Search PubMed⌕ Search

Biomedical subjects

J R Whitaker

Publications and source records attributed to J R Whitaker.

At least 19 recordsLinked to original sources
PubMed1990

Expression and secretion of rice alpha-amylase by Saccharomyces cerevisiae.

We report the high level expression and secretion of rice alpha-amylase isozyme by Saccharomyces cerevisiae. Transcription of this gene was under control of the yeast enolase promoter. The synthesized protein had an approximate molecular size of 45 kDa and a pI of approx 4.7 to 5.0. The rice alpha-amylase signal peptide was recognized and efficiently processed by yeast and the active, glycosylated enzyme was secreted into the culture media. This enzyme was purified to homogeneity by affinity chromatography and its enzymatic properties were characterized. The Km and Vmax were found to be similar to those of alpha-amylases from other organisms. The high level of secretion observed in these studies may be due to the unique features of the rice signal peptide and/or to the glycosylation of the recombinant enzyme.

Amino Acid Sequence↗
PubMed1988

Parameters involved in binding of porcine pancreatic alpha-amylase with black bean inhibitor: role of sulfhydryl groups, chloride, calcium, solvent composition and temperature.

The amylase inhibitor of black (kidney) beans (Phaseolus vulgaris; MW 53,000) forms a 1:1 stoichiometric complex with porcine pancreatic alpha-amylase (MW 52,000) at pH 5.40. The single sulfhydryl group of the inhibitor and the two sulfhydryl groups of alpha-amylase are not involved in recognition and binding. Chloride ions, required for activity of alpha-amylase at both pH 5.40 and 6.90, are important for inhibitor--enzyme binding at pH 6.90 but not at pH 5.40. Calcium-free alpha-amylase binds with the inhibitor. An increase in the ionic strength of the solvent increases the rate of binding of the inhibitor with alpha-amylase; a decrease in the dielectric constant decreases the rate of binding; and decreasing the temperature increases the dissociation constant, Kd, of the complex. These data support the hypothesis that hydrophobic interaction is of primary importance in complex formation. The activation energy, Ea, for complex formation was found to be 12.4 kcal/mol at pH 5.40 and 24.2 kcal/mol at pH 6.90. In the presence of the poor substrate, p-nitrophenyl-alpha-D-maltoside, the Ea for complex formation was 4.1 kcal/mol at pH 6.90.

Animals↗
PubMed1987

The effect of oral stimulation on human parotid salivary flow rate and alpha-amylase secretion.

Unilateral parotid saliva was collected from ten subjects following oral stimulation with water as baseline, and aqueous solutions of starch (2.5, 5.0, and 10%), sucrose (0.1, 0.2, and 0.4 M) sodium chloride (0.075, 0.15, and 0.30 M), and citric acid (0.005, 0.01, and 0.02 M). Salivary flow rate increased with increasing levels of each taste stimulus. At concentrations of equal taste intensity, citric acid evoked the highest flow rate, followed by sodium chloride and sucrose, while starch, in solution, had a minimal effect. Secretion rate patterns for total protein and alpha-amylase mirrored those of flow rate. The total protein and alpha-amylase concentrations of the saliva, and specific activity of alpha-amylase, were influenced by the type but not the concentration of stimulus, with citric acid stimulation resulting in the lowest concentrations and highest specific activity. Sodium ion (Na+) concentration generally increased with increasing stimulated flow rate, while K+, Ca++, and Mg++ concentrations remained relatively constant. Subjects with lower flow rates had a more concentrated saliva than those with high flow, except for Na+ concentration. Oral stimulation resulted in similar changes in protein and alpha-amylase secretion rates for the two groups.

Adult↗
PubMed1985

A longitudinal study of tolerance to cold stress among C57BL/6J mice.

C57BL/6J male mice of different ages were movement-restricted and exposed to 10 degrees C for 3-hr periods every other week while colonic temperature was measured. A longitudinal trend in cold tolerance related to age and to initial colonic temperature was demonstrated. Adaptative thermoregulatory changes during cold exposure occurred during the first two tests. These were similar for all age groups except 30-month-old mice. There was no adaptation of colonic temperature during cold exposure among aged mice with repeated testing; however, their baseline colonic temperatures prior to testing increased after the first two tests. This finding suggests that old animals adjust to repeated cold stress differently than do younger mice. Specifically, younger animals are capable of adjusting their thermoregulatory response during cold stress with no change in baseline (pre-stress) temperature. Old animals do not modify the responses emitted during the stress; however, they do adapt by raising their baseline temperatures. Repeated cold exposure started later in life increased mortality among old animals but did not affect maximum lifespan.

Adaptation, Physiological↗
PubMed1985

Measurement of nicotine in building air as an indicator of tobacco smoke levels.

Humans apparently differ greatly in their sensitivity and tolerance to tobacco smoke, thereby creating conflicts in the workplace. Resolution of conflicts in a large office complex at the authors' institution required an objective measure of smoke levels. A gas chromatographic technique was devised for collection and analysis of nicotine concentrations in the building air as an indicator of tobacco smoke pollution. Segregation of smokers and nonsmokers in the large office complex still resulted in substantial exposure of the nonsmoker to tobacco smoke, although a gradient of exposure was certainly observed. Passive tobacco smoke consumption in the smoking area of the office complex was calculated to be equivalent to 1.1 cigarettes per 8-hr period, and nicotine density in this area was 1.96 microgram/m. The restriction of smoking to a foyer area outside the office complex resulted in a slow but eventual reduction in nicotine concentrations in the office complex. Observed "background" nicotine concentration levels corresponding to 4 to 7% of those encountered in smoking areas demonstrate that central air circulation systems and people movement increase the nicotine level throughout all rooms of a building, regardless of the smoking policies of an individual office complex. Recent documentation of the relationship between passive smoking and cancer, heart disease, pulmonary dysfunction, and allergic responses argues for restriction of smoking to building exteriors.

