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H R Horton

Publications and source records attributed to H R Horton.

At least 19 recordsLinked to original sources
PubMed1992

Anti-IgG immobilized controlled-pore glass. Thionyl chloride-activated succinamidopropyl-glass as a covalent immobilization matrix.

Rabbit anti-bovine IgG was covalently immobilized on thionyl chloride-activated succinamidopropyl controlled-pore glass (CPG) beads (3000 A pore diam; 120/200 mesh). Thionyl chloride-activated beads remained stable for over 1 y retaining full capability of immobilizing protein upon recirculation of protein solution. The immobilized anti-bovine IgG is capable of binding IgG with a dissociation constant (Kd) of 9.45 x 10(-7) M and a capacity of 0.85 g/L of matrix. A column of immobilized anti-IgG is able to remove all detectable contaminating IgG from partially purified enzyme preparations of sulfhydryl oxidase and gamma-glutamyltransferase as determined by ELISA and Western blot. The column matrix could be regenerated by washing with 0.1M acetic acid, pH 2.8.

Animals↗
PubMed1988

Purification and properties of sulfhydryl oxidase from bovine pancreas.

Immunofluorescent studies showed that antibodies prepared against bovine milk sulfhydryl oxidase reacted with acinar cells of porcine and bovine pancreas. A close inspection of the specific location within bovine pancreatic cells revealed that the zymogen granules, themselves, bound the fluorescent antibody. Bovine pancreatic tissue was homogenized in 0.3 M sucrose, then separated into the zymogen granule fraction by differential centrifugation. The intact zymogen granules were immunofluorescent positive when incubated with antibodies to bovine milk sulfhydryl oxidase, and glutathione-oxidizing activity was detected under standard assay conditions. Pancreatic sulfhydryl oxidase was purified from the zymogen fraction by precipitation with 50% saturated ammonium sulfate, followed by Sepharose CL-6B column chromatography. Active fractions were pooled and subjected to covalent affinity chromatography on cysteinylsuccinamidopropyl-glass using 2 mM glutathione as eluant at 37 degrees C. The specific activity of bovine pancreatic sulfhydryl oxidase thus isolated was 10-20 units/mg protein using 0.8 mM glutathione as substrate. Ouchterlony double-diffusion studies showed that antibody directed against the purified bovine milk enzyme reacted identically with pancreatic sulfhydryl oxidase. The antibody also immunoprecipitated glutathione-oxidizing activity from crude pancreatic homogenates. Western blotting analysis indicated a 90,000 Mr antigen-reactive band in both bovine milk and pancreatic fractions while sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single silver-staining protein with an apparent Mr 300,000. Thus, we believe that sulfhydryl oxidase may exist in an aggregated molecular form. Bovine pancreatic sulfhydryl oxidase catalyzes the oxidation of low-molecular-weight thiols such as glutathione, N-acetyl-L-cysteine, and glycylglycyl-L-cysteine, as well as that of a high-molecular-weight protein substrate, reductively denatured pancreatic ribonuclease A.

Animals↗
PubMed1987

Enzymatic enhancement of the catalytic rate of sulfhydryl oxidase.

The rate of oxidation of glutathione by solubilized sulfhydryl oxidase was significantly enhanced in the presence of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). This enhancement was proportional to the amount of active peroxidase in the assay, but could not be attributed solely to the oxidation of glutathione catalyzed by the peroxidase. A change in the Soret region of the horseradish peroxidase spectrum was observed when both glutathione and peroxidase were present. Moreover, addition of glutathione to a sulfhydryl oxidase/horseradish peroxidase mixture resulted in a rapid shift of the absorbance maximum from 403 nm to 417 nm. This shift indicates the oxidation of horseradish peroxidase. Spectra for three isozyme preparations of horseradish peroxidase, two acidic and one basic, all underwent this red-shift in the presence of sulfhydryl oxidase and glutathione. Cysteine and N-acetylcysteine could replace glutathione. Addition of catalase had no effect on the oxidation of peroxidase, indicating that the peroxide involved in the reaction was not derived from that released into the bulk solution by sulfhydryl oxidase-catalyzed thiol oxidation. Further evidence for a direct transfer of the hydrogen peroxide moiety was obtained by addition of glutaraldehyde to a sulfhydryl oxidase/horseradish peroxidase/N-acetylcysteine mixture. Size exclusion chromatography revealed the formation of a high-molecular-weight species with peroxidase activity, which was completely resolved from native horseradish peroxidase. Formation of this species was absolutely dependent on the presence of both the cysteine-containing substrate and sulfhydryl oxidase. The observed enhancement of sulfhydryl oxidase catalytic activity by the addition of horseradish peroxidase supports a bi uni ping-pong mechanism proposed previously for sulfhydryl oxidase.

