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R A Stinson

Publications and source records attributed to R A Stinson.

15 recordsLinked to original sources
PubMed1991

High-molecular-weight alkaline phosphatase in serum has properties similar to the enzyme in plasma membranes of the liver.

Partially purified high-molecular-weight alkaline phosphatase from serum was compared with two other forms of the enzyme from the human liver, enzyme in native plasma membranes and purified alkaline phosphatase as a hydrophilic dimer. In a high-molecular-weight form from serum and plasma membranes, and when treated with 1% (v/v) Triton X-100, alkaline phosphatase showed a major band on gradient gel electrophoresis with a mobility equivalent to 400 kD. Nondetergent-treated material from both sources did not enter the gel and was in the voided volume of a gel permeation column. Stimulation of catalytic activity by four different phospholipids and by albumin yielded similar results for high-molecular-weight alkaline phosphatase and for the enzyme in plasma membranes, but these were different from the hydrophilic form. Inhibitors of alkaline phosphatase had similar effects on all forms. Of the three forms of the enzyme, only the hydrophilic dimer did not become incorporated into liposomes or adsorb to octyl-Sepharose after solubilization with Triton X-100 and removal of the detergent. Km (substrate concentration to give half maximal velocity) values with p-nitrophenylphosphate and heat and sodium dodecyl sulfate stabilities were similar for all forms. In the high-molecular-weight form from serum and in plasma membranes, alkaline phosphatase and 5'-nucleotidase showed similar rates of release by phosphatidylinositol phospholipase C. Three preparations of phospholipase D failed to release alkaline phosphatase from either the high-molecular-weight form or from plasma membranes. Based on these similarities, it is probable that the complex of high-molecular-weight alkaline phosphatase in serum most often originates from fragments of hepatic plasma membranes.

Alkaline Phosphatase↗
PubMed1991

Properties of amphiphilic and hydrophilic forms of alkaline phosphatase from human liver.

Amphiphilic and hydrophilic forms of alkaline phosphatase differed in electrophoretic mobility, sensitivity to heat, activation by phospholipids and albumin, and affinity of monoclonal antibodies, but were similar in substrate Km and inhibitor Ki values, sensitivity to sodium dodecyl sulfate, and electrophoretic behavior on desialylation. Chemical cross-linking experiments failed to conclusively demonstrate an aggregated state of amphiphilic alkaline phosphatase in Triton X-100. Further, attempts to identify a polymeric hybrid between amphiphilic forms of human liver and placental alkaline phosphatase were unsuccessful. We conclude that the covalent attachment of the hydrophobic phosphatidyl-inositol membrane anchor causes the amphiphilic form to behave anomalously on electrophoresis and to affect certain of the enzyme's catalytic and physical properties.

Alkaline Phosphatase↗
PubMed1990

Incorporation of human liver and placental alkaline phosphatases into liposomes and membranes is via phosphatidylinositol.

As assessed by incorporation into liposomes and by adsorption to octyl-Sepharose, the integrity of the membrane anchor for the purified tetrameric forms of alkaline phosphatase from human liver and placenta was intact. Any treatment that resulted in a dimeric enzyme precluded incorporation and adsorption. An intact anchor also allowed incorporation into red cell ghosts. The addition of hydrophobic proteins inhibited incorporation into liposomes to varying degrees. Alkaline phosphatase was 100% releasable from liposomes and red cell ghosts by a phospholipase C specific for phosphatidylinositol. There was no appreciable difference in the rates of release of placental and liver alkaline phosphatases, although both were approximately 250 x slower in liposomes and 100 x slower in red cell ghosts than the enzyme's release from a suspension of cultured osteosarcoma cells. Both enzymes were released by phosphatidylinositol phospholipase C as dimers and would not reincorporate or adsorb to octyl-Sepharose. However, the enzyme incorporated, resolubilized by Triton X-100, and cleansed of the detergent by butanol treatment was tetrameric by gradient gel electrophoresis, was hydrophobic, and could reincorporate into fresh liposomes. A monoclonal antibody to liver alkaline phosphatase inhibited the enzyme's incorporation into liposomes, and abolished its release from liposomes and its conversion to dimers by phosphatidylinositol phospholipase C.

Alkaline Phosphatase↗
PubMed1979

Evidence that three structural genes code for human alkaline phosphatases.

The number of structural gene loci that code for the different molecular forms of human alkaline phosphatase is unknown. Physical properties of the enzymes, immunological data, chemical inhibition and genetic studies suggest that at least three structural genes are involved: one coding for alkaline phosphatase from placenta, another for the enzyme from intestine, and one or more for the enzymes from liver, kidney and bone. Badger and Sussman have shown that alkaline phosphatases from human liver and placenta are products of different structural genes, and Greene and Sussman have shown that alkaline phosphatase from a metastasised bronchogenic carcinoma was nearly identical to the enzyme from placenta. However, other tumour-associated alkaline phosphatases and the enzymes from normal tissue other than placenta and liver have not been identified by conclusive structural criteria, and thus it is not known whether these onco-alkaline phosphatases represent ectopic production or unusual post-translational modification of the enzymes found in normal tissues. We present here, using a sensitive peptide-mapping technique, structural evidence that the enzyme forms from liver, kidney and serum from a patient with Paget's disease of bone (osteitis deformans) are products of the same structural gene and can be easily distinguished from either the intestinal or placental isoenzymes. The technqiue seems to be useful for the classification of tumour-associated alkaline phosphatases on a structural basis.

Alkaline Phosphatase↗
PubMed1979

Inhibition of human alkaline phosphatases by vanadate.

