Purification and properties of a neutral peroxidase isozyme from turnip (Brassica napus L. Var. Purple Top White Globe) roots.

A neutral peroxidase isozyme (pI 7.2) from turnip roots (TNP) was purified to homogeneity and partially characterized. TNP is a monomeric glycoprotein with 9.1% carbohydrate content and a molecular weight of 36 kDa. Optimum pH values for activity using 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and guaiacol as H donors were 4.5 and 5.5, whereas the K(m) values were 0.7 and 3.7 mM, respectively. The ABTS K(m) was approximately 7 times higher than that reported for basic commercial horseradish peroxidase (HRP-C). TNP retained approximately 70% activity after 11 min of heating at 65 degrees C, whereas the activation energy for inactivation (132 kJ/mol) was higher than or comparable to that of other peroxidases. The low ABTS K(m) and high specific activity (1930 units/mg) gave a high catalytic efficiency (500 M(-1) s(-1)). These properties make TNP an enzyme with a high potential as an alternative to HRP in various applications.

Purification and partial characterization of three turnip (Brassicanapus L. var. esculenta D.C.) peroxidases.

Three turnip peroxidases (fractions C1, C2, and C3) were partially purified and characterized, to permit study of their feasibility for use in clinical and enzyme immunoassays. These fractions represented 20% of the initial activity, and fractions C1 and C2 were purified to homogeneity. The optimum pH was between 5.0 and 5.5, while optimum temperature ranged from 40 to 55 degrees C. The ABTS K(m) values for the two acidic fractions (C2 and C3) were 0.70 and 0.42 mM, respectively; about 5 times lower than that reported for the acidic commercial horseradish peroxidase (HRP). Fraction C3 had 4 times higher K(m) value than commercial cationic HRP. The molecular weights determined by SDS-PAGE ranged from 39.2 to 42.5 kDa. Activation energies for inactivation were 113 (C1), 130 (C2), and 172 kJ/mol (C3) which are higher or comparable to other peroxidase isoenzymes reported. Fractions C1 and C3 represent an alternative source of peroxidase because of their higher purification yield and specific activity, when compared to fraction C2.

Biochemical characterization of cystine lyase from broccoli (Brassica oleracea var. italica).

Cystine lyase is the enzyme responsible for off-aroma deterioration in fresh unblanched broccoli. In this research, cystine lyase purification from broccoli has been optimized. Only one protein peak with cystine lyase activity was found during purification. Broccoli cystine lyase was purified 100-fold to homogeneity. L-Cystine, L-cysteine-S-sulfate, L-djenkolic acid, and some S-alkyl-L-cysteines and their sulfoxides are substrates, but the enzyme had negligible activity with L-cystathionine. A K(m) value of 81.2 microM was found for L-cystine. Inhibition and K(i) determinations indicated that L-cysteine is a linear noncompetitive inhibitor with a K(i) of 5 mM and DL-homocysteine is a competitive inhibitor with a K(i) of 1.5 mM. The molecular weight of cystine lyase was determined to be 100 kDa by three methods, with two subunits of 48 kDa each and a carbohydrate content of 3%. Further characterization included cofactor quantification, the effects of temperature and pH on activity and stability, and amino acid composition.

Methionine-containing proteins in two Phaseolus vulgaris cultivars with different methionine bioavailabilities.

The full utilization of legumes as human food is limited by a deficiency of sulfur amino acids, low protein digestibility, low methionine bioavailability and the presence of anti-nutritional factors. A new cultivar of Phaseolus vulgaris (Carioca 80) has 56.8% available methionine, compared with 29.3% availability in the parent cultivar Carioca. The total methionine content, denaturing gel electrophoretic patterns of methionine-containing proteins, and the percentage of phaseolins (the major storage proteins in Phaseolus) relative to the total protein are similar in the two cultivars. Although the digestibility of the two cultivars is similar, the increased biological value of Carioca 80 may indicate that there are differences in overall bean composition that affect protein hydrolysis and utilization. We suggest the tentative explanation that this is due to differences in the distribution of methionine in the methionine-containing proteins of the two cultivars.

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.

Characteristics of the Inhibition of Potato (Solanum tuberosum) Invertase by an Endogenous Proteinaceous Inhibitor in Potatoes.

Effect of several parameters on inhibition of potato (Solanum tuberosum) invertase by its endogenous proteinaceous inhibitor was determined using homogeneous preparations of both proteins. The inhibitor and invertase formed an inactive complex with an observed association rate constant at pH 4.70 and 37 degrees C of 8.82 x 10(2) per molar per second and a dissociation rate constant of 3.3 x 10(-3) per minute. The inhibitor appeared to bind to invertase in more than one step. Initial interaction (measured by loss of invertase activity) was rapid, relatively weak, readily reversible (K(i) of 2 x 10(-6) molar) and noncompetitive with substrate at pH 4.70. Initial interaction was probably followed by isomerization to a tighter (K(i) of 6.23 x 10(-8) molar) complex, which dissociated slowly with a half-time of 3.5 hour. Interaction between enzyme and inhibitor appeared to be of ionic character and essentially pH independent between pH 3.5 and 7.4.

Purification and Partial Characterization of Potato (Solanum tuberosum) Invertase and Its Endogenous Proteinaceous Inhibitor.

Invertase plays an important role in the hydrolysis of sucrose in higher plants, especially in the storage organs. In potato (Solanum tuberosum) tubers, and in some other plant tissues, the enzyme seems to be controlled by interaction with an endogenous proteinaceous inhibitor. An acid invertase from potato tubers (variety russet) was purified 1560-fold to electrophoretic homogeneity by consecutive use of concanvalin A-Sepharose 4B affinity chromatography, DEAE-Sephadex A-50-120 chromatography, Sephadex G-150 chromatography, and DEAE-Sephadex A-50-120 chromatography. The enzyme contained 10.9% carbohydrate, had an apparent molecular weight of 60,000 by gel filtration, and was composed of two identical molecular weight subunits (M(r) 30,000). The enzyme had a K(m) for sucrose of 16 millimolar at pH 4.70 and was most stable and had maximum activity around pH 5. The endogenous inhibitor was purified 610-fold to homogeneity by consecutive treatment at pH 1 to 1.5 at 37 degrees C for 1 hour, (NH(4))(2)SO(4) fractionation, Sephadex G-100 chromatography, DEAE-Sephadex G-50-120 chromatography, and hydroxylapatite chromatography. The inhibitor appears to be a single polypeptide (M(r) 17,000) without glyco groups. The purified inhibitor was stable over the pH range of 2 to 7 when incubated at 37 degrees C for 1 hour.

Synthesis and degradation of nitrate reductase during the cell cycle of Chlorella sorokiniana.

Studies on the diurnal variations of nitrate reductase (NR) activity during the life cycle of synchronized Chlorella sorokiniana cells grown with a 7:5 light-dark cycle showed that the NADH:NR activity, as well as the NR partial activities NADH:cytochrome c reductase and reduced methyl viologen:NR, closely paralleled the appearance and disappearance of NR protein as shown by sodium dodecyl sulfate gel electrophoresis and immunoblots. Results of pulse-labeling experiments with [35S]methionine further confirmed that diurnal variations of the enzyme activities can be entirely accounted for by the concomitant synthesis and degradation of the NR protein.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).