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1.
Satou Y Al-Shawafi HA Sultana S Makita S Sohda M Oda K 《Biochimica et biophysica acta》2012,1822(4):581-588
Hypophosphatasia (HPP), a rare genetic disease characterized by reduced serum alkaline phosphatase (ALP) activity and failure in bone and tooth mineralization, is caused by mutations in tissue-nonspecific ALP (TNSALP) gene. Two missense mutations (C201Y and C489S, standardized nomenclature) of TNSALP, involved in intra-chain disulfide bonds, were reported in patients diagnosed with perinatal HPP (Taillandier A. et al. Hum. Mutat. 13 (1999) 171-172, Hum. Mutat. 15 (2000) 293). To investigate the role of the disulfide bond in TNSALP, we expressed TNSALP (C201Y) and TNSALP (C489S) in COS-1 cells transiently. Compared with the wild-type enzyme [TNSALP (W)], both the TNSALP mutants exhibited a diminished ALP activity in the cells, where a 66kDa immature form was predominant with a marginal amount of a 80kDa mature form of TNSALP. Detailed studies on Tet-On CHO established cell line expressing TNSALP (W) or TNSALP (C201Y) showed that the 66kDa form of TNSALP (C201Y) exists as a monomer in contrast to a dimer of TNSALP (W). Only a small fraction of the TNSALP (C201Y) reached cell surface as the 80kDa mature form, though most of the 66kDa form was found to be endo-β-N-acetylglucosaminidase H sensitive and rapidly degraded in proteasome following polyubiquitination. Collectively, these results indicate not only that the intra-subunit disulfide bonds are crucial for TNSALP to properly fold and assemble into the dimeric enzyme, but also that the development of HPP associated with TNSALP (C201Y) or TNSALP (C489S) is attributed to decreased cell surface appearance of the functional enzyme. 相似文献
2.
Yasuhito SatouHiba A. Al-Shawafi Sara Sultana Saori Makita Miwa SohdaKimimitsu Oda 《生物化学与生物物理学报:疾病的分子基础》2012,1822(4):581-588
Hypophosphatasia (HPP), a rare genetic disease characterized by reduced serum alkaline phosphatase (ALP) activity and failure in bone and tooth mineralization, is caused by mutations in tissue-nonspecific ALP (TNSALP) gene. Two missense mutations (C201Y and C489S, standardized nomenclature) of TNSALP, involved in intra-chain disulfide bonds, were reported in patients diagnosed with perinatal HPP (Taillandier A. et al. Hum. Mutat. 13 (1999) 171-172, Hum. Mutat. 15 (2000) 293). To investigate the role of the disulfide bond in TNSALP, we expressed TNSALP (C201Y) and TNSALP (C489S) in COS-1 cells transiently. Compared with the wild-type enzyme [TNSALP (W)], both the TNSALP mutants exhibited a diminished ALP activity in the cells, where a 66 kDa immature form was predominant with a marginal amount of a 80 kDa mature form of TNSALP. Detailed studies on Tet-On CHO established cell line expressing TNSALP (W) or TNSALP (C201Y) showed that the 66 kDa form of TNSALP (C201Y) exists as a monomer in contrast to a dimer of TNSALP (W). Only a small fraction of the TNSALP (C201Y) reached cell surface as the 80 kDa mature form, though most of the 66 kDa form was found to be endo-β-N-acetylglucosaminidase H sensitive and rapidly degraded in proteasome following polyubiquitination. Collectively, these results indicate not only that the intra-subunit disulfide bonds are crucial for TNSALP to properly fold and assemble into the dimeric enzyme, but also that the development of HPP associated with TNSALP (C201Y) or TNSALP (C489S) is attributed to decreased cell surface appearance of the functional enzyme. 相似文献
3.
