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1.
The alkaline phosphatase from KB cells was purified, characterized, and compared to placental alkaline phosphatase, which it resembles immunologically. Two nonidentical nonomeric subunits of the KB phosphatase were found. The two subunits, which have apparent molecular weights of 64,000 and 72,000, can be separated on polyacrylamide gels containing sodium dodecyl sulfate. The Mr = 64,000 KB subunit appears to be identical in protein structure to the monomer of placental alkaline phosphatase. The Mr = 72,000 KB subunit, while differing in the NH2-terminal amino acid, appears also to be very similar to the placental alkaline phosphatase monomer. Both KB phosphatase subunits bind (32P)phosphate, and bind to Sepharose-bound anti-placental alkaline phosphatase. Native KB phosphatase is identical to the placental isozyme in isoelectric point, pH optimum, and inhibition by amino acids, and has a very similar peptide map. The data presented support the hypothesis that the Mr = 64,000 KB phosphatase subunit may the the same gene product as the monomer of placental alkaline phosphatase. This paper strengthens the evidence that the gene for this fetal protein, normally repressed in all cells but placenta, is derepressed in the KB cell line. In addition, this paper presents the first structural evidence that there are two different subunit proteins comprising the placental-like alkaline phosphatase from a human tumor cell line. 相似文献
2.
1. A heat-stable alkaline phosphatase was purified from Penaeus japonicus, with a final specific activity of 21,280 U/mg of protein. 2. In polyacrylamide-gel electrophoresis under non-denaturing conditions, the purified shrimp alkaline phosphatase was found to have an identical molecular size and surface charge as the human placental enzyme. 3. By using SDS-PAGE, the monomers of shrimp alkaline phosphatase were discovered to have a Mr 55,000 but those of human placental enzyme with a Mr 70,000. Deglycosylation decreases the Mr values of the subunits to 33,000 for shrimp alkaline phosphatase. 4. The purified alkaline phosphatase from shrimp was recovered with both the attachment sites for sialic acids and phosphatidylinositol. 5. The shrimp alkaline phosphatase has an isoelectric point (pI) of 7.6 and the human placental enzyme has a pI of 4.8. 相似文献
3.
Evidence for two structural genes for alkaline phosphatase in Bacillus subtilis. 总被引:9,自引:7,他引:2
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Two secreted alkaline phosphatase proteins were purified from cultures of Bacillus subtilis JH646MS. The two proteins showed slight differences in subunit molecular weight, substrate specificity, and charge characteristics. A total of 62% of the first 22 amino-terminal amino acids were identical. Both sequences showed conservation of structural features identified in Escherichia coli and human alkaline phosphatases. One alkaline phosphatase was a monomer and the other was a dimer. Southern analysis of genomic DNA with degenerative oligomers based on the amino acid sequences suggest that there are two structural genes for alkaline phosphatase in the genome of B. subtilis. 相似文献
4.
Treatment of immature mice with gonadotrophins. The influence of mouse age on the response of ovarian alkaline phosphatase activities to gonadotrophins
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Thomas A. Bramley 《The Biochemical journal》1975,152(2):365-372
Treatment of mice aged 23-25 days with chorionic gonadotrophin induced large amounts of an ovarian alkaline phosphatase activity (phosphatase Ib) kinetically distinct from that of untreated ovaries (phosphatase I). The activities of alkaline phosphatase I and Ib varied with age in untreated mice. Phosphatase Ib appeared when serum luteinizing hormone concentrations increased (days 4-10 and days 35-45), and disappeared when concentrations were low (days 11-35). Injection of human chorionic gonadotrophin induced progressively larger amounts of phosphatase Ib activity between day 19 and day 29. However, gonadotrophin treatment failed to induce this activity on days 10-18 and 30-35. Nevertheless, during the latter period, human chorionic gonadotrophin induced especially large increases in uterine weight. Treatment at different ages with sheep luteinizing hormone plus human pituitary follicle-stimulating hormone induced a pattern of response identical with that induced by human chorionic gonadotrophin, although sheep luteinizing hormone alone was ineffective before 35 days. In contrast, human luteinizing hormone induced a response in the absence of exogenous follicle-stimulating hormone. 相似文献
5.
