Showing papers on "Alkaline phosphatase published in 1981"

Functional properties of hormonally responsive cultured normal and malignant rat osteoblastic cells.

Abstract: Certain metabolic properties of hormonally responsive osteogenic sarcoma cells derived from a transplantable rat tumor have been compared with those of related normal rat bone cells. All studies were carried out on cells grown in monolayer culture. Normal rat bone cells derived by repeated collagenase/trypsin digestion of newborn rat calvaria. Bone cells selected for comparison were thought to be osteoblast-like, as judged by enrichment of alkaline phosphatase and adenylate cyclase responsiveness to parathyroid hormone and prostaglandin E2. The adenylate cyclases of the two cell strains were similarly stimulated by a range of prostanoids and their metabolites and analogs. Morphology showed the two cell strains to be similar; the only obvious difference was a multilayering of cells in the sarcoma cultures, while the normal cultures showed abundant extracellular fibril formation which was not seen in the tumor cells. Investigation of the cAMP-dependent protein kinase isoenzymes showed the presence of two forms in both cell types, one eluting at a low salt concentration and the other at a high salt concentration. There was approximately twice the amount of the first isoenzyme compared to the second isoenzyme. The results indicate the usefulness of the two cell strains to elucidate further the molecular mechanisms of action of parathyroid hormone and prostaglandins.

Inhibition of endogenous tissue alkaline phosphatase with the use of alkaline phosphatase conjugates in immunohistochemistry

Abstract: In mammals there are two forms of alkaline phosphatase, one of which is widely distributed in a variety of tissues, and one of which is confined to intestine. Levamisole (1-tetramisole) inhibits the nonintestinal form of the enzyme, but is without effect on the intestinal form. We have exploited this difference by using conjugates made with calf intestinal alkaline phosphatase for immunohistochemical demonstration of H2 antigens in frozen section of mouse tissues. The alkaline phosphatase staining is performed in the presence of 1 mm levamisole, which inhibits the endogenous tissue enzyme without loss of staining by the conjugate. Endogenous enzyme can be inhibited by other means, such as exposure to 20% acetic acid, but labile antigens may be destroyed.

Amino acid sequence of Escherichia coli alkaline phosphatase.

Abstract: The complete amino acid sequence of the Escherichia coli alkaline phosphatase subunit [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1, isozyme 3] has been determined. The monomer contains 449 amino acid residues in a single unglycosylated polypeptide chain having a calculated Mr of 47,029. Isozyme 1 has an additional arginine residue at the NH2 terminus that presumably results from variability in processing of precursor molecules. Sequence data were obtained from both manual and automatic Edman degradation of the tryptic and cyanogen bromide peptides, as well as other peptides derived therefrom. The two disulfide bonds were determined from analyses of the appropriate peptic peptides. This structure confirms earlier reports of the sequence surrounding the active-site serine and both the NH2- and COOH-terminal cyanogen bromide fragments. A secondary structure prediction places nearly half the residues in alpha-helical segments that have 13% and 16%, respectively, in beta-strand and beta-turn orientations.

Genetic and biochemical characterization of periplasmic-leaky mutants of Escherichia coli K-12.

Abstract: Periplasmic-leaky mutants of Escherichia coli K-12 were isolated after nitrosoguanidine-induced mutagenesis. They released periplasmic enzymes into the extracellular medium. Excretion of alkaline phosphatase, which started immediately in the early exponential phase of growth, could reach up to 90% of the total enzyme production in the stationary phase. Leaky mutants were sensitive to ethylenediaminetetraacetic acid, cholic acid, and the antibiotics rifampin, chloramphenicol, mitomycin C, and ampicillin. Furthermore, they were resistant to colicin E1 and partially resistant to phage TuLa. Their genetic characterization showed that the lky mutations mapped between the suc and gal markers, near or in the tolPAB locus. A biochemical analysis of cell envelope components showed that periplasmic-leaky mutants contained reduced amounts of major outer membrane protein OmpF and increased amounts of a 16,000-dalton outer membrane protein.

Cloning and restriction mapping of the alkaline phosphatase structural gene (phoA) of Escherichia coli and generation of deletion mutants in vitro.

Abstract: The structural gene for alkaline phosphatase (phoA) of Escherichia coli was cloned into the PstI site of pBR322, from a transducing bacteriophage, lambda p(phoA-proC). The restriction map of the plasmid was established. Based upon this information, several phoA deletion plasmids as well as a smaller phoA+ plasmid were constructed. The genetic map and restriction map were correlated by recombination analysis. Cells carrying one of the phoA+ plasmids overproduce alkaline phosphatase 10-fold upon phosphate limitation. However, both regulation and processing of the enzyme were found to be normal.