In the majority of hypophosphatasia patients, reductions in the serum levels of alkaline phosphatase activity are caused by various missense mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. A unique frame-shift mutation due to a deletion of T at cDNA number 1559 [TNSALP (1559delT)] has been reported only in Japanese patients with high allele frequency. In this study, we examined the molecular phenotype of TNSALP (1559delT) using in vitro translation/translocation system and COS-1 cells transiently expressing this mutant protein. We showed that the mutant protein not only has a larger molecular size than the wild type enzyme by approximately 12 kDa, reflecting an 80 amino acid-long extension at its C-terminus, but that it also lacks a glycosylphosphatidylinositol anchor. In support of this, alkaline phosphatase activity of the cells expressing TNSALP (1559delT) was localized at the juxtanucleus position, but not on the cell surface. However, only a limited amount of the newly synthesized protein was released into the medium and the rest was polyubiquitinated, followed by degradation in the proteasome. SDS/PAGE and analysis by sucrose-density-gradient analysis indicated that TNSALP (1559delT) forms a disulfide-bonded high-molecular-mass aggregate. Interestingly, the aggregate form of TNSALP (1559delT) exhibited a significant enzyme activity. When all three cysteines at positions of 506, 521 and 577 of TNSALP (1559delT) were replaced with serines, the aggregation disappeared and instead this modified mutant protein formed a noncovalently associated dimer, strongly indicating that these cysteine residues in the C-terminal region are solely responsible for aggregate formation by cross-linking the catalytically active dimers. Thus, complete absence of TNSALP on cell surfaces provides a plausible explanation for a severe lethal phenotype of a homozygote hypophosphatasia patient carrying TNSALP (1559delT). 相似文献
4.
Hypophosphatasia, a congenital metabolic disease related to the tissue-nonspecific alkaline phosphatase gene (TNSALP), is characterized by reduced serum alkaline phosphatase levels and defective mineralization of hard tissues. A replacement of valine with alanine at position 406, located in the crown domain of TNSALP, was reported in a perinatal form of hypophosphatasia. To understand the molecular defect of the TNSALP (V406A) molecule, we examined this missense mutant protein in transiently transfected COS-1 cells and in stable CHO-K1 Tet-On cells. Compared with the wild-type enzyme, the mutant protein showed a markedly reduced alkaline phosphatase activity. This was not the result of defective transport and resultant degradation of TNSALP (V406A) in the endoplasmic reticulum, as the majority of newly synthesized TNSALP (V406A) was conveyed to the Golgi apparatus and incorporated into a cold detergent insoluble fraction (raft) at a rate similar to that of the wild-type TNSALP. TNSALP (V406A) consisted of a dimer, as judged by sucrose gradient centrifugation, suggestive of its proper folding and correct assembly, although this mutant showed increased susceptibility to digestion by trypsin or proteinase K. When purified as a glycosylphosphatidylinositol-anchorless soluble form, the mutant protein exhibited a remarkably lower Kcat/Km value compared with that of the wild-type TNSALP. Interestingly, leucine and isoleucine, but not phenylalanine, were able to substitute for valine, pointing to the indispensable role of residues with a longer aliphatic side chain at position 406 of TNSALP. Taken together, this particular mutation highlights the structural importance of the crown domain with respect to the catalytic function of TNSALP. 相似文献
5.
Various mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene are responsible for hypophosphatasia characterized by defective bone and tooth mineralization; however, the underlying molecular mechanisms remain largely to be elucidated. Substitution of an arginine at position 433 with a histidine [TNSALP(R433H)] or a cysteine [TNSALP(R433C)] was reported in patients diagnosed with the mild or severe form of hypophosphatasia, respectively. To define the molecular phenotype of the two TNSALP mutants, we sought to examine them in transient (COS-1) and conditional (CHO-K1 Tet-On) heterologous expression systems. In contrast to an 80 kDa mature form of the wild-type and TNSALP(R433H), a unique disulfide-bonded 160 kDa molecular species appeared on the cell surface of the cells expressing TNSALP(R433C). Sucrose density gradient centrifugation demonstrated that TNSALP(R433C) forms a disulfide-bonded dimer, instead of being noncovalently assembled like the wild-type. Of the five cysteine residues per subunit of the wild-type, only Cys102 is thought to be present in a free form. Replacement of Cys102 with serine did not affect the dimerization state of TNSALP(R433C), implying that TNSALP(R433C) forms a disulfide bridge between the cysteine residues at position 433 on each subunit. Although the cross-linking did not significantly interfere with the intracellular transport and cell surface expression of TNSALP(R433C), it strongly inhibited its alkaline phosphatase activity. This is in contrast to TNSALP(R433H), which shows enzyme activity comparable to that of the wild-type. Importantly, addition of dithiothreitol to the culture medium was found to partially reduce the amount of the cross-linked form in the cells expressing TNSALP(R433C), concomitantly with a significant increase in enzyme activity, suggesting that the cross-link between two subunits distorts the overall structure of the enzyme such that it no longer efficiently carries out its catalytic function. Increased susceptibility to proteases confirmed a gross conformational change of TNSALP(R433C) compared with the wild-type. Thus, loss of function resulting from the interchain disulfide bridge is the molecular basis for the lethal hypophosphatasia associated with TNSALP(R433C). 相似文献
6.