Kauko Kouvalainen 《The Histochemical journal》1971,3(1):55-69
Synopsis Alkaline phosphatase activity in lymphatic tissues of guineapig, cat, cow, dog, rabbit, sheep, rat, mouse, hamster, chicken and man was studied with histochemical, biochemical and electrophoretic techniques. The thymus showed decreasing alkaline phosphatase activity from species to species in the order just given. Activity of alkaline phosphatase in other lymphatic tissues did not show such clear species and organ dependence. Spleens of the cat, cow and rabbit and lymph nodes of the cow and sheep gave, however, very characteristic patterns of alkaline phosphatase activity. In the chicken there was no difference between the alkaline phosphatase content of the thymus and that of the bursa of Fabricius. The lymphatic follicles of human tonsils and appendix and in the appendix of the rabbit exhibited alkaline phosphatase activity in the circular cell layer. This was also seen in some follicles in the lymph nodes of certain species. Electrophoretically, the main alkaline phosphatase fraction of the lymphatic tissues closely resembled the main fraction of blood, though it is probably not identical with it. Although the biological function of alkaline phosphatase is unknown, the greatly varying alkaline phosphatase content in different lymphatic organs of different species indicates that immunological studies with one species or with cells derived from a certain lymphatic tissue or with both are probably not directly comparable with studies using other species or cells from other lymphatic tissues. 相似文献
6.
S Seetharam C Ovitt A W Strauss D Rubin D H Alpers 《Biochemical and biophysical research communications》1987,145(1):363-368
We have identified two mRNAs in rat intestinal mucosa by Northern blot analysis, using cloned cDNAs encoding human placental alkaline phosphatase (PLAP). Probes from both the NH2- and COOH-terminal ends of the human PLAP coding region identified, in rat intestine (especially duodenum), an mRNA of nearly identical size (3 kb) to that found in human placenta. A smaller mRNA (2.7 kb), detected only with the COOH-terminal probe, was more prevalent in jejunum. Following feeding of triacylglycerols, the prevalence of the 2.7 kb mRNA increased over 2-fold. The tissue distribution and response of the 2.7 kb mRNA to fat feeding corresponds exactly with the known behavior of the secreted alkaline phosphatase. 相似文献
7.
E Ezra R Blacher S Udenfriend 《Biochemical and biophysical research communications》1983,116(3):1076-1083
Two forms of human placental alkaline phosphatase have been purified to homogeneity utilizing high performance liquid chromatography. Both have the same amino acid composition but they differ in their carbohydrate substituents. Sequence data indicate that the two forms are identical for the first forty two residues from the amino terminus are presented. 相似文献
8.
F J Vanneuville J G Leroy 《Archives internationales de physiologie et de biochimie》1980,88(2):155-161
The kinetic properties of the "constitutive" and the "induced" alkaline phosphatase in diploid fibroblasts are compared with those of the enzymes in crude tissue homogenates. Both the constitutive as the induced enzyme have properties comparable with those of the liver-bone-kidney group. The induced alkaline phosphatase clearly differs from the "constitutive" alkaline phosphatase concerning the effect of high concentrations of L-phenylalanine and the effect of Mg2+ ions. The induced alkaline phosphatase seems to be identical with the enzyme in liver, but the constitutive alkaline phosphatase could not be identified. 相似文献
9.
Expression and Localization of Escherichia coli Alkaline Phosphatase Synthesized in Salmonella typhimurium Cytoplasm 总被引:14,自引:10,他引:4
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The Escherichia coli structural gene for alkaline phosphatase was inserted into Salmonella typhimurium by episomal transfer in order to determine whether this enzyme would continue to be localized to the periplasmic space of the bacterium even though it was formed in a cell that does not synthesize alkaline phosphatase. The S. typhimurium heterogenote synthesized alkaline phosphatase under conditions identical to that observed with E. coli. This enzyme appeared to be identical to that synthesized by E. coli, and was quantitatively released from the bacterial cell by spheroplast formation with lysozyme. These results showed that localization is not a property unique to the E. coli cell and suggested that, in E. coli, enzyme location is related to the structure of the protein. Formation of alkaline phosphatase in the S. typhimurium heterogenote was repressed in cells growing in a medium with excess inorganic phosphate, even though only one of the three regulatory genes for this enzyme is on the episome. Thus, S. typhimurium can supply the products of the other two regulatory genes essential for repression even though this bacterium seems to lack the structural gene for alkaline phosphatase. 相似文献
10.