Protozoan Parasite of Humans: Surface Membrane With Externally Disposed Acid Phosphatase

Abstract: Plasma membranes isolated from the protozoan parasite Leishmania donovani were enriched in acid phosphatase (E.C. 3.1.3.2) activity. Cytochemically, the enzyme was distributed uniformly on the surface of intact cells and was localized on the external face of isolated membranes. Physical characteristics and orientation of the membrane-bound enzyme suggest that the organism is adapted for existence in hydrolytic environments.

Improved method for quantitative determination in serum of alkaline phosphatase of skeletal origin.

Abstract: In this quantitative method for detection of skeletal alkaline phosphatase (EC 3.1.3.1) activity in human serum, intestinal and placental alkaline phosphatase activities are recognized by their susceptibility to inhibition by L-phenylalanine, and skeletal and hepatic alkaline phosphatases are distinguished by their different sensitivities to inactivation by heat. Alkaline phosphatase isoenzymes prepared from organ sources may behave differently from the corresponding isoenzymes in serum. Our procedure allows us to include organ-derived internal standards of skeletal, intestinal, and biliary alkaline phosphatase to minimize between-assay variation. In preliminary applications, we have found that (a) total serum alkaline phosphatase activity is extremely variable in post-menopausal osteoporotic subjects and is not a reliable index of skeletal alkaline phosphatase activity; (b) seven osteoporotic patients responding to therapy with sodium fluoride with increased bone formation showed increased skeletal alkaline phosphatase activity in their serum as compared with age-matched controls (p less than 0.005); and (c) 10 post-menopausal osteoporotic patients responding to therapy with stanozolol with increased total body calcium showed an increase in circulating skeletal alkaline phosphatase activity (p less than 0.001).

Effect of fat feeding on intestinal alkaline phosphatase activity in tissue and serum

Abstract: Serum intestinal alkaline phosphatase activity is increased by fat feeding, but the mechanism of this increase is not fully understood. Fasting rats were fed a single feed of either corn oil (12 kcal) or an isocaloric elemental feed (Vivonex 100 HN). Changes in enzyme activity in the small bowel mucosa and serum were followed for 20 h. Only the fat-fed rats had increased serum enzyme activity, being maximal at 7 h and three times the fasting level. This resulted from an increase in the amount of enzyme protein in the serum and not from an increase in its catalytic efficiency. The serum biological half-life of 125I-labeled intestinal alkaline phosphatase was the same in fasted (2.51 min) and fat-fed rats (2.55 min). Both types of feed caused a quantitatively similar increase in brush-border-bound alkaline phosphatase activity. However, levels of soluble intracellular alkaline phosphatase in intestinal mucosa were affected differently: the elemental diet caused a substantial rise, whereas no significant change was seen after fat feeding. The isoelectric pattern of phosphatase activity in serum after fat feeding was identical to that of soluble intracellular and not membranous alkaline phosphatase. Therefore, serum intestinal alkaline phosphatase activity rises in response to a single fat feed as a result of increased delivery of the enzyme to the blood and not as a result of an increase in its normally short biological half-life. This rise cannot be directly linked to an increase in the amount of brush-border-bound enzyme, and it appears that the serum enzyme is derived directly from a pool of soluble intracellular enzyme in the small bowel mucosa.

Patterns of vascular sprouting in the postnatal development of the cerebral cortex of the rat.

Abstract: Light microscopic histochemistry for alkaline phosphatase was employed in a study of the development of vascular sprouting, with respect to time and distribution, inthe rat cerebral cortex. Sprouts were counted in the full thickness of the cerebral cortex at each day from birth to 21 days of age. Several distinct bursts of sprouting activity were observed at specific times and levels of cortex. From birth to 4 days of age, sprouting was intense in the superficial third of the cortex. At 7 to 8 days, a burst of sprouting was found which was greatest in the middle third. Additional bursts of sprouting appeared at 10 and 14 days. Developing vessels with characteristics of arteries, capillaries, or sprouts were alkaline-phosphatase positive, while veins were not. It is concluded that alkaline phosphatase is a useful marker for identification of both mature and immature vasculature, as it reveals patent and nonpatent vessels, and the sprouts which are precursors of the mature vascular bed. New vessels developing in the cortex arise mainly from blind sprouts of capillaries, evidently in response to the metabolic demands imposed by the maturational process. At birth, the majority of intracortical vessels are capillaries. By 10 days of age, most perforating vessels from the surface have taken on arterial or venous characteristics. The findings are discussed in connection with morphological and biochemical differentiation and the pattern of vascularization in the mature cerebral cortex.