Phytase (myo-inositol hexakisphosphate phosphohydrolase; EC 3.1.3.8 or 3.1.3.26) was purified from rat intestinal mucosa. The purified enzyme preparation exhibited two protein bands on SDS-polyacrylamide gel electrophoresis with estimated molecular masses of 70 kDa and 90 kDa. Rabbit antisera prepared against the 90K subunit cross-reacted with the 70K subunit on immunoblotting. The peptide maps of the 70K and 90K subunits were similar, and the N-terminal amino acid sequences of the two subunit proteins were almost identical. Treatments to remove sugar moieties from the proteins showed that the two subunit proteins had different oligosaccharide chains, although the difference in their molecular masses was not due to the difference in their oligosaccharide compositions. The purified enzyme also showed activity of alkaline phosphatase (orthophosphoric monoester phosphohydrolase; EC 3.1.3.1), but the properties of the two enzyme activities were different; the optimum pH for phytase activity was 7.5, while that for alkaline phosphatase was 10.4. Phytase activity did not necessarily require divalent cations, while Mg2+ was essential for alkaline phosphatase activity. Phenylalanine, a specific inhibitor of intestine-type alkaline phosphatase had no effect on the phytase activity. 相似文献
7.
Mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene are responsible for hypophosphatasia, an inborn error of bone and teeth metabolism associated with reduced levels of serum alkaline phosphatase activity. A missense mutation (c.346G>A) of TNSALP gene, which converts Ala to Thr at position 116 (according to standardized nomenclature), was reported in dominantly transmitted hypophosphatasia patients (A.S. Lia-Baldini et al. Hum Genet. 109 (2001) 99-108). To investigate molecular phenotype of TNSALP (A116T), we expressed it in the COS-1 cells or Tet-On CHO K1 cells. TNSALP (A116T) displayed not only negligible alkaline phosphatase activity, but also a weak dominant negative effect when co-expressed with the wild-type enzyme. In contrast to TNSALP (W, wild-type), which was present mostly as a non-covalently assembled homodimeric form, TNSALP (A116T) was found to exist as a monomer and heterogeneously associated aggregates covalently linked via disulfide bonds. Interestingly, both the monomer and aggregate forms of TNSALP (A116T) gained access to the cell surface and were anchored to the cell membrane via glycosylphosphatidylinositol (GPI). Co-expression of secretory forms of TNSALP (W) and TNSALP (A116T), which are engineered to replace the C-terminal GPI anchor with a tag sequence (his-tag or flag-tag), resulted in the release of heteromeric complexes consisting of TNSALP (W)-his and TNSALP (A116T)-flag. Taken together, these findings strongly suggest that TNSALP (A116T) fails to fold properly and forms disulfide-bonded aggregates, though it is indeed capable of interacting with the wild-type and reaching the cell surface, therefore explaining its dominant transmission. 相似文献
8.
