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Biomedical subjects

J R Whitaker

Publications and source records attributed to J R Whitaker.

At least 37 records · Page 2Linked to original sources
PubMed1982

Dynamics of ligand binding to alpha-chymotrypsin and to N-methyl-alpha-chymotrypsin.

Ks values for binding of selected substrates, competitive inhibitors, and a noncompetitive inhibitor were found to be similar for alpha-chymotrypsin and N-methyl-alpha-chymotrypsin. The rates and steps of binding of a competitive inhibitor and a noncompetitive inhibitor were also found to be similar for alpha-chymotrypsin and N-methyl-alpha-chymotrypsin. Therefore, N-methyl-alpha-chymotrypsin is an appropriate model for alpha-chymotrypsin in studying the dynamics of the binding of substrates by temperature-jump techniques in aqueous solvents. 2-Toluidinylnaphthalene-6-sulfonate, a noncompetitive inhibitor, bound to alpha-chymotrypsin in a single step with rate constants k1 and k-1 of 3.9 X 10(7) M-1 s-1 and 1.9 X 10(3) s-1, respectively, at pH 5.0 (0.2 M acetate, ionic strength of 0.2). Similar values were obtained for N-methyl-alpha-chymotrypsin and chymotrypsinogen A at pH 5.0 and for alpha-chymotrypsin at pH 7.8 [0.1 M tris(hydroxymethyl)aminomethane-0.03 M CaCl2]. Indole, a competitive inhibitor, bound to alpha-chymotrypsin in a single step at pH 5.0 and 7.8, with k1 and k-1 of 1.8 X 10(7) M-1 s-1 and 7.8 X 10(3) s-1, respectively, at pH 5.0 while proflavin, another competitive inhibitor, bound to alpha-chymotrypsin with two observable steps where k1, k-1, k2, and k-2 were 1.0 X 10(7) M-1 s-1, 7 X 10(2) s-1, 1.0 X 10(3) s-1, and 7 X 10(2) s-1, respectively, at pH 5.0. The specific substrate N-acetyl-L-3,5-dinitrotyrosine ethyl ester bound to N-methyl-alpha-chymotrypsin at pH 5.0 in three observable steps where k1, k-1, k2, k-2, k3, and k-3 were 3.7 X 10(7) M-1 s-1, 6.2 X 10(4) s-1, 1.2 X 10(3) s-1, 3.5 X 10(2) s-1, 3 X 10(2) s-1, and 4 X 10(2) s-1, respectively. Preliminary data indicated that the third step of this reaction is probably absent when Met192 of N-methyl-alpha-chymotrypsin is oxidized to methionine sulfoxide. These results confirm the validity of data obtained from reactions at subzero temperatures in 65% dimethyl sulfoxide in indicating multiple steps in the binding of substrates to alpha-chymotrypsin. The methodology described should make it possible to measure quantitatively the contribution of the binding process to enzyme catalysis (the Circe effect).

Animals↗
PubMed1981

[3H]Dopamine accumulation and release from striatal slices in young, mature and senescent rats.

Examinations of [3H]dopamine ([3H]DA) release following KCl or amphetamine administration in striatal slices from young (7 month), and senescent (24 month) Wistar rats showed no age-related changes. Further, the amount of [3H]DA accumulated in the striatal slices showed no changes with age. Thus, previously reported age-related deficits in motor behavior (i.e. rotational) are not produced by changes in striatal DA accumulation or release.

Aging↗
PubMed1981

Trypsin activation of enterotoxin from Clostridium perfringens type A: fragmentation and some physicochemical properties.

Clostridium perfringens type A enterotoxin was activated about 3-fold by treatment with trypsin, without an observed change in molecular weight. On denaturation in 8 M urea, the trypsinated enterotoxin lost a small peptide of about 4000 daltons. The single cysteine residue of enterotoxin was in the small peptide together with seven out of nine residues of proline. Trypsin activation, without removal of the small peptide, increased the 'outside' number of amino groups from eight to eleven. The trypsin treatment of the enterotoxin did not change the antigenic properties of the protein. Glycine was the C-terminal residue of the native enterotoxin while the dansyl alpha-amino acid of the N-terminal could not be identified.

Amino Acids↗
PubMed1980

Improved method for purification of enterotoxin from Clostridium perfringens type A.

The purification procedure of Clostridium perfringens type A enterotoxin has been improved. The cell sonic extract was precipitated twice with ammonium sulfate, first 40% saturated to concentrate the enterotoxin and then 15% saturated. The two precipitations were followed by gel filtration on Sephadex G-100. The enterotoxin appeared to be homogeneous on 7% polyacrylamide gel electrophoresis after this three-step purification procedure, with a recovery of 56% and a 12.3-fold purification. The solubility properties at different pH values, temperatures, and ammonium sulfate concentrations are also given as basis for the purification procedure.

