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D Bhatnagar

Publications and source records attributed to D Bhatnagar.

At least 163 records · Page 9Linked to original sources
PubMed1987

Conversion of a new metabolite to aflatoxin B2 by Aspergillus parasiticus.

A new metabolite which could be converted to aflatoxin (AF) B2 was detected during cofermentation analysis of two nonaflatoxigenic strains (SRRC 2043 and SRRC 163) of Aspergillus parasiticus. SRRC 2043, which accumulates the xanthone O-methylsterigmatocystin (OMST), a late precursor in the AFB1 pathway, was observed to accumulate another chemically related compound (HOMST; molecular weight, 356); SRRC 163 is blocked early in the pathway and accumulates averantin. During cofermentation of the two strains, levels of OMST and HOMST were observed to be greatly reduced in the culture, with simultaneous production of AFB1, AFB2, and AFG1. Intact cells of SRRC 163 were able to convert pure OMST or its precursor, sterigmatocystin, to AFB1 and AFG1 without AFB2 accumulation; the same cells converted isolated HOMST to AFB2 with no AFB1 or AFG1 production. The results indicate that AFB2 is produced from a separate branch in the AF biosynthetic pathway than are AFB1 and AFG1; AFB2 arises from HOMST, and AFB1 and AFG1 arise from sterigmatocystin and OMST.

Aflatoxins↗
PubMed1987

Identification of O-methylsterigmatocystin as an aflatoxin B1 and G1 precursor in Aspergillus parasiticus.

An isolate of Aspergillus parasiticus CP461 (SRRC 2043) produced no detectable aflatoxins, but accumulated O-methylsterigmatocystin (OMST). When sterigmatocystin (ST) was fed to this isolate in a low-sugar medium, there was an increase in the accumulation of OMST, without aflatoxin synthesis. When radiolabeled [14C]OMST was fed to resting mycelia of a non-aflatoxin-, non-ST-, and non-OMST-producing mutant of A. parasiticus AVN-1 (SRRC 163), 14C-labeled aflatoxins B1 and G1 were produced; 10 nmol of OMST produced 7.8 nmol of B1 and 1.0 nmol of G1, while 10 nmol of ST produced 6.4 nmol of B1 and 0.6 nmol of G1. A time course study of aflatoxin synthesis in ST feeding experiments with AVN-1 revealed that OMST is synthesized by the mold during the onset of aflatoxin synthesis. The total amount of aflatoxins recovered from OMST feeding experiments was higher than from experiments in which ST was fed to the resting mycelia. These results suggest that OMST is a true metabolite in the aflatoxin biosynthetic pathway between sterigmatocystin and aflatoxins B1 and G1 and is not a shunt metabolite, as thought previously.

Aflatoxin B1↗
PubMed1987

Appearance of enzyme activities catalyzing conversion of sterigmatocystin to aflatoxin B1 in late-growth-phase Aspergillus parasiticus cultures.

Two activities involved in terminal pathway conversion of sterigmatocystin to aflatoxin B1 were isolated from an aflatoxin-nonproducing mutant of Aspergillus parasiticus (avn-1), and the time course of appearance of the activities in culture was determined. Subcellular fractionation of fungal mycelia resolved the two activities into a postmicrosomal activity which catalyzed conversion of sterigmatocystin to O-methylsterigmatocystin and a microsomal activity which converted O-methylsterigmatocystin to aflatoxin B1. The two activities were absent in 24-h-old cells, increased to optimum levels during the stationary phase, and then declined.

Aflatoxin B1↗
PubMed1987

Individual reaction requirements of two enzyme activities, isolated from Aspergillus parasiticus, which together catalyze conversion of sterigmatocystin to aflatoxin B1.