The use of potent inhibitors of alkaline phosphatase to investigate the role of the enzyme in intestinal transport of inorganic phosphate

Abstract: In an investigation of the link between Pi transport and alkaline phosphatase in mammalian small intestine, the characteristics of Pi uptake by brush-border membrane vesicles prepared from rat intestine were compared with the properties of the tissue alkaline phosphatase. The NaCl-dependent Pi uptake had a Km of 0.1 mM at pH 7.5 and was inhibited totally by 1 mM-arsenate and by 1 mM-vanadate. These compounds are also potent competitive inhibitors of the alkaline phosphatase activity of the vesicles, with Ki values less than 5 microM at pH 7.5. When the effect on Pi uptake of several other potent inhibitors of alkaline phosphatase, including phosphonates and phosphate analogues, was tested, however, it was found that there was little, if any, inhibition of transport under conditions in which the inhibition of phosphatase activity was total. Incubation of the vesicles for 20 min with oxidized adenosine 5'-[beta gamma-imido]triphosphate followed by rapid gel filtration to remove the inhibitor resulted in an irreversible loss of phosphatase activity, but left Pi transport unimpaired. Conversely, a similar prolonged incubation with adenosine 5'-[beta-thio]diphosphate or adenosine 5'-[gamma-thio]triphosphate had no effect on alkaline phosphatase activity but resulted in a permanent partial loss of transport capability. The failure to demonstrate an inhibition of Pi transport resulting from inhibition of alkaline phosphatase and the different responses of enzymic activity and Pi transport to irreversible inhibition make it very unlikely that the enzyme is directly involved in the transport system.

Effect of zinc supplementation on serum testosterone level in adult male sickle cell anemia subjects.

Abstract: Previously, we have documented primary testicular failure in adult male subjects with sickle cell anemia. We have also reported the occurrence of zinc deficiency and suggested that androgen deficiency may be related to zinc deficiency in such patients. In this study, we present data with respect to the efferent of oral zinc supplementation on serum testosterone levels in adult male patients with sickle cell anemia. An increase in serum testosterone, neutrophil zinc, and neutrophil alkaline phosphatase activity ws observed in the zinc-supplemented group in comparison with the group on placebo. Additionally, body weight increased and serum lactic dehydrogenase activity decrease in response to zinc supplementation. We conclude that androgen deficiency in adult male subjects with sickle cell anemia is correctable with zinc supplementation and that the determination of neutrophil zinc and alkaline phosphatase activity in the neutrophils may be utilized as good indicators of body zinc status in such subjects.

Properties of a series of tegumental membrane-bound phosphohydrolase activities of Schistosoma mansoni.

Abstract: 1. Incubation of Schistosoma mansoni for 5 min in a phosphate-buffered medium, pH 7.4, released tegumental material containing the following phosphohydrolase activities: alkaline phosphatase, 5'-nucleotidase, glycerol-2-phosphatase, glucose 6-phosphatase, phosphodiesterase and ATPase. 2. Maximum activity of these enzymes was measured at pH 9.5; however, the phosphodiesterase and ATPase activities were also appreciable at pH 7.0. 3. Solubilization of the released tegumental material in 1% Triton X-100 followed by gel filtration distinguished three peaks of enzyme activity: an ATPase (mol.wt. greater than 1000 000), a phosphodiesterase (mol.wt. 1 000 000) and an alkaline phosphomonoesterase with broad specificity (mol.wt. 232 000). 4. The ATPase activity was highly activated by 10 mM-Mg2+ or 1 mM-Ca2+ and was inhibited by chelating agents. Ouabain, Na+ and K+ had little effect on enzyme activity, whereas activity was increased by 50% in the presence of calmodulin. The phosphodiesterase activity was highest in the presence of 100 mM-Na+ or -K+, and 10 mM-Mg2+ or -Ca2+. Alkaline phosphatase activity was also stimulated by 100 mM-Na+ or -K+, and 10 mM-Mg2+; however Ca2+ inhibited at greater than 1 mM. 5. Surface iodination of parasites followed by detergent solubilization and gel filtration of the released tegumental membranes indicated that these enzymes were not accessible. A major surface component, apparent mol.wt. 80 000, was iodinated. 6. Rabbit anti-(mouse liver 5'-nucleotidase) antibodies did not inhibit the phosphohydrolase activities. However, an immunoglobulin G fraction from sera of mice chronically infected with S. mansoni partially inhibited alkaline phosphatase activity, but was without effect on the phosphodiesterase and ATPase activities. 7. The location of the enzymes in the double membrane of the tegument and their significance in host-parasite interactions is discussed.