Characterization of a bone-specific alkaline phosphatase in chick limb mesenchymal cell cultures 总被引:1,自引:0,他引:1
Previous morphometric and biochemical studies suggested that osteoblasts develop in cultures derived from phenotypically unexpressive stage 24 chick limb mesenchymal cells. Others have shown that osteoblast expression is marked by an increase in bone-specific alkaline phosphatase activity. Our results indicate that chick limb mesenchymal cells develop alkaline phosphatase activity that is identical to that of the chick embryonic bone-specific isoenzyme. The alkaline phosphatase isozymes were partially purified from samples of chick intestine, liver, stage 38 embryonic limbs, and cultures of stage 24 limb mesenchymal cells. These tissues were separately extracted with butanol, acetone precipitated, redissolved, and passed over a DEAE-Sephacel ion-exchange column and ion-filtration column (Sephadex A-25). From the data obtained during this purification scheme, we conclude that the alkaline phosphatase from stage 38 limbs (bones) and Day 4 cultures are identical, and this activity is different from the enzyme purified from intestine and liver. The cell culture isozyme has an apparent Km, heat lability, response to specific inhibitors, electrophoretic mobility, and molecular weight similar to those of bone-specific alkaline phosphatase. These observations support the view that osteoblastic progenitor cells are present in the stage 24 limb mesenchyme and that under specific culture conditions, bone development can be uniquely observed in vitro. 相似文献
11.
An attempt has been made to show that the increase in enzyme activities in sera of pregnant women found with glucose-6-phosphate and adenosine 5'-monophosphate as substrates (described as glucose-6-phosphatase and 5'-nucleotidase) was due to the increase in alkaline phosphatase. The three enzyme activities has pH optima and heat stability characteristics of alkaline phosphatase. The response to the action of inhibitors and activators was typical for alkaline phosphatase. There was an identical increase in all three enzyme activities during pregnancy. As a control similar investigations were made with liver and placental tissue extracts. 相似文献
12.
Treatment of immature mice with gonadotrophins. Effects on some enzymic activities of unfractionated ovarian homogenates 总被引:2,自引:2,他引:0
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Thomas A. Bramley 《The Biochemical journal》1974,140(3):451-460
Treatment of immature mice with both follicle-stimulating hormone and human chorionic gonadotrophin in vivo resulted in large increases in the specific activities of ovarian alkaline phosphatase and alkaline nucleotidase. The specific activities of other ovarian enzymes studied were not altered by gonadotrophin treatment. A simultaneous change in the Michaelis constant of ovarian alkaline phosphatase accompanied the increase in specific activity. These changes commenced 6-8h after injection of human chorionic gonadotrophin plus follicle-stimulating hormone. Injection of human chorionic gonadotrophin induced the change in Michaelis constant and increased ovarian alkaline phosphatase activity. Treatment with follicle-stimulating hormone had no effect on ovarian alkaline phosphatase. However, follicle-stimulating hormone synergistically augmented the response to human chorionic gonadotrophin. A latent period of about 24h elapsed before this augmentation was expressed. Augmentation of ovarian alkaline phosphatase was directly related to the dose of follicle-stimulating hormone at a fixed dose of chorionic gonadotrophin. No response of ovarian alkaline phosphatase was observed after treatment of immature mice in vivo with oestrogens, progesterone, growth hormone or prolactin. Unlike chorionic gonadotrophin, sheep luteinizing hormone over a wide dose range induced no response within 24h. However, a response in ovarian alkaline phosphatase was observed when sheep luteinizing hormone was administered in combination with follicle-stimulating hormone. The specific activity and K(m) of ovarian alkaline phosphatase increased during normal maturation. The Michaelis constant ceased to increase as sexual maturity was reached. The changes in alkaline phosphatase activity were of a similar magnitude to those induced by gonadotrophin treatment. It is concluded that the changes induced acutely by treatment in vivo with unphysiological doses of gonadotrophins occur in the maturing mouse under the influence of endogenous, homologous gonadotrophins at physiological concentrations. 相似文献
13.
