During purification, the phosphatase was separated into at least three fractions, all of which exhibited very similar properties and an apparent molecular weight of 40 000 upon gel filtration. The enzyme dephosphorylated phosphotyrosine (P-Tyr)-containing carboxymethylated and succinylated (CM-SC) phosphorylase with an apparent Km of 0.8 microM, as well as P-Tyr containing casein and epidermal growth factor (EGF) receptor kinase, but did not dephosphorylate P-Ser-phosphorylase. The phosphatase was inhibited by Zn2+ at micromolar concentrations (K0.5 with EGF receptor kinase = 5 X 10(-6) M; with CM-SC phosphorylase = 3.3 X 10(-5) M) but not by millimolar concentrations of EDTA and NaF. No inhibition was seen with 1 mM tetramisole, a specific inhibitor of alkaline phosphatases. P-Tyr inhibited the enzyme by 50% at 0.4 X 10(-3) M, while Tyr, Pi, PPi, and p-nitrophenyl phosphate, an excellent substrate for alkaline phosphatases and structurally very similar to P-Tyr, exerted partial inhibition at concentrations above 10(-3) M. The pH optimum was found to be 6.5-7, depending on the substrate used. Very little activity was seen below pH 5 and above pH 8.5. These properties clearly distinguish this enzyme from alkaline phosphatases, as well as the neutral and acidic protein phosphatases so far described, and therefore define it as a new enzyme of the phosphatase family--a phosphotyrosyl-protein phosphatase. 相似文献