Air Pollutants↗
PubMed1984

Age-related decrease in apomorphine modulation of acetylcholine release from rat striatal slices.

Release of [3H]acetylcholine ( [3H]ACh) was assessed in striatal slices from mature, middle-aged and senescent Wistar rats 8, 12 and 24 months of age, respectively. There was an age-related decline in basal release of [3H]ACh as a function of age which was correlated with a decline in accumulation of [3H]ACh. However, the most striking finding was the failure of apomorphine to inhibit KCl-induced [3H]ACh release in the senescent (24 months) animals. Striatal dopaminergic receptor losses in senescence apparently produce several subsequent changes in striatal function which ultimately result in the decline of motor-behavioral function.

Acetylcholine↗
PubMed1984

Some aspects of the mechanism of complexation of red kidney bean alpha-amylase inhibitor and alpha-amylase.

Bovine pancreatic alpha-amylase binds 1 mol of acarbose (a carbohydrate alpha-amylase inhibitor) per mol at the active site and also binds acarbose nonspecifically. The red kidney bean alpha-amylase inhibitor-bovine pancreatic alpha-amylase complex retained nonspecific binding for acarbose only. Binding of p-nitrophenyl alpha-D-maltoside to the final complex of red kidney bean alpha-amylase inhibitor and bovine pancreatic alpha-amylase has a beta Ks (Ks') value that is 3.4-fold greater than the Ks (16 mM) of alpha-amylase for p-nitrophenyl alpha-D-maltoside alone. The initial complex of alpha-amylase and inhibitor apparently hydrolyzes this substrate as rapidly as alpha-amylase alone. The complex retains affinity for substrates and competitive inhibitors, which, when present in high concentrations, cause dissociation of the complex. Maltose (0.5 M), a competitive inhibitor of alpha-amylase, caused dissociation of the red kidney bean alpha-amylase inhibitor--alpha-amylase complex. Interaction between red kidney bean (Phaseolus vulgaris) alpha-amylase inhibitor and porcine pancreatic alpha-amylase proceeds through two steps. The first step has a Keq of 3.1 X 10(-5) M. The second step (unimolecular; first order) has a forward rate constant of 3.05 min-1 at pH 6.9 and 30 degrees C. alpha-Amylase inhibitor combines with alpha-amylase, in the presence of p-nitrophenyl alpha-D-maltoside, noncompetitively. On the basis of the data presented, it is likely that alpha-amylase is inactivated by the alpha-amylase inhibitor through a conformational change. A kinetic model, in the presence and absence of substrate, is described for noncompetitive, slow, tight-binding inhibitors that proceed through two steps.

Animals↗
PubMed1984

31P NMR chemical shifts of phosphate covalently bound to proteins.

31P nuclear magnetic resonance (NMR) spectroscopy for characterizing the nature of covalently bound phosphate in proteins is relatively unexploited by the biochemist. 31P NMR chemical shifts of phosphate covalently bound to naturally occurring phosphoproteins, phosphorylated enzyme intermediates and chemically phosphorylated proteins have been compiled in this review. The chemical shifts (31P NMR) of selected reference compounds are reported to assist in the assignment of 31P resonances of phosphate covalently attached to proteins. 31P NMR chemical shifts of phosphate and phospho compounds non-covalently bound to selected proteins as well as the pH dependence of 31P NMR resonance have also been compiled.

Animals↗
PubMed1984

Age-related decline in cold tolerance can be retarded by brain stimulation.

The sharp decline in capacity of old C57BL/6J male mice to maintain body temperature during 3-hr cold exposure can be delayed and even partly restored after 15, 30-min, daily sessions of hypothalamic self-stimulation. In control experiments it was demonstrated that electrical stimulation of "rewarding" areas of hypothalamus itself is sufficient for improvement of the age-related deterioration of thermoregulatory ability.

Acclimatization↗
PubMed1983

Chemical and physical modification of proteins by the hydroxide ion.

Proteins are exposed to alkaline conditions during solubilization and/or purification, during food storage and processing, in removal of toxic constituents, and for characterization. During alkali treatment, there are changes in solubility and aggregation, hydrolysis, elimination reactions involving the side chains of certain amino acids, racemization of amino acid residues, addition of compounds to proteins, fragmentation of the peptide chain, as well as modification or elimination of nonprotein constituents. The rates of these reactions are affected by pH, temperature, cations (in some cases), ionic strength (in some cases), protein concentration, and to some extent by the specific nature of the protein. The general mechanisms and stoichiometry of these reactions are described. Other constituents of high protein foods also undergo reactions in alkaline solutions and the products of these reactions may in turn react with proteins. We have described the effect of alkali on enediol formation and fragmentation of carbohydrates, the hydrolysis of lipids in alkaline solution and effect on rate of peroxidation of the polyunsaturated fatty acids, the oxidation of amino acid residues, especially methionine, the oxidation of phenols to benzoquinones, and the catalytic effect of metal ions in alkaline solutions. Alkali treatment is also used in the specific modification of proteins to distinguish between O-glycosyl and amide-linked glycosyl groups, to effect specific cleavage of peptide bonds via beta elimination, in the formation of anhydrotrypsin, anhydrochymotrypsin, anhydrosubtilisin and thiol-subtilisin, and in formation of intrachain crosslinking in proteins.

Amides↗
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↗