Outer membrane protein e of Escherichia coli K-12 is co-regulated with alkaline phosphatase. 总被引:41,自引:23,他引:18
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Outer membrane protein e is induced in wild-type cells, just like alkaline phosphatase and some other periplasmic proteins, by growth under phosphatase limitation. nmpA and nmpB mutants, which synthesize protein e constitutively, are shown also to produce the periplasmic enzyme alkaline phosphatase constitutively. Alternatively, individual phoS, phoT, and phoR mutants as well as pit pst double mutants, all of which are known to produce alkaline phosphatase constitutively, were found to be constitutive for protein e. Also, the periplasmic space of most nmpA mutants and of all nmpB mutants grown in excess phosphate was found to contain, in addition to alkaline phosphatase, at least two new proteins, a phenomenon known for individual phoT and phoR mutants as well as for pit pst double mutants. The other nmpA mutants as well as phoS mutants lacked one of these extra periplasmic proteins, namely the phosphate-binding protein. From these data and from the known positions of the mentioned genes on the chromosomal map, it is concluded that nmpB mutants are identical to phoR mutants. Moreover, some nmpA mutants were shown to be identical to phoS mutants, whereas other nmpA mutants are likely to contain mutations in one of the genes phoS, phoT, or pst. 相似文献
14.
Asparagine-linked carbohydrate does not determine the cellular location of yeast vacuolar nonspecific alkaline phosphatase 总被引:6,自引:0,他引:6
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The nonspecific alkaline phosphatase of Saccharomyces sp. strain 1710 has been shown by phosphatase cytochemistry to be exclusively located in the vacuole, para-Nitrophenyl phosphate-specific alkaline phosphatase is not detected by this procedure because the activity of this enzyme is sensitive to the fixative agent, glutaraldehyde. To determine whether the oligosaccharide of nonspecific alkaline phosphatase is necessary to transport the enzyme into the vacuole, protoplasts were derepressed in the absence or in the presence of tunicamycin, an antibiotic which interferes with the glycosylation of asparagine residues in proteins. The location of the enzyme in the tunicamycin-treated protoplasts, as determined by electron microscopy and subcellular fractionation, was identical to its location in control protoplasts. In addition, carbohydrate-free alkaline phosphatase was found in vacuoles from tunicamycin-treated protoplasts. Our findings indicate that the asparagine-linked carbohydrate moiety does not determine the cellular location of the enzyme. 相似文献
15.
Human liver alkaline phosphatase, purification and partial sequencing: homology with the placental isozyme 总被引:2,自引:0,他引:2
E Garattini J C Hua Y C Pan S Udenfriend 《Archives of biochemistry and biophysics》1986,245(2):331-337
Human liver alkaline phosphatase (AP) has been purified to homogeneity. The enzyme has a molecular weight of 150,000 in its native state and consists of two identical subunits of Mr 75,000. After treatment with endoglycosidase F the molecular weight is reduced to 50,000 indicating a high degree of glycosylation. The amino-terminal sequence up to 22 residues was found to be Leu-Val-Pro-Glu-Lys-Glu-Lys-Asp-Pro-Lys-Tyr-(Ala)-Arg-Asp-Gln-Ala-Gln-?- Thr-Leu-Lys-Tyr. The amino-terminal portions of human and bovine liver AP are identical. The amino termini of the human liver and human placental AP isozymes have appreciable homology. Conformationally the amino termini are very similar. 相似文献
16.
The distribution of reaction for acid and alkaline phosphatases in the proximal cartilage of the os penis and the mandibular condylar cartilage has been compared. The distribution of acid phosphatase in the two structures seems to be identical, whereas the distribution of alkaline phosphatase in the os penis cartilage seems to differ from that in the mandibular condylar cartilage and, by this, from all other studied growth cartilages. 相似文献
17.
