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Biomedical subjects

I B Wilson

Publications and source records attributed to I B Wilson.

At least 91 records · Page 5Linked to original sources
PubMed1983

The inhibition of acetylcholinesterase by arsenite and fluoride.

The effect of fluoride on the rate of reaction of acetylcholinesterase with arsenite, on the rate of dissociation of the enzyme-arsenite complex, and on the equilibrium between enzyme and arsenite was studied. Fluoride decreases the rate of the reaction between acetylcholinesterase and arsenite and changes the apparent equilibrium dissociation constant between the enzyme and arsenite, but even at concentrations as high as 0.2 M has no effect on the rate of dissociation of the enzyme-arsenite complex. The binding of fluoride and arsenite with the enzyme is highly anticooperative and may well be mutually exclusive. These results are consistent with a model in which the binding sites overlap and in which the same functional groups are involved.

Acetylcholinesterase↗
PubMed1983

Polyacrylamide gels which contain a novel mixed disulfide compound can be used to detect enzymes that catalyze thiol-producing reactions.

The synthesis of N-[5-(hydroxyethyl)dithio-2-nitrobenzoylaminoethyl] acrylamide (I) is described. If the disulfide bond in this compound is reduced with thiol reagents, an intense yellow color develops (epsilon 412 V 13,600 at pH 7.4) due to essentially the same chromophore as 5-thio-2-nitrobenzoic acid, the reduced form of 5,5'-dithiobis(2-nitrobenzoic acid)(Ellman's reagent). Polyacrylamide gels were prepared that were crosslinked with N,N'-methylenebisacrylamide and which contained I as an integral part of the polymerized acrylamide chain. Acetylcholinesterase (from electric eel and human brain tissue slices) and alkaline phosphatase (from Escherichia coli and calf intestine) were subjected to electrophoresis and then the gels were immersed in an appropriate thiol-substrate buffer (acetylthiocholine and cysteamine-S-phosphate, respectively). A yellow band developed rapidly in the acrylamide gel at the site of enzyme activity. Electrophoresis on the mixed disulfide-polyacrylamide gel proved to be a rapid and sensitive technique to detect very small amounts of enzyme (approximately 0.02 fmol acetylcholinesterase) and should have wide application for detecting other enzymes that hydrolyze thiol substrates.

Acetylcholinesterase↗
PubMed1983

Glutamic acid is an active site residue of angiotensin I-converting enzyme. Use of the Lossen rearrangement for identification of dicarboxylic acid residues.

A set of chemical reactions was used to show that one glutamic acid residue at the active site of bovine lung angiotensin I-converting enzyme is esterified with the alkylating agent p-[N,N-bis(chloroethyl)amino] phenylbutyryl-L-Pro (chlorambucyl-L-Pro), an affinity label for this enzyme (Harris, R. B., and Wilson, I. B. (1982) J. Biol. Chem. 257, 811-815). The same procedure was used to confirm that a glutamic acid residue of carboxypeptidase A alpha is esterified by reaction with bromoacetyl-N-methyl-L-phenylalanine (Haas, G. M., and Neurath, H. (1971) Biochemistry 10, 3535-3546). In the procedure described in this paper, the esterified residue at the active site is converted to the hydroxamic acid by reaction with hydroxylamine and the hydroxamic acid is subject to the Lossen rearrangement. If a glutamic acid residue was esterified, 1 eq of 2,4-diaminobutyric acid will be formed. Aspartyl esters will give 2,3-diaminopropionic acid. The diamino acids can be quantitatively measured using the short column of an amino acid analyzer if the amount of lysine and histidine is largely decreased by modification with suitable side chain protecting groups. With carboxypeptidase A, the reactions were done on the whole undigested enzyme. With the converting enzyme, we first cleaved the esterified enzyme with cyanogen bromide. Twenty-nine cleavage peptides were separated on high performance liquid chromatography and one of these contained all of the bound radioactive inhibitor. This active site peptide was then subjected to the derivatization and Lossen procedures, and 1 eq of 2,4-diaminobutyric acid was obtained.

