Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this report we demonstrate how the recently developed biotinylated affinity label biotinyl-Phe-Ala-diazomethane (Bio-Phe-Ala-CHN2) [Cullen, McGinty, Walker, Nelson, Halliday, Bailie & Kay (1990) Biochem. Soc. Trans. 18, 315-316; Walker, Cullen, Kay, Halliday, McGinty & Nelson (1992) Biochem. J. 283, 449-453] can be used for the detection of a precursor form of a cathepsin B-like enzyme produced by breast-tumour cells in culture. Thus the cell lines MDA-MB-436, ZR-75-1 and T47-D produce a soluble protein that can be allowed to react with the biotinylated affinity label to yield an SDS-resistant complex; this can be revealed with a streptavidin/alkaline phosphatase label after PAGE and Western blotting. This protein (molecular mass 47 kDa) can also be detected by immunoblotting using sheep anti-(cathepsin B) antibodies in conjunction with a donkey anti-sheep IgG label. None of the cell lines studied produced any mature cathepsin B-like activity, as gauged by the lack of turnover of the fluorogenic substrate benzyloxycarbonyl-Arg-Arg-4-methylcoumarin-7-ylamide (Cbz-Arg-Arg-NH-Mec). However, treatment of medium samples with pepsin resulted in the generation of such activity. When the pepsin-catalysed activation step was analysed by SDS/PAGE, the protein of 47 kDa was completely converted into two species of very similar molecular masses of 30.5 kDa and 29 kDa. Both these proteins can incorporate the biotinylated probe and, in common with the 47 kD species, they can be detected with the streptavidin/alkaline phosphatase label and immunoblotting. We propose that the 47 kD form is the pepsin-activable proform of these lower-molecular-mass species. The release of the proform from the oestrogen-receptor (ER)-positive breast-tumour cell lines ZR-75-1 and T47-D is stimulated 5-10-fold when these cells are grown in medium containing epidermal growth factor (EGF) at a concentration of 10 ng/ml. In contrast, there is no modulation in the amount of proform released by the ER-negative cell line MDA-MB-436, over a range of EGF concentrations from 0 to 100 ng/ml.
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PMID:The application of a novel biotinylated affinity label for the detection of a cathepsin B-like precursor produced by breast-tumour cells in culture. 157 92

The toxic effect and anti-tumor activity of B-3839, a new molecular combination of pyrimidine antimetabolite 5-fluorouracil (5-FU) with the alkylating agent N-Chloroethyl-N-nitrosourea (BCNU), was compared to that of BCNU and 5-FU given alone and in physical combination. The tumor inhibitory effect of B-3839 was similar to that of BCNU given alone or combined with a low dose of 5-FU in the i.m. Walker tumor model. Furthermore, the bone marrow toxicity of BCNU was not significantly altered by either form of combination with 5-FU. The intestinal side effects, evaluated by measuring the decrease of marker enzyme (thymidine kinase, xanthine oxidase, alkaline phosphatase, sucrase, maltase) activities in isolated enterocytes, were dose-dependent and moderate. A significant, more than 30%, decrease occurred only if BCNU and 5-FU were given simultaneously or as B-3839. The molecular combination of the two drugs does not provide any additional advantage over their physical combination.
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PMID:Comparison of tumor growth inhibitory and toxic effects of a new fluorouracil--nitrosourea derivative (B-3839). 297 32

The effect of the growth of the Walker 256 carcinoma on the level of 5'-nucleotidase and alkaline phosphatase in the whole liver and in an isolated hepatocyte membrane preparation of its host was investigated. Alkaline phosphatase activities of whole liver and plasma membrane were increased approximately 5-fold by tumor growth. A 50% decrease in whole liver 5'-nucleotidase activity was observed in tumor-bearing rats while the 5'-nucleotidase activity per milligram membrane protein was unaltered. Tumor growth would therefore appear to affect a pool of 5'-nucleotidase which is not associated with the plasma membrane.
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PMID:Alterations in hepatic 5'-nucleotidase in the tumor-bearing rat. 627 89

