Purification and characterization of S-adenosylhomocysteine deaminase from streptonigrin-producing Streptomyces flocculus.

An S-adenosylhomocysteine deaminase has been isolated and purified from streptonigrin-producing Streptomyces flocculus ATCC 13257. Deamination represents the major metabolic route of S-adenosylhomocysteine in this organism. The protein was found to be monomeric with a molecular weight of 56,100 +/- 1,600. The activity was optimal at pH 7.0 and 37 degrees C, and the deaminase was inactivated by p-chloromercuribenzoate but not by metal chelators. The Km for S-adenosylhomocysteine is 2.5 mM, and the Ki for inhibition by deoxycoformycin is 1.6 nM.     

Thermodynamic investigations of proteins. III. Thermodynamic description of lysozyme.

Standard functions of enthalpy, entropy and the Gibbs energy of native and denatured lysozyme in the range of 0-100 degrees C and pH 1.5-7.0 are represented in three-dimensional projections. The denaturational Gibbs energy change reaches 16 kcal mol-1 at conditions of maximal protein stability (0 degrees C, pH 4.5-7.0) and equals 14.5 kcal mol-1 at 25 degrees C and neutral pH. This result was found to be in agreement with the data reported from guanidine hydrochloride denaturation studies. Partial thermodynamic functions of the conformational and ionizational changes of the protein are obtained from entropy and Gibbs-energy changes in denaturation. The conformational partial entropy and Gibbs-energy change are found to be independent of pH. The pH-dependent partial ionizational entropy and Gibbs-energy changes are induced by normalization of the ionization behaviour of buried groups and cause a decrease of protein stability.     

An acidic protease from the grass carp intestine (Ctenopharyngodon idellus)

The acidic Protease was extracted from the intestine of the grass carp (Ctenopharyngodon idellus) by 0.1 M sodium phosphate buffer, pH 7.0 at 4 degrees C after neat intestine was defatted with acetone, and partially purified by ammonium sulfate precipitation, gel filtration chromatography and ionic exchange chromatography. SDS-PAGE electrophoresis showed that the enzyme was homogeneous with a relative molecular mass of 28,500. Substrate-PAGE at pH7.0 showed that the purified acidic protease has only an active component. Specificity and inhibiting assays showed that it should be a cathepsin D. The optimal pH and optimal temperature of the enzyme were pH2.5 and 37 degrees C, respectively. It retained only 20% of its initial activity after incubating at 50 degrees C for 30 min. The enzyme lost 81% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride (PMSF). Its V(max) and K(m) values were determined to be 3.57 mg/mL and 0.75 min(-1), respectively.     

Purification and characterization of an endochitinase produced by Colletotrichum gloeosporioides

The phytopathogenic fungus Colletotrichum gloeosporioides was analyzed for chitinase activity, the best production occurring at the fourth day. A 43 kDa endochitinase with specific activity of 413 U microg(-1) protein was purified corresponding to a 75% yield. The optima of temperature and pH for the enzyme were 50 degrees C and pH 7.0, respectively. The enzyme showed a high stability at 50 degrees C and pH 7.0. Values of pH from 5.0 up to 7.0 gave, at least, 50% of maximum activity, suggesting a biotechnological application. Further studies are in progress to determine the possible use of this endochitinase in biological control.     

Kinetic and thermodynamic analysis of thermal unfolding of recombinant erythropoietin

Thermal stress was used to assess the stability of recombinant human erythropoietin (EPO) derived from Chinese hamster ovary cells. In 20 mm phosphate at pH 7.0, this protein had a highly reversible thermal unfolding as observed by far UV circular dichroism (CD) and native gel analysis, with no indication of protein aggregation. It had a relatively low melting temperature at 53 degrees C. Assuming a two-state transition, the observed reversibility permits thermodynamic analysis of the unfolding of EPO, which shows that the free energy of unfolding at 25 degrees C is only 6-7 kcal/mol. Upon heating to 79 degrees C over 30 min, however, this protein does undergo aggregation as assessed by native gel. In 20 mm phosphate and citrate at pH 7.0, the results are similar, i.e., EPO suffered a substantial aggregation, while it showed little aggregation in 20 mm Tris or histidine at pH 7.0 and 20 mm glycine at pH 6.3 under identical heat treatment.     