Watanabe H Takinami H Goseki-Sone M Orimo H Hamatani R Ishikawa I 《Biochemical and biophysical research communications》2005,327(1):124-129
Hypophosphatasia (HOPS) is a clinically heterogeneous heritable disorder characterized by defective skeletal mineralization, deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity, and premature loss of deciduous teeth. To date, various mutations in the TNSALP gene have been identified. Especially, A115V located in exon 5 has been detected in a Japanese patient with severe periodontitis and adult-type HOPS. In this study, we have characterized the protein translated from the mutant A115V gene. Wild-type and A115V mutant-type TNSALP cDNA expression vector pcDNA3 have been constructed and transfected to COS-1 cells by lipofectin technique. After 48-h transfection, the cells were subjected to assay ALP activity. In order to identify possible dominant effect of the mutation, we performed co-transfections of wild-type and mutated cDNA, and evaluated the residual activities of each mutation. Detection of TNSALP synthesized by COS-1 cells transfected with the wild- or the mutated-type was also performed by using an immunofluorescent method. ALP activity of cell transfected with the mutant cDNA (A115V) plasmid after 48-h transfection exhibited 0.399+/-0.021 U/mg. As the enzymatic activity of the wild type was taken as 100%, the value of the mutant was estimated as 16.9%. When co-transfected this mutant showed no inhibition of the wild-type enzyme. TNSALP in COS-1 cells with transfected with the mutant exhibited strong fluorescence at the surface of cells as wild-type. This study indicated that the mutant (A115V) TNSALP gene produced the defective ALP enzyme and it could be recessively transmitted and be a disease-causing mutation of the adult-type hypophosphatasia. 相似文献
9.
A missense mutation in the gene of tissue-nonspecific alkaline phosphatase, which replaces aspartic acid at position 289 with valine [TNSALP (D289V)], was reported in a lethal hypophosphatasia patient [Taillandier, A. et al. (1999) Hum. Mut. 13, 171-172]. To define the molecular defects of TNSALP (D289V), this mutant protein in transiently transfected COS-1 cells was analyzed biochemically and morphologically. TNSALP (D289V) exhibited no alkaline phosphatase activity and mainly formed a disulfide-linked high molecular mass aggregate. Cell-surface biotinylation, digestion with phosphatidylinositol-specific phospholipase C and an immunofluorescence study showed that the mutant protein failed to appear on the cell surface and was accumulated intracellularly. In agreement with this, pulse/chase experiments demonstrated that TNSALP (D289V) remained endo-beta-N-acetyl- glucosaminidase H-sensitive throughout the chase and was eventually degraded, indicating that the mutant protein is unable to reach the medial-Golgi. Proteasome inhibitors strongly blocked the degradation of TNSALP (D289V), and furthermore the mutant protein was found to be ubiquitinated. Besides, another naturally occurring TNSALP with a Glu(218)-->Gly mutation was also found to be polyubiquitinated and degraded in the proteasome. Since the acidic amino acids at positions 218 and 289 of TNSALP are thought to be directly involved in the Ca(2+) coordination, these results suggest the critical importance of calcium binding in post-translational folding and assembly of the TNSALP molecule. 相似文献
10.
The preparation of monoclonal antibodies to human bone and liver alkaline phosphatase and their use in immunoaffinity purification and in studying these enzymes when present in serum. 总被引:1,自引:1,他引:0
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E M Bailyes R N Seabrook J Calvin G A Maguire C P Price K Siddle J P Luzio 《The Biochemical journal》1987,244(3):725-733
1. Liver and bone alkaline phosphatase isoenzymes were solubilized with the zwitterionic detergent sulphobetaine 14, and purified to homogeneity by using a monoclonal antibody previously raised against a partially-purified preparation of the liver isoenzyme. Both purified isoenzymes had a specific activity in the range 1100-1400 mumol/min per mg of protein with a subunit Mr of 80,000 determined by SDS/polyacrylamide gel electrophoresis. Butanol extraction instead of detergent solubilization, before immunoaffinity purification of the liver enzyme, resulted in the same specific activity and subunit Mr. The native Mr of the sulphobetaine 14-solubilized enzyme was consistent with the enzyme being a dimer of two identical subunits and was higher than that of the butanol-extracted enzyme, presumably due to the binding of the detergent micelle. 2. Pure bone and liver alkaline phosphatase were used to raise further antibodies to the two isoenzymes. Altogether, 27 antibody-producing cell lines were cloned from 12 mice. Several of these antibodies showed a greater than 2-fold preference for bone alkaline phosphatase in the binding assay used for screening. No antibodies showing a preference for liver alkaline phosphatase were successfully cloned. None of the antibodies showed significant cross-reaction with placental or intestinal alkaline phosphatase. Epitope analysis of the 27 antibodies using liver alkaline phosphatase as antigen gave rise to six groupings, with four antibodies unclassified. The six major epitope groups were also observed using bone alkaline phosphatase as antigen. 3. Serum from patients with cholestasis contains soluble and particulate forms of alkaline phosphatase. The soluble serum enzyme had the same size and charge as butanol-extracted liver enzyme on native polyacrylamide-gel electrophoresis. Cellulose acetate electrophoresis separated the soluble and particulate serum alkaline phosphatases as slow- and fast-moving forms respectively. In the presence of sulphobetaine 14 all the serum enzyme migrated as the slow-moving form on cellulose acetate electrophoresis. Monoclonal anti-(alkaline phosphatase) immunoadsorbents did not bind the particulate form of alkaline phosphatase in cholestatic serum but bound the soluble form. In the presence of sulphobetaine 14 all the cholestatic serum alkaline phosphatase bound to the immunoadsorbents. 4. The electrophoretic and immunological data are consistent with both particulate and soluble forms of alkaline phosphatase in cholestatic serum being derived from the hepatocyte membrane. 相似文献
11.