A cell line from the Walker carcinosarcoma 256 of the rat has been established in suspension culture in medium with 5% bovine calf serum for over 350 generations, with an average population doubling time of 17 h, a plating efficiency of 56%, a colony forming efficiency of 32%, and a good capacity to form colonies in soft agar. The cells are morphologically indistinguishable from those in the solid tumor and ascites as checked by transmission and scanning electron microscopy. The karyotype is characterized by a modal number of 65 chromosomes and by the presence of a marker metacentric chromosome. The cells express thymidine kinase, gamma-glutamyl transpeptidase, and alkaline phosphatase; are agglutinable by concanavalin A; and can be synchronized by the triple thymidine block. They induce primary tumors, both subcutaneously (solid) and intraperitoneally (ascitic), in the rat; are able to metastasize upon injection by the tail vein; and invade the chorioallantoic membrane of the chick embryo. Cells in suspension can be transferred to monolayers, considerably decreasing their tumorigenicity without affecting the other parameters studied, and can be switched back to suspension culture. DNA-mediated transfection showed that DNA from these cells can transform the NIH-3T3 line. Upon growth of the monolayers in a BrdUr-containing medium, a sub-line was established that was cloned into a thymidine kinase-deficient line unable to grow in HAT medium and with properties otherwise similar to those of the parental wild type cells.
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PMID:Establishment and characterization of cell lines from the Walker carcinoma 256 able to grow in suspension culture and deficient in thymidine kinase. 646 74

A preliminary investigation of enzymes functioning at the cell surface indicates that when Walker 256 carcinoma cells growing ascitically are induced to grow as a solid tumour there is a marked increase in the activities of Na+, K(+)-ATPase, Mg(2+)-ATPase, 5'-nucleotidase, alkaline phosphatase and phosphodiesterase I; 5'-nucleotidase and alkaline phosphatase being particularly affected. These enzyme changes occur in the absence of any major alteration in the protein composition of the plasma membrane.
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PMID:Protein and enzyme content of plasma membranes derived from Walker 256 carcinoma cells grown as ascitic or solid tumours. 838 3

The objective of the present work has been to study some aspects of bone and intestinal compartments in rats with Walker 256 carcinosarcoma, an experimental model of humoral hypercalcemia of malignancy (HHM). The results have been compared to those obtained in control animals and, also, to those obtained in Yoshida sarcoma-bearing rats, which were used as tumoral controls without hypercalcemia. Urinary hydroxyproline/creatinine ratio (OHProl/creat) is increased, in both Walker 256 and Yoshida tumor-bearing animals, showing the nonspecifity of this bone marker. However, serum tartrate-resistant acid phosphatase (TRAP) levels are increased in Walker 256 tumor-bearing animals, but they are normal in Yoshida tumor-bearing animals, indicating that TRAP is a better index of bone resorption than OHProl/creat in the HHM syndrome. The decrease of bone calcium content in Walker 256 tumor-bearing rats, not shown by Yoshida-bearing rats, also reflects an increased bone resorption due to HHM. Serum and bone osteocalcin levels are similar in control, Walker 256 and Yoshida tumor-bearing rats, but we observed a decrease in serum alkaline phosphatase levels in Walker 256 and Yoshida tumor-bearing animals, which could also be a nonspecific tumor effect, due to the presence of the neoplasia. Our results support the convenience of the employment of a nonhypercalcemic tumor group as control in the HHM study, in addition to the healthy controls. We have also observed higher 1,25-dihydroxyvitamin D serum levels in Walker 256 tumor-bearing rats than in control and Yoshida tumor-bearing rats. On the other hand, we have found normal levels in the fractional rate of intestinal calcium absorption in Walker-256 tumor-bearing rats, in spite of their high calcium levels, and a significant decrease of this parameter in Yoshida sarcoma-bearing animals. These results support the concomitant contribution of intestinal compartment to hypercalcemia, in the experimental model of HHM studied.
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PMID:Osseous and intestinal compartments in the humoral hypercalcemia of malignancy associated to Walker 256 tumor in rats. 845 Oct 38