Acetylcysteine↗
PubMed1986

Requirement for a sulfhydryl group for sulfhydryl oxidase activity.

Sulfhydryl oxidase was isolated from bovine skim milk membranes using a transient covalent affinity chromatographic method. This preparation exhibited two chemically reactive sulfhydryl groups in the native enzyme and three in the denatured form, based on a subunit weight of 85 kdaltons. The kinetics of inactivation by carboxymethylation with iodoacetate indicated that modification of one sulfhydryl group per enzyme subunit caused complete loss of activity. These results, together with the enzyme's attachment to cysteinylsuccinamidopropyl-glass and the observed initial rate enzyme kinetics, strongly implicate a substituted-enzyme kinetic mechanism with a mixed disulfide as the intermediate enzyme form.

Animals↗
PubMed1985

Some kinetic characteristics of immobilized protomers and native dimers of mitochondrial malate dehydrogenase: an examination of the enzyme mechanism.

Some kinetic characteristics of immobilized native mitochondrial malate dehydrogenase dimers and immobilized protomers, prepared by direct immobilization under conditions yielding complete dissociation without substantial unfolding, were compared to those of native soluble enzyme. Enzyme was covalently immobilized to derivatized porous glass by using a technique which permitted subsequent release of bound enzyme with 0.2 M hydroxylamine at room temperature and pH 7. Kinetic properties of enzyme released from both immobilized dimers and protomers were the same as those for native soluble enzyme, indicating that the immobilization reaction per se did not affect the structure. Both immobilized native dimers and the immobilized protomers exhibited activity with a pH dependence similar to that of native soluble enzyme. The effects of diffusional inhibition were demonstrated for both forms of the immobilized enzyme, especially for the NADH----NAD+ reaction direction. Intrinsic Michaelis constants of both immobilized forms, obtained by extrapolation of apparent values, were similar to those of the soluble enzyme. Furthermore, the effects of inhibitors and effectors with the immobilized forms were the same as those with native soluble enzyme. For example, substrate inhibition was observed with oxalacetate, the inhibitor hydroxymalonate was competitive with ketomalonate and uncompetitive with L-malate, and inhibition was observed with citrate in the NADH----NAD+ direction. Thus, immobilization did not appear to suppress the conformational equilibria of either protomers or dimers. More significantly, the kinetic characteristics of the immobilized protomer were indistinguishable from those of the dimer. Hence, a reciprocating mechanism involving subunit interactions cannot be invoked to explain the allosteric behavior of this dimeric enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Glycylglycyl-L-cysteine as a substrate for renal sulfhydryl oxidase (glutathione oxidase).

Covalent chromatographically isolated bovine kidney sulfhydryl oxidase was found to catalyze the oxidation of cysteine and cysteine-containing substrates as determined by assaying with 5,5'-dithiobis(2-nitrobenzoate). Monitoring the time-course of substrate disappearance and product formation by means of high-pressure liquid chromatography revealed that such partially purified renal sulfhydryl oxidase preparations catalyze the direct oxidation of glycylglycyl-L-cysteine to its disulfide form with no other detectable metabolic products. Accordingly, Gly-Gly-Cys appears to be better suited for routine assays of sulfhydryl oxidase activity than is the traditionally employed substrate, glutathione, whose oxidation can be initiated by gamma-glutamyltransferase-catalyzed cleavage of the gamma-peptide bond, leading to falsely 'positive' assays in the absence of sulfhydryl oxidase per se.