Mammalian DNA ligases. Catalytic domain and size of DNA ligase I. 总被引:14,自引:0,他引:14
A E Tomkinson D D Lasko G Daly T Lindahl 《The Journal of biological chemistry》1990,265(21):12611-12617
DNA ligase I is the major DNA ligase activity in proliferating mammalian cells. The protein has been purified to apparent homogeneity from calf thymus. It has a monomeric structure and a blocked N-terminal residue. DNA ligase I is a 125-kDa polypeptide as estimated by sodium dodecyl sulfate-gel electrophoresis and by gel chromatography under denaturing conditions, whereas hydrodynamic measurements indicate that the enzyme is an asymmetric 98-kDa protein. Immunoblotting with rabbit polyclonal antibodies to the enzyme revealed a single polypeptide of 125 kDa in freshly prepared crude cell extracts of calf thymus. Limited digestion of the purified DNA ligase I with several reagent proteolytic enzymes generated a relatively protease-resistant 85-kDa fragment. This domain retained full catalytic activity. Similar results were obtained with partially purified human DNA ligase I. The active large fragment represents the C-terminal part of the intact protein, and contains an epitope conserved between mammalian DNA ligase I and yeast and vaccinia virus DNA ligases. The function of the N-terminal region of DNA ligase I is unknown. 相似文献
12.
Placental alkaline phosphatase has a binding site for the human immunoglobulin-G Fc portion. 总被引:3,自引:0,他引:3
Affinity chromatography of human plasma on placental-alkaline-phosphatase-Sepharose columns (placental alkaline phosphatase, PLAP) yielded consistently a pure protein which was identified as IgG on the basis of electrophoretical and immunological comparisons with authentic human IgG. SDS/PAGE of the protein revealed, under reducing conditions, two polypeptides of 55 kDa and 25 kDa. The N-terminal amino acid sequence (12 residues) of the 55-kDa subunit presented high similarity (83-100%) with known sequences of immunoglobulin gamma chains. The IgG binds by its Fc portion to a fully exposed domain in the plasma-membrane-anchored PLAP. Scatchard analysis of the interaction gave a dissociation constant of 3.68 microM, a value close to those found for haematopoietic cells and syncytiotrophoblast Fc receptors. The latter was affinity purified from human placenta as the major IgG-binding component and presented cross-immunoreactivity with anti-PLAP antibodies, indicating that PLAP and the putative placental Fc receptor could be identical molecules. 相似文献
13.
We developed a method for selective preparation of two forms of alkaline phosphatase from rat tissues. The enzyme was extracted by n-butanol treatment at pH 5.5 and pH 8.5 as soluble and aggregated (membranous) forms, respectively. The soluble form prepared from liver was found to be identical with the serum enzyme. Complete solubilization of the membrane-bound enzyme without detergents had a great advantage in its purification. Rat hepatoma AH-130 cells enriched in alkaline phosphatase were first used for purification of the liver-type enzyme. The hepatoma enzyme, purified by chromatographies on concanavalin-A-Sepharose, Sephacryl S-300 and hydroxyapatite was used for production of antibodies specific for the liver-type isozyme. An immunoaffinity column, prepared with anti-(hepatoma-enzyme) IgG was utilized for the enzyme purification from other tissues including the membranous form. Analyses of amino acid composition of the purified enzymes revealed that all the liver-type enzymes from hepatoma, liver, kidney and serum had the same composition, whereas the intestinal type consisted of the composition distinctly different from that in the liver type. In addition, there was no significant difference in amino acid composition between the soluble and membranous forms, suggesting a possible involvement in the membranous form of a hydrophobic component other than its polypeptide domain. The present method for selective preparation of the soluble and membranous forms of alkaline phosphatase will be useful for a further investigation on the interaction of the enzyme with membranes. 相似文献
14.