The 60-kDa Ca2+-ATPase from Flavobacterium odoratum is kinetically and mechanistically similar to other P-type ATPases, suggesting its use as a model system for structure-function studies of ion transport. A portion of the gene was amplified by polymerase chain reaction of genomic DNA with degenerate oligonucleotide primers, one based on the N-terminal amino acid sequence of the purified protein and the other based on a consensus sequence for the phosphorylation site of P-type ATPases. This gene fragment was used to screen a lambda library of F. odoratum 29979 DNA. Clone "C" is 3.3 kilobases in length and contains one complete and part of a second open reading frame, the first of which encodes a 58-kDa protein containing the exact N-terminal amino acid sequence of the purified protein. We have named this gene cda, for calcium-dependent ATPase. Escherichia coli, transformed with clone C, demonstrates high levels of calcium-dependent and vanadate-sensitive ATP hydrolysis activity, forms a 60-kDa phosphointermediate, and cross-reacts with antibodies to the purified Ca2+-ATPase. The gene has almost no sequence homology to even the highly conserved regions characteristic of P-type ATPases but does possess significant homology to a protein with alkaline phosphatase activity (PhoD) from Zymomonas mobilis. The putative phosphorylation site is a Walker A (P-loop) ATP binding sequence and is modified relative to P-type ATPases, suggesting that the F. odoratum Ca2+-ATPase may represent an ancestral link between the F- and the P-type ATPases or perhaps a new class of ATPases.
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PMID:Cloning and expression of the unique Ca2+-ATPase from Flavobacterium odoratum. 861 88

Advanced tumor osteopathy is characterized by abnormal bone turnover. Using a rat model of parathyroid hormone-related peptide (PTHrP)-mediated tumor osteolysis, the aim of the present study was to define the sequential changes in, and the association between, biochemical and histomorphometric indices of bone metabolism during the early stages of developing tumor osteopathy. Eight-month-old Wistar rats (n = 48) were subcutaneously inoculated with either 2 x 10(6) cells of the Walker carcinosarcoma 256, or saline on day 0, and treated with either saline or the bisphosphonate ibandronate until killing on day 8. Serum calcium (sCa), alkaline phosphatase (sTAP), and osteocalcin (sOC) and urinary calcium (uCa), deoxypyridinoline (uDPD), and pyridinoline (uPYD) were measured daily. In a second semilongitudinal experiment (n = 70), the number of osteoclasts and osteoblasts (N.Oc, N.Ob), trabecular bone volume (BV/TV), and osteoid volume (O.Ar) were assessed by histomorphometry. In untreated tumor-bearing animals, osteoclast numbers increased by 74% on day 3 (5.4 +/- 2.4 vs. 3.1 +/- 1.5/mm(2), p < 0.05), and trabecular bone volume fell by 24% on day 4 (12.5 +/- 2.0 vs. 15.8 +/- 1.2%, p < 0.05). Both time course and magnitude of these changes were closely reflected by an increase in uDPD (0.46 +/- 0.14 vs. 0. 31 +/- 0.15 nmol/12 h, p < 0.05) and uPYD on day 4 (1.44 +/- 0.25 vs. 1.03 +/- 0.3 nmol/12 h, p < 0.05), sCa (3.8 +/- 0.52 vs. 3.0 +/- 0. 13 mmol/L, p < 0.01), and uCa (0.13 +/- 0.08 vs. 0.03 +/- 0.01 mmol/12 h, p < 0.001) on day 6, and sTAP (254 +/- 127 vs. 120 +/- 40 U/L, p < 0.001) on day 7 (mean +/- SD), whereas sOC remained unchanged until day 8. When combining the results of the two experiments, a high correlation was found between the number of osteoclasts and the urinary excretion of PYD (r = 0.91) and DPD (r = 0.89). Treatment with ibandronate delayed hypercalcemia, abolished hypercalciuria, and accelerated bone resorption. We conclude that osteoclast activation is an early event in PTHrP-mediated osteolysis, which is closely reflected by the renal excretion of pyridinium cross-links of type I collagen. Therefore, specific biochemical markers of collagen breakdown may be useful as early indicators of developing tumor osteopathy.
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PMID:Association between histomorphometry and biochemical markers of bone turnover in a longitudinal rat model of parathyroid hormone-related peptide (PTHrP)-mediated tumor osteolysis. 1077 87