Ammonium Sulfate↗
PubMed1978

A method for improving the nutritional value of food proteins: covalent attachment of amino acids.

Casein was modified by use of a series of active N-hydroxy-succinimide esters of amino acids in order to study the effects of new covalently linked hydrophobic or hydrophilic groups on its physical and nutritional properties. Tryptophan was used to determine the best conditions for the chemical reaction and to study the stability of the newly formed amide linkage (isopeptide bond). Casein was also modified with glycine, alanine, methionine, N-acetyl-methionine and aspartic acid. In vitro hydrolysis studies using bovine chymotrypsin, pancreatine and rat bile-pancreatic juice indicated that digestibility of the modified casein derivatives was lower than that of the untreated protein. Since solubility was not significantly changed (except for tryptophyl-casein), the decreased in vitro digestibility is probably due to other factors such as steric hindrance as well as decrease in lysine residues available to trypsin in pancreatin and rat pancreatic juice. Plasma amino acid patterns for rats fed a 10% protein diet of highly modified glycyl-casein or methionyl-casein suggest that the epsilon-aminolysyl derivatives are readily hydrolyzed in vivo. This was confirmed by the growth response of rats fed the following isonitrogenous diets (protein source listed only): casein, casein + free methionine, methionyl-casein, casein + free N-acetyl-methionine, N-acety-methionyl-casein. Covalently attached methionine appeared to be as readily available as the free amino acid; bound N-acetyl-methionine was also available but to a slightly lower extent. Although this study is preliminary, the covalent attachment of amino acids to proteins appears to be a promising method for improving the biological value of food proteins.

Amino Acids↗
PubMed1978

Effect of reductive alkylation of the epsilon-amino group of lysyl redsidues of casein on its nutritive value in rats.

Casein epsilon-aminolysyl residues were converted to the methyl (and dimethyl), isopropyl or cyclopentyl derivatives in high yield with formaldehyde, acetone or cyclopentanone, respectively, in the presence of sodium borohydride. When incorporated into diets at 10% as the sole protein source, the chemically modified caseins failed to support growth of young rats. Methyl casein did, however, support limited growth after about 5 days. Plasma threonine levels increased and lysine levels decreased markedly in rats fed the alkyl caseins. The respective alkyllsine derivatives were present in plasma and urine. In another experiment, nearly normal or normal growth was obtained by feeding lysine-supplemented methyl or isopropyl casein, respectively. A preparation of partially methylated casein, containing approximately equal amounts of monomethyl- and dimethyllysines, supported normal rat growth. These results demonstrate that lysine deficiency was produced by feeding highly alkylated caseins. Digestibility of the chemically modified caseins in vivo was not affected, although in vitro studies with trypsin and alpha-chymotrypsin showed lowered digestibility. Since no apparent toxicity was observed limited methylation of food proteins may be useful for protection of lysyl residues against deteriorative reactions during processing and storage.

Acetone↗
PubMed1977

Isolation and characterization of sheep pepsin.

Sheep pepsin was isolated (approx. 120-fold purification) from aqueous abomasal homogenates by (1) pH fractionation, (2) chromatography on Sepharose 4B-poly-L-lysine columns and (3) gel filtration on Sephadex G-100. The enzyme had mol.wt. approx. 34000, N-terminal valine and C-terminal alanine. The amino acid composition of sheep pepsin was generally similar to that of pig and ox pepsins, with a very low content of basic residues and a high content of acidic and hydroxy-amino acids. The pH optimum for NN-dimethyl-casein and NN-dimethyl-haemoglobin as substrates was approx. 1.8. The Km and kcat. for NN-dimethyl-haemoglobin were 46micronM and 1100min-1 respectively, and for NN-dimethyl-casein the corresponding parameters were 50micronM and 420min-1. These values were generally similar to those for pig and ox pepsins. At the pH optimum of 4.6, the sheep pepsin was about 50% as active on benzyloxycarbonyl-L-histidyl-L-phenyl-alanyl-L-tryptophan ethyl ester as was pig pepsin. The pH optimum for the hydrolysis of N-acetyl-L-phenylalanyl-L-di-iodotyrosine by sheep, ox and pig pepsins was approx. 1.85.

Amino Acids↗
PubMed1975

Selection of strain, growth conditions, and extraction procedures for optimum production of lactase from Kluyveromyces fragilis.