Detection of enzyme polymorphism by using monoclonal antibodies

Abstract: Six monoclonal antibodies against human placental alkaline phosphate [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1], a highly polymorphic enzyme, were tested for reactivity against a panel of 295 placental extracts that had been typed electrophoretically. The products of the three common alleles as well as several rare alleles could be discriminated by the various antibodies. In some cases differences between allelic products were reflected by essentially "all-or-none" reactions, but in other cases the differences were smaller and demonstrable only by quantitative analysis of the binding. Evidence for allelic differences not detectable electrophoretically was also obtained.

Immunoperoxidase study of alkaline phosphatase in testicular tumor

Abstract: Indirect immunoperoxidase staining was carried out on human testicular tumors using monospecific antibodies against placental (Regan) and intestinal isoenzymes of alkaline phosphatase (ALPase). The very high incidence of seminoma (approximately 90%) revealed positive staining of placental ALPase mainly on the cell membrane of tumor cells, whereas none of the seminoma showed presence of intestinal isoenzyme. Placental isoenzyme was not recognized in any embryonal carcinoma and interstitial cell tumor. The epithelial cells of the glandular elements of teratoma occasionally exhibited strong staining for intestinal ALPase and weak staining for placental ALPase. The appearance of Regan isoenzyme in seminomas might be considered possible conversion of hepatic to placental isoenzyme, a consequence of malignant transformation of spermatogenic cells. Regan isoenzyme appears to be a new tumor marker for seminoma and the frequent identification of Regan isoenzyme in seminoma may disclose a unique biologic characteristic of this germinal tumor.

Colostral transfer of gamma glutamyl transpeptidase in calves.

Abstract: Serum gamma glutamyl transpeptidase (GGT) levels in blood samples taken from normal calves which bad suckled colostrum were much higher than those found in healthy adult cattle. Levels of over 60 times the normal adult level were observed. These high levels of GGT took approximately 5 weeks to decline to adult values. Calves which appeared to have not received or absorbed colostrum had GGT levels which would be considered normal in adult cattle. A calf with serum gamma globulin levels which indicated an intermediate amount of colostrum absorption had a level of GGT which was intermediate between that expected for normal adult cattle and that found in calves which had more fully absorbed colostrum. The mean GGT level observed in colostrum from 6 newly-calved cows was over 800 times the mean serum GGT level of the same 6 cows. It therefore appears most likely that GGT is concurrently absorbed with colostrum by calves and this gives rise to the very high levels seen in normal calves. Calves with very high levels of serum GGT also had raised levels of alkaline phosphatase (AP) which slowly declined but never reached normal adult levels within the 53-day period of observation. It therefore appears that high levels of both GGT and AP are achieved by calves at the time of colostrum absorption and it is concluded that clinical interpretation of serum GGT and AP levels in young calves is closely dependent upon parallel knowledge of their serum gamma globulin levels.

Enzyme activities in the dog: tissue analyses, plasma values, and intracellular distribution.

Abstract: Activities of 14 enzymes were determined in psoas muscle, smooth muscle, diaphragm, heart, brain, liver, kidney, spleen, pancreas, salivary glands, zygomatic gland, intestinal mucosa, subcellular fractions, and plasma of the dog. In pups, plasma activity of most enzymes was high, except iditol dehydrogenase (ID), glutamate dehydrogenase (GLD), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and D-fructose-1,6-diphosphate aldolase (ALS). Alkaline phosphatase (ALP), ALS, cholinesterase (CHS), creatine kinase (CK), alpha-hydroxybutyrate dehydrogenase (HBD), lactate dehydrogenase (LD), and malate dehydrogenase (MD) decreased significantly (P less than 0.01) with increasing age, but in dogs greater than 7 months, all enzymes except CK, HBD, and ALT revealed reasonably constant plasma values. Enzymes ALT, GLD, CHS, and ID are specific for liver, CK and ALS for muscle, HBD to some degree for myocardium, and alpha-amylase for pancreas. The ALP and gamma-glutamyltransferase were located in microsomes, GLD in mitochondria, MD and AST in mitochondria and cytoplasm, and isocitric dehydrogenase, LD, and the other enzymes only in cytoplasm.