Animals↗
PubMed1985

Renaturation of soluble and immobilized ribonuclease: are the polypeptide folding pathways for structure formation the same for soluble proteins and for proteins associated with a surface?

During the refolding and oxidation of reductively denatured ribonuclease A in solution, there is a marked lag in appearance of enzymatic activity as compared to the oxidation of sulfhydryl groups, whether such oxidation is spontaneous or is catalyzed by sulfhydryl oxidase. However, if ribonuclease is covalently attached to a derivatized glass surface, a lag period is not observed during the reformation of native structure from the completely reduced, denatured state. These results suggest that, in solution, intermolecular interactions alter the pathway of polypeptide chain folding and disulfide bond formation, leading to nonnative disulfides which do not rapidly interchange to form native pairings. The isolation of refolding polypeptide chains by covalent immobilization prevents such interactions. Presumably, such intermolecular interactions would be similarly prevented by "isolation" of nascent polypeptide chains during protein synthesis on ribosomes.

Animals↗
PubMed1984

Kinetic mechanism and specificity of bovine milk sulphydryl oxidase.

Sulphydryl oxidase is known to catalyse the synthesis de novo of disulphide bonds in a variety of thiol-containing compounds. Reduced glutathione is the best thiol substrate; however, D- and L-cysteine, cysteamine and N-acetyl-L-cysteine, as well as cysteine-containing peptides and proteins, are also effectively oxidized. In contrast, oxidation of the thiol groups of mercaptoethanol, mercaptopyridine, dithiothreitol, dithioerythritol, mercaptoacetate, mercaptopropionate or lipoic acid is not detectably catalysed. In bovine milk, sulphydryl oxidase is closely associated with another glutathione-metabolizing enzyme, gamma-glutamyltransferase. Covalent chromatography of crude preparations on cysteinylsuccinamidopropyl-glass resolves the oxidase from the transferase, thus permitting the kinetic characterization of glutathione oxidation. Initial-rate data imply a Ter Bi substituted-enzyme mechanism, and the observed substrate inhibition by thiols suggest that O2 binds first. Independent, non-kinetic, data, namely the immobilization of sulphydryl oxidase on cysteinyl-matrices, support formation of a mixed-disulphide intermediate between the thiol and enzyme, as predicted by the proposed mechanism. The enzyme-catalysed reaction appears not to be mediated via a superoxide intermediate, since O2 consumption is not affected by the presence of Nitro Blue Tetrazolium. FAD, NAD+, NADP+ and Nitro Blue Tetrazolium are all inactive as electron acceptors for sulphydryl oxidase catalysis.

Acetylcysteine↗
PubMed1984

Immunological similarity of milk sulfhydryl oxidase and kidney glutathione oxidase.

A radioimmunoassay for sulfhydryl oxidase, a membrane enzyme, was developed using antibodies raised to the bovine milk enzyme which had been purified by transient covalent affinity chromatography on a cysteinylsuccinamidopropyl-glass matrix. Bovine milk sulfhydryl oxidase and bovine kidney sulfhydryl oxidase ("glutathione oxidase") appear to be immunologically identical as evidenced by parallel responses in radioimmunoassays. Antibodies raised to the purified milk sulfhydryl oxidase can immunoprecipitate glutathione oxidase activity, but not gamma-glutamyltransferase ("transpeptidase") activity, from bovine kidney preparations.

Animals↗
PubMed1984

Tissue distribution of mammalian sulfhydryl oxidase.

Sulfhydryl oxidase activity is present in cow, goat, sow, human, and rat milks, and can also be measured in several rat tissues following homogenization in 1% polyoxyethylene-9-lauryl ether. These include lactating mammary tissue, kidney, and pancreas. Bovine kidney homogenates also exhibit sulfhydryl oxidase activity; however, no activity could be detected in rat thymus, brain, heart, liver, spleen, lung, or small intestinal tissue homogenates. Indirect immunofluorescent staining of tissue sections using rabbit antibodies directed against highly purified bovine milk sulfhydryl oxidase preparations revealed that the enzyme is closely associated with the plasma membrane of lactating cow and rat mammary tissues and the basal-lateral membrane of rat kidney cortex. In addition, the oxidase appears to be associated with endothelial cells lining the capillaries of rat kidney, heart, and small intestine, and centroacinar cells in pancreatic tissue slices also stain for sulfhydryl oxidase. In contrast, liver, brain, and thymus tissues do not exhibit fluorescent staining and appear to be devoid of sulfhydryl oxidase activity.