Forty-one strains of Kluyveromyces fragilis (Jörgensen) van der Walt 1909 varied 60-fold in ability to produce lactase (beta-galactosidase). The four best strains were UCD No. 55-31 (Northern Regional Research Center NRRL Y-1196), UCD No. C21(-), UCD No. 72-297(-), and UCD No. 55-61 (NRRL Y-1109). Biosynthesis of lactase during the growth of K. fragilis strain UCD No. 55-61 was followed on both lactose and sweet whey media. Maximum enzyme yield was obtained at the beginning of the stationary phase of growth. Bets lactase yields from K. fragilis UCD No. 55-61 were obtained with 15% lactose and an aeration rate of at least .2 mmol oxygen/liter per min. Supplementary growth factors were unneccessary for good lactase yeilds when yeast was grown on whey media. Best extraction of lactase from fresh yeast cells was obtained by toluene autolysis (2% vol/vol) at 37 C in .1 M potassium phosphate buffer, pH 7.0, containing .1 mM manganese chloride and .5 mM magnesium sulfate. The enzyme was concentrated and purified partially by acetone precipitation. At least 95% of the enzyme activity of the concentrated solution was retained after storage for 7 days at 22 C, for 3 wk at 4 C, and for 6 wk at -20 C.

Aerobiosis↗
PubMed1974

The identification of intermediates in the reaction of pig heart lactate dehydrogenase with its substrates.

Pig heart lactate dehydrogenase was studied in the direction of pyruvate and NADH formation by recording rapid changes in extinction, proton concentration, nucleotide fluorescence and protein fluorescence. Experiments measuring extinction changes show that there is a very rapid formation of NADH within the first millisecond and that the amplitude of this phase (phase 1) increases threefold over the pH range 6-8. A second transient rate (phase 2) can also be distinguished (whose rate is pH-dependent), followed by a steady-state rate (phase 3) of NADH production. The sum of the amplitudes of the first two phases corresponds to 1mol of NADH produced/mol of active sites of lactate dehydrogenase. Experiments that measured the liberation of protons by using Phenol Red as an indicator show that no proton release occurs during the initial very rapid formation of NADH (phase 1), but protons are released during subsequent phases of NADH production. Fluorescence experiments help to characterize these phases, and show that the very rapid phase 1 corresponds to the establishment of an equilibrium between E(NAD) (Lactate) right harpoon over left harpoon H(+)E(NADH) (Pyruvate). This equilibrium can be altered by changing lactate concentration or pH, and the H(+)E(NADH) (Pyruvate) species formed has very low nucleotide fluorescence and quenched protein fluorescence. Phase 2 corresponds to the dissociation of pyruvate and a proton from the complex with a rate constant of 1150s(-1). The observed rate constant is slower than this and is proportional to the position of the preceding equilibrium. The E(NADH) formed has high nucleotide fluorescence and quenched protein fluorescence. The reaction, which is rate-limiting during steady-state turnover, must then follow this step and be involved with dissociation of NADH from the enzyme or some conformational change immediately preceding dissociation. Several inhibitory complexes have also been studied including E(NAD+) (Oxamate) and E(NADH) (Oxamate') and the abortive ternary complex E(NADH) (Lactate). The rate of NADH dissociation from the enzyme was measured and found to be the same whether measured by ligand displacement or by relaxation experiments. These results are discussed in relation to the overall mechanism of lactate dehydrogenase turnover and the independence of the four binding sites in the active tetramer.

Acid-Base Equilibrium↗
PubMed1973

Purification and some properties of two polyphenol oxidases from bartlett pears.

Two polyphenol oxidases (enzymes A and B) from Bartlett pear (Pyrus communis) peelings were purified to electrophoretic homogeneity according to polyacrylamide gel by a combination of Sephadex gel filtration, diethylaminoethyl cellulose chromatography and hydroxyl apatite chromatography. While the two enzymes differ electrophoretically at pH 9.3, chromatographically on hydroxyl apatite, and in the effect of ionic strength on activity, they are similar with respect to chromatography on diethylaminoethyl cellulose, substrate specificity, pH activity relations, inhibition by p-coumaric and benzoic acids, and heat stability. The two enzymes are o-diphenol oxidases with no detectable monophenolase or laccase activities. Pyrocatechol, 4-methyl catechol, chlorogenic acid, and d-catechin are good substrates of the enzymes with K(m) values in the range of 2 to 20 mm. Dependences of activity on oxygen and chlorogenic acid concentrations indicate a sequential mechanism for binding of these substrates to enzyme B. V(max) and K(m) values for oxygen and chlorogenic acid were 103 mumoles O(2) uptake per minute per milligram of enzyme, 0.11 mm and 7.2 mm, respectively, for enzyme B at pH 4.0. Both enzymes had maximum activity at pH 4.0 on chlorogenic acid. K(m) values for chlorogenic acid were independent of pH from 3 to 7; the V(max) values for both enzymes gave bell-shaped curves as a function of pH. p-Coumaric acid is a simple, linear noncompetitive inhibitor with respect to chlorogenic acid at pH 6.2 with K(i) values of 0.38 and 0.50 mm for enzymes A and B, respectively. Benzoic acid is a linear competitive inhibitor with respect to chlorogenic acid at pH 4.0 with K(i) values of 0.04 and 0.11 mm for enzymes A and B, respectively.

Journal Article↗