Orthovanadate was shown to be a potent competitive inhibitor (Ki less than 1 microM) of purified alkaline phosphatase from human liver, intestine of kidney. Inhibition was reversed and full enzymic activity restored in the presence of 1mM-adrenaline. Phosphate and vanadate competed for the same binding site on the enzyme.

Alkaline Phosphatase↗
PubMed1979

Affinity elution from a phosphonic acid-Sepharose derivative in the purification of human liver alkaline phosphatase.

The compound p-aminobenzylphosphonic acid has been coupled via an azo linkage to tyraminyl-Sepharose 4B. This derivative at pH 6.0 bound most of the protein and all of the alkaline phosphatase in a crude preparation from human liver. The phosphatase was selectively eluted with the substrate 2-naphthylphosphate and a purification of 400-fold obtained. This step, when incorporated into a procedure for the purification of human liver alkaline phosphatase, yielded essentially pure enzyme.

Alkaline Phosphatase↗
PubMed1979

Phosphoester specificity of purified human liver alkaline phosphatase.

Kinetic parameters for the hydrolysis of a number of physiologically important phosphoesters by purified human liver alkaline phosphatase have been determined. The enzyme was studied at pH values of 7.0 to 10.0. The affinity of the enzyme for the compounds was determined by competition experiments and by their direct employment as substrates. Phosphodiesters and phosphonates were not hydrolysed but the latter were inhibitors. Calcium and magnesium ions inhibited the hydrolysis of ATP and PP1 and evidence is presented to show that the metal complexes of these substrates are not hydrolysed by alkaline phosphatase. A calcium-stimulated ATPase activity could not be demonstrated for the purified enzyme or the enzyme in the presence of a calcium-dependent regulator protein. Nevertheless, the influence of magnesium and calcium ions on the ATPase activity of alkaline phosphatase means that precautions must be taken when assaying for Ca2+-ATPase in the presence of alkaline phosphatase. The low substrate Km values and the hydrolysis which occurs at pH 7.4 mean that the enzyme could have a significant phosphohydrolytic role. However, liver cell phosphate concentrations, if accessible to the enzyme, are sufficient to strongly inhibit this activity.

Adenosine Triphosphate↗
PubMed1978

Anion binding to yeast phosphoglycerate kinase.

The single thiol of yeast phosphoglycerate kinase was labelled with the chromophoric sulfhydryl reagent, 2-chloromercuri-4-nitrophenol. Sequential additions of individual anions to this modified enzyme brought about a decrease in absorbance at 410 nm that reflected the degree of saturation of the enzyme with anion. The binding curves were analyzed to determine the dissociation constants of a number of anions with charges varying from--1 to--4.1. A linear relationship was found between the charge of the anion and the negative logarithm of the dissociation constant for the labelled enzyme-anion complex. The highly charged anions, such as ATP, bound more tightly than did anions with less charge, such as Cl-. The average number of binding sites for those anions for which accurate results could be obtained was 1.06 mol per 47000 g of enzyme. Several lines of evidence suggested that titration of the active center was not being monitored. Anions bound to phosphoglycerate kinase decreased the rate of reaction between the enzyme thiol and 5,5'-dithiobis(2-nitrobenzoic acid). The relationship between the degree of saturation of the anion binding site and the reaction rate constant was used to calculate the dissociation constant between anion and enzyme. Dissociation constants determined in this manner were in good agreement with those determined by titration of the enzyme-mercurial complex.

Anions↗
PubMed1977

Use of isoelectric focusing and a chromophoric organomercurial to monitor urea-induced conformational changes of yeast phosphoglycerate kinase.

The effects of urea in concentrations from 0 to 6M on the following properties of yeast phosphoglycerate kinase were studied: the kinetics of inactivation of the enzyme, the spectrum of 2-chloromercuri-4-nitrophenol bound to the single thiol group of the enzyme, the rate of reaction between the mercurial and enzyme, and the isoelectric point. The enzyme was inactivated by as much as 30% in 1M-urea, and the other data were interpreted as a possible 'tightening' of enzyme structure. The catalytic behaviour of the enzyme in 2M-urea was time-dependent, the initial effects being similar to those in 1M-urea. Polyacrylamide-gel isoelectric focusing of the enzyme in the presence of 2M-urea showed a single species of enzyme with an isoelectric point intermediate between those in 1M- and 3M-urea; a species with an identical isoelectric point was obtained after an 11-day exposure at 4 degrees C to the denaturant at 2M. The enzyme was rapidly inactivated in 3M-urea, with the thiol group fully exposed and the isoelectric point 0.9pH unit higher than in the absence of urea. No further conformational changes could be demonstrated with urea concentrations of 4M or greater. It is suggested that the equilibrium species that exists in 2M-urea has one of two buried lysine residues exposed. The second lysine residue is exposed in 3M or greater concentrations of the denaturant.

Chloromercurinitrophenols↗
PubMed1976

Affinity purification and some molecular properties of human liver alkaline phosphatase.

Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The isoelectric point of the protein was determined to be 4.0. Sephadex-gel filtration gave a mol.wt. of 146000, although a higher value was obtained in the presence of 100mM-NaC1. The subunit mol.wt. 76700, was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Neuraminidase treatment resulted in two enzyme-activity bands on isoelectric-focused gels with isoelectric points of 6.6 and 6.8. The desialylated enzyme gave only one protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a subunit molecular weight indistinguishable from that of the non-neuraminidase-treated protein. The desialylated enzyme was more readily denatured by sodium dodecyl sulphate in the presence of mercaptoethanol than was the native enzyme.

Alkaline Phosphatase↗