Amino Acids↗
PubMed1983

Soluble pig intestinal cell membrane components with affinities for E. coli K88+ antigen.

Pig intestinal brush borders (BB) were radiolabeled by iodination using the lactoperoxidase-hydrogen peroxide procedure. The BB were then detergent solubilized, centrifuged to remove particulate material, and chromatographed on Sepharose CL-4B. The fractions were incubated with K88+ E. coli using an in vitro binding assay. Binding of the iodinated membranes to K88+ E. coli occurred throughout a wide range of molecular weight components, in excess of 690K daltons to near 25K daltons. The system utilizing intact K88+ E. coli and solubilized BB was shown to be saturable. Prior contact of K88+ E. coli with nonradiolabeled membranes or specific antibodies to K88+ pili inhibited binding of the radiolabeled BB. Simple sugars were tested for their ability to block binding of the labeled BB; partial inhibition occurred with galactose (17.9%), galactosamine (32%), glucose (10.6%), and N-acetylglucosamine (32%). Calcium enhanced binding with as little as 10 microM. A 10 x increase in binding occurred with 500 microM calcium. Affinity chromatography using K88+ pili coupled on agarose beads avidly bound the labeled BB. The receptor membranes were eluted with high molar concentrations of salt, however considerable degradation occurred. Despite low yields from the affinity system, receptor membranes with higher binding activities were recovered. Protein: glycoprotein ratios were 1:4. Elution with SDS and electrophoresis on 12.5% polyacrylamide gels in the presence of a reducing agent produced two major subunits 35--32K and 23K daltons. These components were recovered from the gels and retained their binding activity. This information suggests that the intestinal receptor responsible for binding of K88+ E. coli is a glycoprotein, that in the native state exists in multimeric forms.

Animals↗
PubMed1982

Irreversible inhibition of bovine lung angiotensin I-converting enzyme with p-[N,N-bis(chloroethyl)amino]phenylbutyric acid (chlorambucil) and chlorambucyl L-proline and with evidence that an active site carboxyl group is labeled.

Bovine lung angiotensin I-converting enzyme is rapidly and irreversibly inactivated by p-[N,N-bis(chloroethyl)amino]phenylbutyric acid (chlorambucil) and by the chlorambucil derivative of L-proline (chlorambucyl-proline). Chlorambucil is a nitrogen mustard alkylating agent that is used as an antineoplastic drug. At any one concentration, the inactivation is pseudo-first order with time. Inhibition by both substances is active site directed as suggested by the formation of a reversible enzyme-inhibitor complex prior to the alkylation reaction and by the fact that L-Phe-L-Pro, a reversible inhibitor which is competitive with substrate, is also competitive with both irreversible inhibitors in protecting the enzyme against inactivation. The second order rate constant for inactivation increases in the pH range 5-8 and reaches a value of 3.5 X 10(3) M-1 . min-1 for chlorambucil and 4.8 X 10(2) M-1 . min-1 for chlorambucyl-proline. Chlorambucyl [U-14C]L-proline reacts 1:1 with the converting enzyme and the uptake of radioactivity paralleled the loss of enzyme activity with and without protection by Phe-Pro. Once bound, the radioactive chlorambucyl proline was released (as the dihydroxy derivative) by hydroxide ion with a second order rate constant of 2.2 M-1 . min-1 at 25 degrees C. The radioactive label is also removed by hydroxylamine at pH 10. The lability of the irreversibly bound inhibitor in alkali and in hydroxylamine indicates that an ester bond is formed by the alkylation of an aspartic acid or glutamic acid side chain.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1982

Physicochemical characteristics of homogeneous bovine lung angiotensin I-converting enzyme. Comparison with human serum enzyme.

