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Biomedical subjects

L G Butler

Publications and source records attributed to L G Butler.

At least 37 records · Page 2Linked to original sources
PubMed1985

The active-site and amino-terminal amino acid sequence of bovine intestinal alkaline phosphatase.

The active site of bovine intestinal alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) was labeled with [32P]Pi, a radioactive CNBr peptide was isolated and the amino acid sequence was determined. The sequence of the active-site peptide has limited homology (26%) with the active-site sequence of Escherichia coli alkaline phosphatase except for the ten residues immediately flanking the active-site serine (70%). A possible amino acid sequence deduced from the amino acid composition of an active-site tryptic peptide from human placental alkaline phosphatase is very similar to the bovine intestinal active-site sequence. The amino-terminal sequence of bovine intestinal alkaline phosphatase is homologous (69%) with the human placental enzyme but not with the E. coli phosphatase.

Alkaline Phosphatase↗
PubMed1985

The relationship of alkaline phosphatase, CaATPase, and phytase.

Alkaline phosphatase, highly purified from bovine intestinal mucosa, has significant hydrolytic activity against phytate and CaATP. Phytase and CaATPase activities require quite different assay conditions than those which are optimal for conventional alkaline phosphatase substrates such as 4-nitrophenyl phosphate. We have used affinity chromatography and antibody recognition to demonstrate that the phytase and CaATPase activities are not due to contaminating enzymes, but are intrinsic activities of intestinal alkaline phosphatase. All of the phytase and CaATPase activities present in crude extracts of bovine intestinal mucosa can be accounted for by alkaline phosphatase. Apparently neither phytase nor CaATPase exist in this tissue as independent enzymes. Specific substrates which require assay conditions quite different from the conventional 4-nitrophenyl phosphate substrate may account for the physiological function of "alkaline phosphatase."

6-Phytase↗
PubMed1985

Amino acid sequence of the active site peptide of bovine intestinal 5'-nucleotide phosphodiesterase and identification of the active site residue as threonine.

We report here the identification of the amino acid residue which forms the covalent intermediate in the catalytic mechanism of bovine intestinal 5'-nucleotide phosphodiesterase and the sequence of the neighboring amino acids. The active site of 5'-nucleotide phosphodiesterase was labeled using thymidine 5'-[alpha-32P]triphosphate as substrate. A single labeled cyanogen bromide peptide was isolated using reversed-phase high performance liquid chromatography. After subdigestion with endoproteinase Lys-C and chymotrypsin, the entire amino acid sequence of the 60-residue active site peptide was obtained using automated Edman degradation. All of the radioactivity of the active site peptide was localized to a hexapeptide with sequence Thr-Phe-Pro-Asn-His-Tyr. Phosphoamino acid analysis of this peptide indicated that the labeled residue was threonine. We are not aware of any other enzymes in which threonine is phosphorylated as a covalent intermediate in the catalytic mechanism.

Amino Acid Sequence↗
PubMed1983

The catalytic mechanism of bovine intestinal 5'-nucleotide phosphodiesterase. pH and inhibition studies.

Extensive kinetic studies of bovine intestinal 5'-nucleotide phosphodiesterase as a function of pH have confirmed and amplified the catalytic mechanism previously proposed on the basis of isolation of a covalent phosphorylated intermediate (Landt, M., and Butler, L.G. (1978) Biochemistry 17, 4130-4135). An enzyme-ionizing group with apparent pKa = 6.85 controls the rate-determining step. Electrostatic interactions between anionic substrate and two or more ionic groups on the enzyme have a major role in substrate binding. Binding of strongly inhibitory 5'-AMP is controlled by an ionizing group, probably on the enzyme, with pKa less than or equal to 5.9. At pH 6.0, imidazole is a classic uncompetitive inhibitor, in agreement with independent evidence that it stabilizes the covalent intermediate form of the enzyme. KI values for phosphonate analogs, which are competitive inhibitors, indicate that phosphodiesterase binds its products and product analogs more strongly than it binds substrate analogs. Some of the results presented here can be interpreted as indicating that 5'-nucleotide phosphodiesterase is the evolutionary precursor of alkaline phosphatase, with which it has many structural and catalytic properties in common, and which is found in relatively large amounts in the same tissue.

Animals↗
PubMed1983

A family of phosphohydrolases from bovine intestinal mucosa: 5'-nucleotidase.