Isolation and purification of protein kinase C from human leukemia ML-1 cells phosphorylation of human leukemia RNA polymerase II in vitro 总被引:1,自引:0,他引:1
Protein kinase C (PKC) was purified to near homogeneity from human leukemia ML-1 cells. The purified enzyme showed a single polypeptide band of 80 kDa on SDS-polyacrylamide gel after electrophoresis, and was totally dependent on Ca2+/phospholipid for activity. Diacylglycerol and the tumor-promoting on Ca2/phospholipid for activity. Diacylglycerol and the tumor-promoting phorbol esters stimulated the enzyme activity. Autophosphorylation of PKC purified from phenyl-Sepharose column showed both 80- and 37 kDa polypeptides. Further fractionation of PKC on a hydroxyapatite column revealed two peaks of enzyme activity, indicating that there may be two different forms of protein kinase C present in human leukemia cells. The purified PKC was used to phosphorylate RNA polymerase II of human leukemia cells in vitro and the autoradiogram showed that RNA polymerase II large subunits (240, 220 and 150 kDa) were phosphorylated in a time-dependent manner. 相似文献
15.
Two protein phosphatases of 103 and 29 kDa as determined by gel filtration, were purified from hen oviducts. The 103 -kDa phosphatase was purified 7300-fold to near homogeneity and dissociated into two polypeptides in the presence of SDS. Molecular masses of these polypeptides were estimated to be 60 and 38 kDa by SDS-polyacrylamide slab gel electrophoresis using the buffer system of Laemmli, but 68 and 35 kDa using the buffer system of Weber and Osborn. The stoichiometry of these polypeptides was approx 1:1 according to the densitometric analysis of gels at 550 nm. The 29 -kDa phosphatase was purified 2900-fold. Both phosphatases dephosphorylated the alpha-subunit of phosphorylase kinase more rapidly than the beta-subunit. 相似文献
16.
Furuhashi K 《Archives of microbiology》2008,189(2):151-156
The plasmodia of Physarum polycephalum grow as multinucleated cells in the presence of sufficient humidity and nutriment. Under non-illuminating conditions, stresses
such as low temperature or high concentrations of salts transform the plasmodia into spherules whereas dehydration induces
sclerotization. Some phosphatases including protein phosphatase and acid phosphatase have been purified from the plasmodia,
but alkaline phosphatase remains to be elucidated. Phosphatase of the plasmodia, spherules and sclerotia was visualized by
electrophoresis gel-staining assay using 5-bromo-4-chloro-3-indolyl phosphate. Insoluble fractions of the sclerotia were abundant
in phosphatase activity. The phosphatase which was extracted by nonionic detergent was subjected to column chromatography
and preparative electrophoresis. Purified phosphatase showed the highest activity at pH 8.8, indicating that this enzyme belongs
to alkaline phosphatase. The apparent molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis under
non-reducing condition was estimated to be 100 kDa whereas that under reducing was 105 kDa. An amount of 1% sodium dodecyl
sulfate or 0.5 M NaCl had no effects on the activity although the phosphatase showed heat instability, Mg2+-dependency and sensitivity to 2-glycerophosphate or NaF. The extracting conditions and enzymatic properties suggest that
this alkaline phosphatase which is in a membrane-bound form plays important roles in phosphate metabolism. 相似文献
17.