Angiotensin I-converting enzyme was purified to electrophoretic homogeneity (12 units/mg) from bovine lung tissue and from human serum using an affinity gel described previously (Harris et al., (1981) Anal. Biochem. 111, 227-234). The isoelectric point (4.5), molecular weight (145 000), S20,W (8.1), amino acid composition and carbohydrate content of the lung enzyme are all similar to the values obtained for the human serum enzyme. The NH2-terminus of the lung enzyme (Ala) is different from that of the serum enzyme (Tyr) but the COOH-terminal sequences are identical (-Leu-Ser-OH). Pure bovine lung enzyme was reduced and carboxyamidomethylated with iodo (14C1) acetamide to the extent predicted by the number of cysteine residues. Since no radioactivity was incorporated into denatured enzyme that was not reduced, all of the cysteine residues must be in the form of disulfide bonds. Reverse-phase HPLC was used to separate peptides obtained from the lung enzyme after degradation with either trypsin or cyanogen bromide. The number of peptides resolved (42 after trypsin, 31 after cyanogen bromide), were only 20% fewer than the number predicted from the amino acid analysis and therefore the possibility that the converting enzyme (a single polypeptide chain) might be a fused dimer is excluded.

Amino Acid Sequence↗
PubMed1981

The uptake of amines by polymorphonuclear leukocytes.

Muscarinic and beta-adrenergic ligands associate with polymorphonuclear leukocytes to show high-affinity, saturable accumulation. This association can be distinguished from specific receptor binding by its temperature dependence, sensitivity to pH, requirement of an energy source, inhibition by ionophores, and inhibition by a variety of permeable basic amines. Our results suggest that these amines accumulate in acidic lysosomes which are plentiful in these cells. This permeable amine effect can be inhibited without affecting specific receptor binding.

Alprenolol↗
PubMed1980

Phosphorylated intermediate of alkaline phosphatase.

We have measured the phosphorylation of the subunits of alkaline phosphatase in the steady state with several substrates and at several pH values. Our results vary from 80% phosphorylation of both subunits at pH7 to only 9% at pH 10. There is no evidence of anticooperativity. With the measurement of kcat, we are able to evaluate rate constants in a minimal scheme. The results show that the main rate influencing steps ar chemical dephosphorylation and dissociation of phosphate. The predominates at pH 7.0 but declines in importance as the pH is raised. Our rate constants for dissociation of phosphate are in agreement with recent NMR studies.

Alkaline Phosphatase↗
PubMed1978

Phosphate content of Escherichia coli alkaline phosphatase isozymes. The effect of phosphate and zinc on the separation of isozymes.

Alkaline phosphatase from Escherichia coli was isolated as two major isoenzyme forms that were separated by DEAE-cellulose chromatography. Each form contained 2 equiv of endogenous phosphate. The endogenous phosphate, although difficult to remove, readily exchanges with phosphate. The forms also were separable by polyacrylamide gel electrophoresis. Apoenzyme prepared from native enzyme by the removal of zinc (and phosphate) also contains electrophoretically distinct enzyme forms which are indistinguishable from the native forms on gel electrophoresis. The isozymes were also found to have similar affinities for inorganic phosphate and susceptibilities to inactivation by EDTA. These results are not consistent with the notion that the formation or separation of isoenzyme forms is dependent upon different amounts of bound phosphate. They are consistent with the suggestion that a difference in amino acid composition is the basis for the occurrence and separation of these isoenzymes.

Alkaline Phosphatase↗
PubMed1978

On the mechanism of the acceleration of methanesulfonylation of acetylcholinesterase with cationic accelerators.

1. In order to elucidate some features of the mechanism of the acceleration of methanesulfonylation of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) with cationic accelerators, the methanesulfonylation of this enzyme by high concentrations of methanesulfonylfluoride, in the absence and presence of accelerators decamethonium and tetraethylammonium, was studied. 2. The results showed that the accelerator accelerates the reaction by electrostatically improving the binding between acetylcholinesterase and methanesulfonylfluoride without effecting the rate of the decomposition of the enzyme-inhibitor complex into the methanesulfonylated enzyme and product.

Acetylcholinesterase↗