Cytochrome bd is a respiratory quinol oxidase in Escherichia coli. Besides the structural genes (cydA and cydB) encoding the oxidase complex, the cydD and cydC genes, encoding an ABC-type transporter, are required for assembly of this oxidase. Recently, cysteine has been identified as a substrate (allocrite) that is transported from the cytoplasm by CydDC, but the mechanism of cysteine export to the periplasm and its role there remain unknown. To initiate an understanding of structure-function relationships in CydDC, its membrane topography was analysed by generating protein fusions between random and selected residues in the two polypeptides with both alkaline phosphatase and beta-galactosidase. CydD and CydC are experimentally shown each to have six transmembrane segments, two major cytoplasmic loops and three minor periplasmic loops; both termini of each protein face the cytoplasm. The cydD1 allele is shown to have two point mutations (G319D, G429E) within the ATP-binding domain of CydD; either mutation alone is sufficient to cause loss or severe reduction of cytochrome bd assembly. A comparative sequence analysis prompted the targeting of residues in CydD for site-directed mutational analysis, which identified (i) the 'start' methionine residue, (ii) essential residues in the ATP-binding site (Walker sequence A) and (iii) a duplicated positively charged heptameric motif, R-G/T-L/M-X-T/V-L-R, in CydD cytoplasmic loop II. The replacement of arginines in these motifs with glycines resulted in Cyd- phenotypes; however, activity could be restored at these positions by replacing the glycine with lysine or histidine and hence returning the positive charge. The conservation of these charges in CydD-like proteins indicates functional importance. Evolutionary aspects of bacterial cyd genes are discussed.
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PMID:Membrane topology and mutational analysis of Escherichia coli CydDC, an ABC-type cysteine exporter required for cytochrome assembly. 1547 Jan 19

Leucine can modulate skeletal muscle metabolism by enhancing protein synthesis and decreasing proteolysis. In this study, we investigated the effects of leucine on the ubiquitin-proteasome system in skeletal muscle of pregnant tumour-bearing rats fed a leucine-rich diet. Pregnant Wistar rats were distributed into three groups that were fed a semi-purified control diet (C, control; W, Walker tumour-bearing; P, pair-fed) and three other groups of pregnant rats fed a semi-purified leucine-rich diet (L, leucine; WL, Walker tumour-bearing; PL, pair-fed). The tumour-bearing rats were injected subcutaneously with a suspension of Walker 256 tumour cells. Protein synthesis and degradation were measured in gastrocnemius muscle; the total protein content and tissue chymotrypsin-like and alkaline phosphatase enzyme activities were also determined. Muscle protein extracts were run on SDS-PAGE to assess the expression of the myosin heavy chain (MHC), 20S alpha proteasome subunit, 19S MSSI ATPase regulator subunit and 11S alpha subunit. Although tumour growth decreased the incorporation of [3H]-Phe, the concomitant feeding of a leucine-rich diet increased the rate of protein synthesis. Muscle proteolysis in both tumour-bearing groups was increased more than in the respective control groups. Conversely, the leucine-rich diet caused less protein breakdown in the WL group than in the W group. Only the W group showed a significant reduction (71%) in the myosin content. In WL rats, the 20S proteasome content (32 kDa band) was reduced, while the expression of the 19S subunit was 3-fold less than in the W group and the 11S proteasome subunit reduced, to around 32% less than in the W group. These findings clearly indicate that leucine can stimulate protein synthesis and inhibit protein breakdown in pregnant rats, probably by modulating the activation of the ubiquitin-proteasome system during tumour growth.
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PMID:Proteasome activity is altered in skeletal muscle tissue of tumour-bearing rats a leucine-rich diet. 1561 61


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