Our recently described purification scheme for rat brain protein kinase C yields an enzyme consisting of a 78/80-kilodalton (kDa) doublet upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (submitted for publication). Antisera against this preparation were raised in two rabbits. One of the antisera detected only the 80-kDa component by immunoblotting of purified protein kinase C and immunoprecipitated an 80-kDa [35S]methionine-labeled protein from a variety of human, rodent, and bovine cells, which was shown to represent protein kinase C by comparative one-dimensional peptide mapping. In contrast, the second antiserum detected both 78- and 80-kDa enzyme forms by immunoblotting and immunoprecipitated a [35S]methionine-labeled 78/80-kDa doublet from mammalian cells. One-dimensional peptide maps of these 78- and 80-kDa proteins were similar to those derived from the 78- and 80-kDa forms of purified protein kinase C, respectively. The two forms were not related by either partial proteolysis or differential phosphorylation, showing that two distinct forms of this enzyme exist in mammalian cells. Treatment of mouse B82 L cells with 2.5 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) per ml for 18 h resulted in complete loss of immunoprecipitable protein kinase C with a half time of disappearance of 48 min. Since the normal half-life of protein kinase C was greater than 24 h and the biosynthetic rate of the protein was not decreased after 18 h by TPA treatment, TPA induces down-regulation by increasing the degradation rate of the enzyme. Treatment of cells with 50 ng of TPA per ml followed by resolution of the membrane and cytosol in the presence of ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) promoted an apparent translocation of both 78- and 80-kDa proteins from the cytosol to the membrane fraction. A similar translocation was effected by cell lysis in the presence of Ca2+, indicating the subcellular localization of protein kinase C to be sensitive to the presence of both activators and micromolar amounts of Ca2+. 相似文献
18.
Induced alkaline phosphatase has been extracted from osteosarcoma cells grown in tissue culture medium. The extracted enzyme has been purified. Using electrophoresis, inhibition studies, and thermolability, the enzyme was categorized as alkaline phosphatase of osseous origin. Antibodies to this enzyme were reacted against alkaline phosphatase extracted from cadaveric bone, liver, intestine, kidney and fresh placenta. The antibodies were specific against alkaline phosphatase of osseous origin only. No cross-reaction occurred with the enzyme extracted from other sources. The data derived from these studies indicate that alkaline phosphatase of bone is a specific enzyme of osseous tissue. Furthermore, the enzyme has specific antigenic and other properties which distinguish it from alkaline phosphatases from other sources. A model for in vitro production of a specific alkaline phosphatase of bone is presented. 相似文献
19.
Purification and characterization of human platelet phospholipase A2 which preferentially hydrolyzes an arachidonoyl residue 总被引:3,自引:0,他引:3
A phospholipase A2 with an arachidonoyl residue preference was purified about 11,700-fold from human platelet soluble fraction to near homogeneity. The purified phospholipase A2 exhibited a molecular mass of about 90 kDa on SDS polyacrylamide gel electrophoresis and hydrolyzed phospholipids with an arachidonoyl residue more effectively than those with a linoleoyl residue. The catalytic activity of the purified enzyme detected with phosphatidylcholine as a substrate increased sharply between 3 x 10(-7) and 10(-6) M free calcium ion. Thus, the 90-kDa phospholipase A2 is considered to be a novel enzyme, distinct from the 14-kDa one previously purified from human platelets. The 90-kDa phospholipase A2 may participate mainly in arachidonate metabolism of platelets. 相似文献
20.
Biochemical properties of a termostable alkaline phosphatase obtained from the mycelium extract of A. caespitosus were described. The enzyme was purified 42-fold with 32% recovery by DEAE-cellulose and concanavalin A-Sepharose chromatography. The molar mass estimated by Sephacryl S-200 or by 7% SDS-PAGE was 138 kDa and 71 kDa, respectively, indicating a homodimer. Temperature and pH optima were 80 degrees C and pH 9.0. This enzyme was highly glycosylated (approximately 74% saccharide content). The activity was enhanced by Mg2+ (19-139%), NH4+ (64%), Na+ (51%) and Mn2+ (38%). 4-Nitrophenyl phosphate (4-NPP) was preferentially hydrolyzed, but glucose 1-phosphate (93%), UTP (67%) and O-phosphoamino acids also acted as substrates. V(lim) and K(m) were 3.78 nkat per mg protein and 270 micromol/L in the absence of Mg2+ and 7.35 nkat per mg protein and 410 micromol/L in the presence of Mg2+, using 4-NPP as substrate. The purified alkaline phosphatase removed the 5'-phosphate group of a linearized plasmid without showing DNAase activity, indicating its potential for recombinant DNA technology. 相似文献