Redox and flavin-binding properties of recombinant flavodoxin from Desulfovibrio vulgaris (Hildenborough).

Flavodoxin from Desulfovibrio vulgaris (Hildenborough) has been expressed at a high level (3-4% soluble protein) in Escherichia coli by subcloning a minimal insert carrying the gene behind the tac promoter of plasmid pDK6. The recombinant protein was readily isolated and its properties were shown to be identical to those of the wild-type protein obtained directly from D. vulgaris, with the exception that the recombinant protein lacks the N-terminal methionine residue. Detailed measurements of the redox potentials of this flavodoxin are reported for the first time. The redox potential, E2, for the couple oxidized flavodoxin/flavodoxin semiquinone at pH 7.0 is -143 mV (25 degrees C), while the value for the flavodoxin semiquinone/flavodoxin hydroquinone couple (E1) at the same pH is -440 mV. The effects of pH on the observed potentials were examined; E2 varies linearly with pH (slope = -59 mV), while E1 is independent of pH at high pH values, but below pH 7.5 the potential becomes less negative with decreasing pH, indicating a redox-linked protonation of the flavodoxin hydroquinone. D. vulgaris apoflavodoxin binds FMN very tightly, with a value of 0.24 nM for the dissociation constant (Kd) at pH 7.0 and 25 degrees C, similar to that observed with other flavodoxins. In addition, the apoflavodoxin readily binds riboflavin (Kd = 0.72 microM; 50 mM sodium phosphate, pH 7.0, 5 mM EDTA at 25 degrees C) and the complex is spectroscopically very similar to that formed with FMN. The redox potentials for the riboflavin complex were determined at pH 6.5 (E1 = -262 mV, E2 = -193 mV; 25 degrees C) and are discussed in the light of earlier proposals that charge/charge interactions between different parts of the flavin hydroquinone play a crucial role in determining E1 in flavodoxin.     

Reassessment of the cold-labile nature of phosphofructokinase from a hibernating ground squirrel.

This study reassesses the proposal that cellular conditions of low temperature and relative acidosis during hibernation contribute to a suppression of phosphofructokinase (PFK) activity which, in turn, contributes to glycolytic rate suppression during torpor. To test the proposal that a dilution effect during in vitro assay of PFK was the main reason for activity loss (tetramer dissociation) at lower pH values, the influence of the macromolecular crowding agent, polyethylene glycol 8000 (PEG), on purified skeletal muscle PFK from Spermophilus lateralis was evaluated at different pH values (6.5, 7.2 and 7.5) and assay temperatures (5, 25 and 37degrees C). A 78 +/- 2.5% loss of PFK activity during 1 h incubation at 5 degrees C and pH 6.5 was virtually eliminated when 10% PEG was present (only 7.0 +/- 1.5% activity lost). The presence of PEG also largely reversed PFK inactivation at pH 6.5 at warmer assay temperatures and reversed inhibitory effects by high urea (50 or 400 mM). Analysis of pH curves at 5 degrees C also indicated that approximately 70% of activity would remain at intracellular pH values in hibernator muscle. The data suggest that under high protein concentrations in intact cells that the conditions of relative acidosis, low temperature or elevated urea during hibernation would not have substantial regulatory effects on PFK.     

Optimal assay conditions for liver UDP-glucuronosyltransferase from the rainbow trout, Salmo gairdneri

Several kinetic characteristics and assay dependence of UDP-glucuronosyltransferase were studied with microsomal preparations made from liver of rainbow trout. The optimal enzyme assay, performed by incubating less than 5 mg microsomal protein/ml assay buffer for 20 min at 25 degrees C and in pH 7.0, contains 2.5 X 10(-5) M p-nitrophenol (p-NP) and 2.5 X 10(-3) M UDP-glucuronic acid (UDPGA). Apparent Km values revealed that the affinity of trout enzyme for p-NP and UDPGA is, respectively, about 70 and 10 times higher than that of rat enzyme. The optimized method will be used for aquatic bioassays, e.g. when assessing the influencing of toxic effluents from the pulp and paper industry.     