Bovine intestinal 5'-nucleotidase has been partially purified and characterized for comparison with two other phosphohydrolases from the same tissue, alkaline phosphatase and 5'-nucleotide phosphodiesterase, which are closely related structurally and mechanistically. Kinetic studies with a variety of nucleotides and phosphonate analogs show that, although 5'-nucleotidase is a monoesterase like alkaline phosphatase, it more closely resembles 5'-nucleotide phosphodiesterase in its high affinity and specificity for nucleotide binding. 5'-Nucleotidase is bound very strongly by an affinity column containing a bound phosphonate analog of ADP but is not bound by an affinity column containing a non nucleotide phosphonate which selectively binds alkaline phosphatase. 5'-Nucleotidase is strongly bound by immobilized antibodies prepared against 5'-nucleotide phosphodiesterase, and is less strongly bound by immobilized antibodies prepared against alkaline phosphatase. We conclude that 5'-nucleotidase is structurally more similar to 5'-nucleotide phosphodiesterase than to another monoesterase, alkaline phosphatase.

5'-Nucleotidase↗
PubMed1982

Induction of ovulation and cyclic activity in anestrous ewes with testosterone treated wethers and ewes.

An experiment was conducted to evaluate the effectiveness of wethers and ewes treated with testosterone preparations to induce ovulation and breeding activity in anestrous ewes. The testosterone was administered three times at weekly intervals. Wethers and ewes treated with 105 mg testosterone propionate and wethers treated with 100 mg testosterone from testosterone cyclopentyl propionate were as successful as vasectomised rams in inducing ovulation and cyclic activity in ewes. Seven days after the last injection the concentrations of testosterone in peripheral bloods were not significantly different from that in the vasectomised rams. By day 28 the concentrations were significantly lower than in the rams. The testosterone preparations tested are suitable for the induction of male sexual behavior and are rapidly excreted by sheep.

Journal Article↗
PubMed1981

The specificity of proanthocyanidin-protein interactions.

The proanthocyanidins or condensed tannins, phenolic polymers which are synthesized by many plants, characteristically bind and precipitate proteins. The specificity of the interaction was investigated using a competitive binding assay to compare directly the affinities of various proteins and synthetic polymers for the tannin obtained from Sorghum bicolor (Lin.) Moench. At pH 4.9, the relative affinities range over more than 4 orders of magnitude, indicating that this proanthocyanidin interacts quite selectively with protein and protein-like polymers. The affinity for tannins is an inverse function of the size of the polymer, and peptides with less than six residues interact very weakly with tannin. Proteins are precipitated by proanthocyanidins most efficiently at pH values near their isoelectric points. Proline-rich proteins and polymers have very high affinities for tannin. Tightly coiled globular proteins have much lower affinities for tannin than conformationally loose proteins.

Amino Acids↗
PubMed1980

5'-Nucleotide phosphodiesterase: features of the substrate binding site as deduced from specificity and kinetics of some novel substrates.

Phosphonate monoesters and phosphate diesters with systematically varied substituents and leaving groups were synthesized and tested as substrates for homogeneous 5'-nucleotide phosphodiesterase from bovine intestine. The enzyme was shown to hydrolyze phosphorothioate and phosphonoamidate compounds but at significantly lower rates than comparable oxy compounds. The effects of bulk and structure of the ester or phosphonate substituents were also investigated. Dibenzyl phosphate, an ester of an aliphatic alcohol, was a poor substrate. The enzyme did not hydrolyze aliphatic monoesters of phosphonates, regardless of bulk. Kinetic parameters of several nitrophenyl phosphonomonoesters and phosphodiesters are presented. The results suggest that synthetic nonnucleotide substrates can bind in two different modes, only one of which is productive. Incidence of nonproductive binding, with consequent kinetic effects, is increased by increasing the symmetry of the substrates.

Animals↗
PubMed1979

Hydrophobic esters of cellulose: properties and applications in biochemical technology.

We have investigated the utility for enzyme immobilization of several hydrophobic cellulose esters, as a function of solvent composition, extent of esterification, and enzyme. Phenoxyacetyl cellulose was also used for immobilization of rat liver microsomes, hydrophobic chromatography of proteins, and removal of Triton X-100 from protein solutions. Phenoxyacetyl groups esterified to cellulose were much less subject to enzymatic hydrolysis than soluble phenoxyacetyl esters.

Binding Sites↗
PubMed1978

Alkaline phosphatase: affinity chromatography and inhibition by phosphonic acids.

Five phosphonic acid derivatives were synthesized, coupled to agarose, and tested for affinity chromatographic binding of alkaline phosphatase from bovine intestine. Agarose coupled to L-histidyldiazobenzylphosphonic acid was found to be a highly effective adsorbent. In order to understand the large differences in binding capacity observed with derivatized agaroses, inhibition of alkaline phosphatase by phosphonic acid ligands, and related phosphonic acids, was measured. The results of affinity chromatography and inhibition studies were in good agreement, demonstrating that phosphonic acids with large aromatic/hydrophobic, carboxylate substituents bind strongly and competitively to the enzyme active site.