Animals↗
PubMed1984

Preparation and characterization of thionyl chloride-activated succinamidopropyl-glass as a covalent immobilization matrix.

The reaction of succinamidopropyl-glass with nonaqueous thionyl chloride followed by washing with water produces an activated surface which reacts with amino and thiol groups under nondenaturing conditions. Subsequent treatment of the covalently immobilized species with dilute hydroxylamine at pH 7 and room temperature releases about 80% of those molecules attached through amino groups and about 50% of those attached through thiol groups. The succinamidopropyl group appears to be a minimum requirement for such surface reactivity. The derivatized glass beads are stable during storage, and immobilization is achieved by simply contacting the surface with a solution of the protein or biochemical species to be attached. Moreover, the activated succinamidopropyl sites can be dispersed within an inert surface of glycerolpropyl sites. Such dispersion improves the subsequent release of immobilized protein and should provide a useful technique for preparation of a wide variety of specific matrices for affinity chromatography. It was possible to obtain, in solution, molecules which had been refolded from a completely denatured state in an immobilized form; however, extensive unfolding of proteins in strong denaturants reduced by roughly 50% the amount of protein which could be subsequently released.

Binding Sites↗
PubMed1983

Resolution of sulphydryl oxidase from gamma-glutamyltransferase in bovine milk by covalent chromatography on cysteinylsuccinamidopropyl-glass.

1. Sulphydryl oxidase from bovine milk was purified by covalent affinity chromatography on cysteinylsuccinamidopropyl-glass. Selective immobilization of the oxidase occurs through formation of a mixed disulphide between the enzyme and the substrate cysteinyl-glass matrix. Reductive elution of the bound protein can be accomplished with small thiols such as reduced glutathione (GSH), dithiothreitol or cysteine. This method leads to approx. 4000-fold purification of the enzyme from whey. Furthermore, complete resolution of sulphydryl oxidase from gamma-glutamyltransferase was achieved with this procedure. 2. Antibodies prepared against this purified enzyme quantitatively precipitated 95% of the GSH-oxidative activity from detergent-solubilized skim-milk membranes, whereas 100% of the transferase activity remained in the supernatant fraction; these findings confirmed the distinction between these two enzymes. 3. Reverse-phase high-pressure-liquid-chromatographic analyses of assay mixtures containing both enzymes revealed an array of GSH derivatives generated by a combination of the oxidative and hydrolytic activities. However, purified sulphydryl oxidase yielded only GSSG with concomitant stoichiometric loss of GSH. 4. The chromatographic method described is simple and reproducible, and may be applicable to isolation of sulphydryl oxidase from other tissues.

Animals↗
PubMed1982

Enzymatic removal of formalin-fixed tissues from polyester arteries, and its effect on physical properties of fibers.

A method is presented that can be utilized for the removal of formalin-fixed tissues from arterial grafts without affecting fiber material properties. Such a method could prove highly useful for studying the changes in the structure and properties of graft fiber as functions of important clinical variables. Information on what actually happens to the arterial graft material in situ with passage of time is greatly lacking in the current graft literature. The method proposed involves treatment of the formalin-fixed fiber-tissue complex with an enzymatic preparation containing enzymes derived from the porcine pancreas (pancreatin) in solution with tris(hydroxymethyl) aminomethane buffer at pH 7.6. Studies with two different grafts--a Microknit Dacron Bifurcation from a clinical patient 10 1/4 years after implantation and stored in formalin for 11 months, and a Sauvage Filamentous Velour of Dacron from an animal 24 h after implantation and stored in formalin for nine months--showed that the treatment was highly effective in completely removing the tissues at concentrations of pancreatin as low as 2.5%. The same treatment given to the virgin yarns of grafts revealed no significant effects on tensile, dimensional, and morphological properties.

Animals↗