Use of a genetically engineered Escherichia coli strain to produce 1,2-dihydroxy-4'-chlorobiphenyl.

Genetically engineered kanamycin-resistant Escherichia coli HB101 containing the mutant chimeric plasmid pAW6194-T17 specifying biphenyl dioxygenase and dihydrodiol dehydrogenase and lacking the ability to produce active 3-phenylcatechol dioxygenase was used to produce 1,2-dihydroxy-4'-chlorobiphenyl (DHCB) from 4-chlorobiphenyl. Resting-cell suspensions of genetically engineered E. coli in mineral salts medium (pH 7.0) containing 880 microM 4-chlorobiphenyl produced 110 microM DHCB. The Km for 4-chlorobiphenyl was 3.3 mM. Biotransformation of DHCB from 4-chlorobiphenyl was maximum when cells (2.5 mg of protein per ml) were incubated with shaking (150 rpm) at pH 7.0 and 30 degrees C for 6 h. The enzymatically produced DHCB was a suitable substrate for assaying 3-phenylcatechol dioxygenase activity. Biologically produced DHCB showed UV and mass spectra similar to those of chemically synthesized DHCB. The bioconversion rate of ortho-substituted chlorobiphenyl was slower than that of the para- or meta-substituted chlorobiphenyl.     

The role of epsilon-amino groups of lysine of human serum albumin in heat-induced protein denaturation. Increased stability of glycosylated and alkylated albumin in aggregation during heating

Human serum albumin was glycosidated by prolonged protein incubation in phosphate buffer, pH 6.8-7.0, with excess glucose at 37 degrees C. epsilon-amino groups of lysine residues of the albumin molecule were alkylated by pyridoxal-5-phosphate in the presence of NaBH4. The solutions of glycosidated and alkylated serum albumin were incubated at different temperature values in the range of 20 to 80 degrees C in phosphate buffer, pH 7.0, over 30 min. The nondenatured monomer and the resulting aggregated were isolated by TSK-HW-55-gel column chromatography and polyacrylamide gel electrophoresis. The stability of modified proteins elevated in parallel to the increase in the number of the ligand molecules covalently bound to albumin amino groups. The 1-3% aqueous solutions of glycosidated serum albumin containing 3-4 glucose residues and those of alkylated albumin containing 6-7 residues of pyridoxal-5-phosphate were stable on heating up to 80 degrees C and did not form aggregates. Under these conditions the initial serum albumin completely aggregated. Preincubation of the aggregated albumin with glucose at 37 degrees C resulted in protein "renaturation" to the monomeric form with a small number of dimers and trimers.     

Nuclear-magnetic-resonance studies of ferrocytochrome c. pH and temperature dependence

The pH dependence and the temperature dependence of the nuclear magnetic resonance spectrum of horse ferrocytochrome c are described. This protein is very stable; it maintains an ordered structure over the pH range 4 to 12 at 25 degrees C and over the temperature range 4 degrees C to 97 degrees C at pH 7.0. The dynamic characteristics of the conformation of ferrocytochrome c were investigated. Particular emphasis was laid on the aromatic resonances and resonances of methyl groups shifted far upfield. Tyr-48 and Phe-46 were found to be relatively immobile whilst a region of the protein close to Ile-57 was found to be relatively flexible.     

The salting-out behavior of human plasma fibronectin and its possible correlation with heparin-induced cryoprecipitation of the protein

The solubility of human plasma fibronectin in concentrated ammonium sulfate solutions was measured at pH 7.0 and varying temperatures as well as at 25 degrees C and varying pHs. The salting-out parameters, KS and beta were found to increase linearly with temperature in the range 5 degrees-50 degrees C. KS-pH and beta-pH profiles were found to have maxima at pH 7.0. The dependence of both of the solubility parameters of plasma fibronectin on temperature and pH was thus found to be anomalous. The possibility of a correlation between the heparin-induced cryoprecipitation of fibronectin and the dependence of its solubility parameters on pH and temperature is considered. It is suggested that heparin-induced precipitation of human plasma fibronectin at low temperatures is caused by (i) a cold effect and (ii) conformational change in the protein due to heparin binding.     