Alkaline Phosphatase↗
PubMed1977

Enzymic hydrolysis of phosphonate esters. Reaction mechanism of intestinal 5'-nucleotide phosphodiesterase.

The mechanism of bovine intestinal 5'-nucleotide phosphodiesterase was investigated by determining kinetic constants of systematically varied substrates, with emphasis on esters of phosphonic acids (which have much higer Vmax values than conventional phosphodiester substrates), and by pre-steady-state kinetics using bis(4-nitrophenyl) phosphate as substrate. The results suggest a ping-pong type mechanism, with participation of a covalent enzyme intermediate.

Animals↗
PubMed1977

Multiple roles of metal ions in the reaction catalyzed by yeast inorganic pyrophosphatase.

Yeast inorganic pyrophosphatase has three roles for metal ions in its reaction: activator, substrate and structural. Out of a wide variety of metal ions tested, only Mg2+, Zn2+, mn2+ and Co2+ can fulfill both the activator and substrate roles. Several other metal ions inhibit the Mg2+-stimulated activity; the strong inhibition by Ca2+ (and probably Cd2+) is due to interference with both activator and substrate roles, while the weaker inhibition by Sr2+ (and possibly Cu2+ and Ni2+) is due to interference with only the substrate role. Rare earth ions strongly stimulate nonenzymic PPi hydrolysis but do not activate the enzyme. Despite its ability to fulfill both the activator and substrate roles. Zn2+ causes inactivation of the enzyme, probably by interference with the "structural" Mg2+. The results suggest that the three roles for metal ions are independent (an individual metal ion can satisfy only one at a time) and that the metal ion specificity for the three roles declines in the order: structural greater than substrate greater than activator.

Catalysis↗
PubMed1976

Cr(III) complexes as active site probes of yeast inorganic pyrophosphatase.

The inert coordination complex of Cr(III) with PP1 (CrPP1) is not a substrate for yeast inorganic pyrophosphatase and does not inhibit the Mg2+-dependent activity of this enzyme. These results, when compared with the effects of CrPP1 on other enzymes and with the effects of other divalent metal ions on this enzyme, suggest that yeast inorganic pyrophosphatase binds substraather than the phosphate groups. Mg2+ partially protects against a slow inactivation of the enzyme by Cr(H2O)6(3)+; this exchange inert ion may provide a means of labeling the metal ion binding site of this enzyme.

Binding Sites↗
PubMed1975

Hydrolysis of phosphonate esters catalyzed by 5'-nucleotide phosphodiesterase.

4-Nitrophenyl and 2-napthyl monoesters of phenylphosphonic acid have been synthesized, and an enzyme catalyzing their hydrolysis was resolved from alkaline phosphatase of a commerical calf intestinal alkaline phosphatase preparation by extensive ion-exchange chromatography, chromatography on L-phenylalanyl-Sepharose with a decreasing gradient of (NH4) 2SO4, and gel filtration. Detergent-solubilized enzyme from fresh bovine intestine was purified after (NH4)2SO4 fractionation by the same technique. The purified enzyme is homogeneous by polyacrylamide gel electrophoresis and sedimentation equilibrium centrifugation. It has a molecular weight of 108,000, contains approximately 21% carbohydrate, and has an amino acid composition considerably different from that reported from alkaline phosphatase from the same tissue. The homogeneous intestinal enzyme, an efficient catalyst of phosphonate ester hydoolysis but not of phosphate monoester hydrolysis, was identified as a 5'-nucleotide phosphodiesterase by its ability to hydrolyze 4-nitrophenyl esters of 5'-TMP but not of 3'-TMP. Also consistent with this identification was the ability of the enzyme to hydrolyze 5'-ATP to 5'-AMP and PPi, NAD+ to 5'-AMP and NMN, TpT to 5'-TMP and thymidine, pApApApA to 5'-AMP, and only the single-stranded portion of tRNA from the 3'-OH end. Snake venom 5'-nucleotide phosphodiesterase also hydrolyzes phosphonate esters, but 3'-nucleotide phosphodiesterase of spleen and cyclic 3',5'-AMP phosphodiesterase do not. Thus, types of phosphodiesterases can be conveniently distinguished by their ability to hydrolyze phosphonate esters. As substrates for 5'-nucleotide phosphodiesterases, phosphonate esters are preferable to the more conventional esters of nucleotides and bis(4-nitrophenyl) phosphate because of their superior stability and ease of synthesis. Furthermore, the rate of hydrolysis of phosphonate esters under saturating conditions is greater than that of the conventional substrates. At substrate concentrations of 1 mM the rates of hydrolysis of phosphonate esters and of nucleotide esters are comparable and both superior to that of bis(4-nitrophenyl) phosphate.

Amino Acids↗