Role of alkaline phosphatase in phosphate uptake into brush border membrane vesicles from human intestinal mucosa 总被引:1,自引:0,他引:1
In order to elucidate the physiological function of intestinal alkaline phosphatase, the characteristics of human intestinal alkaline phosphatase bound to brush border membrane vesicles were compared under optimal and physiological pHs. The Km value of this enzyme towards p-nitrophenylphosphate at the physiological pH was lower than that at the optimal pH. At the physiological pH, phosphate, arsenate and vanadate competitively inhibited the alkaline phosphatase activity, as they did at optimal pH, and the K1 values of these inhibitors at the physiological pH were also lower than those at the optimal pH. The effects of various inhibitors and antibody to human intestinal alkaline phosphatase on phosphate uptake into brush border membrane vesicles were investigated. The results indicated that phosphate uptake was affected by various inhibitors and the antibody to human intestinal alkaline phosphatase, but L-homoarginine, levamisole, and ouabain had no effect. From the above findings, it is strongly suggested that human intestinal alkaline phosphatase may function as a phosphate binding protein at low phosphate concentrations under physiological conditions. 相似文献
18.
Alkaline phosphatase activity in human placental cells transformed by a tsA mutant of simian virus 40 (SV40) can be greatly induced by growing these cells at 40 degrees C, the temperature at which the tsA transformants regain their nontransformed phenotype. The induction of alkaline phosphatase in these cells requires the synthesis of both RNA and protein. The induced alkaline phosphatase from a SV40 tsA30 mutant-transformed term placental cell line (TPA30-1) was purified, characterized, and compared with alkaline phosphatase from term placenta and first trimester placenta. The form of alkaline phosphatase found in TPA30-1 cells differs from the phosphatase of term placenta in physiochemical and immunological properties. The TPA30-1 phosphatase is, however, indistinguishable from the alkaline phosphatase of human first trimester placenta by several criteria, including electrophoretic mobility, apparent molecular weight (Mr = 165,000), size of monomeric subunit (Mr = 77,000), heat lability, and sensitivity to inhibition by amino acids and EDTA. In addition, alkaline phosphatase from both TPA30-1 cells and first trimester placenta can be inactivated by antiserum to liver alkaline phosphatase but not by antiserum to term placental alkaline phosphatase. The induction of first trimester phosphatase in cells derived from term placenta provides a system for the study of alkaline phosphatase gene regulation in human placenta. 相似文献
19.
S Unakami T Komoda M Watanabe Y Tanimoto Y Sakagishi H Ikezawa 《Comparative biochemistry and physiology. B, Comparative biochemistry》1987,88(1):111-118
1. Activities of alkaline phosphatase, liver-membranous, liver-soluble and serum-soluble, were dramatically induced in dogs by treatment with both phenobarbital and brovanexine. The treatment induced a 17-fold increase in membranous, a 155-fold increase in soluble, and a 105-fold increase in serum alkaline phosphatases. 2. There was no difference in the enzymatic behavior of the three forms of alkaline phosphatase, on heat stability, amino acid inhibition and optimum pH. 3. When the three alkaline phosphatases were treated initially with n-butanol, their apparent molecular size was identical. After treatment with phosphatidylinositol-specific phospholipase C, the liver-soluble and serum-soluble alkaline phosphatase were of the same molecular size. Liver-membranous alkaline phosphatase, however, was larger in molecular size than the other two forms, suggesting a difference between soluble and membranous alkaline phosphatase forms. 4. In terms of the sugar moiety of the three alkaline phosphatase forms, the membranous enzyme showed more of the higher affinity fraction and less of the lower affinity fraction of concanavalin A, compared with the soluble enzymes. 5. Consequently, it is possible that the membranous enzyme may be solubilized by an enzyme such as phosphatidylinositol-specific phospholipase C and modify further the sugar moiety of alkaline phosphatase molecules, resulting in serum alkaline phosphatase transfer from the soluble enzyme in liver. 相似文献
20.
Structural comparisons of two allelic variants of human placental alkaline phosphatase 总被引:2,自引:0,他引:2
A simple immunosorbent purification scheme based on monoclonal antibodies has been devised for human placental alkaline phosphatase. The two most common allelic variants, S and F, have similar amino acid compositions with identical N-terminal amino acid sequences through the first 13 residues. Both variants have identical lectin binding properties towards concanavalin A, lentil-lectin, wheat germ agglutinin, phytohemagglutinin and soybean agglutinin, and identical carbohydrate contents as revealed by methylation analysis. CNBr fragments of the variants demonstrate identical high performance liquid chromatography patterns. The carbohydrate containing fragment is different from the 32P-labeled active site fragment and the N-terminal fragment. 相似文献