Use of a genetically engineered Escherichia coli strain to produce 1,2-dihydroxy-4'-chlorobiphenyl.

Genetically engineered kanamycin-resistant Escherichia coli HB101 containing the mutant chimeric plasmid pAW6194-T17 specifying biphenyl dioxygenase and dihydrodiol dehydrogenase and lacking the ability to produce active 3-phenylcatechol dioxygenase was used to produce 1,2-dihydroxy-4'-chlorobiphenyl (DHCB) from 4-chlorobiphenyl. Resting-cell suspensions of genetically engineered E. coli in mineral salts medium (pH 7.0) containing 880 microM 4-chlorobiphenyl produced 110 microM DHCB. The Km for 4-chlorobiphenyl was 3.3 mM. Biotransformation of DHCB from 4-chlorobiphenyl was maximum when cells (2.5 mg of protein per ml) were incubated with shaking (150 rpm) at pH 7.0 and 30 degrees C for 6 h. The enzymatically produced DHCB was a suitable substrate for assaying 3-phenylcatechol dioxygenase activity. Biologically produced DHCB showed UV and mass spectra similar to those of chemically synthesized DHCB. The bioconversion rate of ortho-substituted chlorobiphenyl was slower than that of the para- or meta-substituted chlorobiphenyl.     

Some properties of cathepsins chemically fixed to carriers.

An insoluble preparation of rat liver cathepsin D was obtained by coupling the enzyme to Enzacryl Polyacetal (EPA-cathepsin) and to CNBr-activated Sepharose 4B. EPA-cathepsin was active toward the synthetic hexapeptides (Gly-Phe-Leu)2 and did not split hemoglobin. The optimum pH of splitting was displaced upward by 1.5 units to pH 5.0. The enzyme exhibited maximum activity at 60 degrees C. No appreciable loss of activity was seen on storage of the enzyme for 4 months or after repeated use of the preparations. Coupling of rat liver cathepsin D to activated Sepharose gave preparations active towards both protein and synthetic substrates. The preparations were totally inactive in acid media and exhibited maximum activity at pH 7.0, that is, under physiological conditions. Optimum temperature was 65 degrees. The specific activity of the preparations (pH 7.0, 65 degrees) was 60-110 percent that of the free enzyme in acid media. Proteolytic activity of the Sepharose-coupled cathepsin D was not inhibited by pepstatin, whereas that of the free enzyme was fully inhibited by this reagent. A sarcoma cathepsin, similar in some of its properties to the rat liver enzyme, was also coupled to CNBr-activated Sepharose 4B. The preparation split protein substrates at pH 7.0 and possessed enhanced thermostability. The enzymes fixed on Sepharose showed increased stability.     

Lipid peroxidation in buffalo (Bubalus bubalis L.) spermatozoa: effect of added vitamin C and glucose

Addition of 2.5 mM vitamin C or 40 mM of glucose to washed buffalo spermatozoan suspensions in Ca2(+)-free Kreb's Ringer Hanseliet saline buffer (pH 7.0) resulted in significant lower malonaldehyde concentration and higher spermatozoan motility and liver spermatozoa compared to control levels after 45 min of aerobic incubation at 37 degrees C or pre-incubation levels.     

Sublethal heat stress of Vibrio parahaemolyticus.

When Vibrio parahaemolyticsu ATCC 17802 was heated at 41 degrees C for 30 min in 100 mM phosphate-3% NaCl buffer (pH 7.0), the plate counts obtained when using Trypticase soy agar containing 0.25% added NaCl (0.25 TSAS) were nearly 99.9% higher than plate counts using Trypticase soy agar containing 5.5% added NaCl (5.5 TSAS). A similar result was obtained when cells of V. parahaemolyticus were grown in a glucose salts medium (GSM) and heated at 45 degrees C. The injured cells recovered salt tolerance within 3 h when placed in either 2.5 TSBS or GSM at 30 degrees C. The addition of chloramphenicol, actinomycin D, or nalidixic acid to 2.5 TSBS during recovery of cells grown in 2.5 TSBS indicated that recovery was dependent upon protein, ribonucleic acid (RNA, and deoxyribonucleic acid (DNA) synthesis. Penicillin did not inhibit the recovery process. Heat-injured, GSM-grown cells required RNA synthesis but not DNA synthesis during recovery in GSM. Chemical analyses showed that total cellular RNA decreased and total cellular DNA remained constant during heat injury. The addition of [6-3H]uracil, L-[U-14C]leucine, and [methyl-3H]thymidine to the recovery media confirmed the results of the antibiotic experiments.     

Kinetic studies on thermal denaturation of C-phycocyanin

The kinetics of thermal denaturation of a biliprotein, C-phycocyanin (C-PC) isolated from Spirulina platensis were studied at different pH values, ranging from 4.0 to 8.0. The denaturation of C-PC follows the first order kinetics and rate constant at pH 5.0 and temperature 55 degrees C is found to be 4.37 x 10(-5) s(-1), which increases to 5.46 x 10(-1) s(-1) at pH 7.0. The denaturation rate is much higher at 65 degrees C and pH 7.0 (7.96 x 10(-4)), as compared to at pH 5.0 (1.46 x 10(-4)). The thermal stability of C-PC is more at pH 5.0, as compared to other pH values. The observed differences in entropy values at pH 5.0, as compared to other pH values indicate a considerably close fit structure of the protein at pH 5.0, which increases the stability of native structure, even at higher temperature (65 degrees C).     

Purification and properties of an alpha-D-xylosidase from Aspergillus niger

Two components of alpha-D-xylosidase (alpha-D-xylosidase I and II) were detected in the culture filtrate of Aspergillus nigher grown in a medium containing Sanzyme 1000-treated Glyloid 2A. The major component (alpha-D-xylosidase I) was purified to an electrophoretically pure state. The purified enzyme showed approximately 540-fold increase in specific activity over the original culture filtrate. The purified enzyme was shown to be an oligomeric protein consisting of four subunits, each of which had a molecular weight of 123,000. The enzyme showed the highest activity at pH 2.5-3.0 and 45 degrees C, and was stable in the pH range from 3.0 to 7.0 and at the temperatures up to 60 degrees C. The isoelectric point of this enzyme was pH 5.6. The purified enzyme was highly specific for p-nitrophenyl alpha-D-xylopyranoside and isoprimeverose (6-O-alpha-D-xylopyranosyl-D-glucopyranose). The apparent Km and Vmax values of the enzyme for p-nitrophenyl alpha-D-xylopyranoside and isoprimeverose were 10.5 mM and 40.8 mumol/min/mg protein, and 2.2 mM and 30 mumol/min/mg protein, respectively. The purified enzyme could also split off the alpha-D-xylopyranosyl residue on the non-reducing terminal of the backbone of oligoxyloglucans such as alpha-D-xylopyranosyl-(1----6)-beta-D-glucopyranosyl- (1----4)-[(alpha-D-xylopyranosyl-(1----6)-]-beta-D-glucopyranosyl- (1----4)-] 2-D-glucopyranose.     

The purification and characterization of a dextranase from Lipomyces starkeyi

Dextranase produced by Lipomyces starkeyi was purified 43-fold, by carboxymethyl-Sepharose chromatography followed by agarose gel-filtration chromatography. The purified enzyme showed four bands by SDS/polyacrylamide gel electrophoresis with estimated mass 74 kDa, 71 kDa, 68 kDa and 65 kDa. This preparation exhibited multiple isoelectric points between 5.6 and 6.1. All the isoelectric forms were active and catalytically similar. The dextranase contained a carbohydrate moiety (8%). The physical properties of the enzyme were pH and temperature optima of 5.0 and 55 degrees C, respectively. This dextranase was stable between pH 2.5 and 7.0 at temperatures below 40 degrees C. Lipomyces dextranase was a typical endodextranase with the final product of dextran hydrolysis being isomalto-oligosaccharides from glucose to isomaltotetrose.