Individual reaction requirements were determined for each of two enzyme activities present in Aspergillus parasiticus mycelia which together catalyze conversion of sterigmatocystin (ST) to aflatoxin B1 (AFB1). A postmicrosomal activity (PMA) catalyzed conversion of ST to O-methylsterigmatocystin (OMST) and a microsomal activity (MA) catalyzed conversion of OMST to AFB1. PMA was stimulated two- to three-fold in the presence of S-adenosylmethionine. Addition of NADPH promoted the maximum MA; this activity was not detected when FAD, FMN, NAD, or NADH were utilized individually as cofactors in reaction mixtures. A substantial amount (62%) of MA was lost during isolation of the microsomal fraction, but the activity was completely restored by reconstitution with a heat-treated (100 degrees C) postmicrosomal fraction. The reaction catalyzed by MA was optimum at pH 7.0 and at 17-23 degrees C, whereas the PMA reaction was optimum at pH 8.0-8.5 and at 35-40 degrees C. Apparent Km values of approximately 2.6 X 10(-6) M (for ST) and 6.6 X 10(-7) M (for OMST) were determined for PMA and MA, respectively.

Aflatoxin B1↗
PubMed1987

Does the measurement of serum fructosamine accurately reflect levels of glycated albumin in insulin-dependent diabetes?

We have measured serum glycated albumin (GSA) by affinity chromatography and immunoturbidimetry, and serum fructosamine using a Cobas FARA analyser in blood samples from 37 type I diabetics and 21 healthy controls. Random blood glucose and glycated haemoglobin levels were also measured. Glycated haemoglobin (HbA1) correlated with glycated albumin and fructosamine in the diabetic group. A less clear relationship was found between glycated albumin and fructosamine. HbA1, GSA and fructosamine correlated poorly with random blood glucose levels. These data indicate that serum fructosamine levels do not accurately reflect those of glycated albumin, as has recently been suggested, in type I insulin-dependent diabetics where glycaemic control fluctuates more than in type II diabetics. It is postulated that the two methods reflect varying glycaemic levels to a different degree, thereby accounting for the disparity.

Adolescent↗
PubMed1985

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: fluorescent affinity labeling of the catalytic subunit from bovine skeletal muscle with o-phthalaldehyde.

The catalytic subunit of adenosine cyclic 3',5'-monophosphate dependent protein kinase from bovine skeletal muscle was rapidly inactivated by o-phthalaldehyde at 25 degrees C (pH 7.3). The reaction followed pseudo-first-order kinetics, and the second-order rate constant was 1.1 X 10(2) M-1 s-1. Absorbance and fluorescence spectroscopic data were consistent with the formation of an isoindole derivative (1 mol/mol of enzyme). The reaction between the catalytic subunit and o-phthalaldehyde was not reversed by the addition of reagents containing free primary amino and sulfhydryl functions following inactivation. The reaction, however, could be arrested at any stage during its progress by the addition of an excess of cysteine or less efficiently by homocysteine or glutathione. The catalytic subunit was protected from inactivation by the presence of the substrates magnesium adenosine triphosphate and an acceptor serine peptide substrate. The decrease in fluorescence emission intensity of incubation mixtures containing iodoacetamide- or 5'-[p-(fluorosulfonyl)benzoyl]adenosine-modified catalytic subunit and o-phthalaldehyde paralleled the loss of phosphotransferase activity. Catalytic subunit denatured with urea failed to react with o-phthalaldehyde. Inactivation of the catalytic subunit by o-phthalaldehyde is probably due to the concomitant modification of lysine-72 and cysteine-199. The proximal distance between the epsilon-amino function of the lysine and the sulfhydryl group of the cysteine residues involved in isoindole formation in the native enzyme is estimated to be approximately 3 A. The molar transition energy of the catalytic subunit-o-phthalaldehyde adduct was 121 kJ/mol and compares favorably with a value of 127 kJ/mol for the 1-[(beta-hydroxyethyl)thio]-2-(beta-hydroxyethyl)isoindole in hexane, indicating that the active site lysine and cysteine residues involved in formation of the isoindole derivative of the catalytic subunit are located in a hydrophobic environment. o-Phthalaldehyde probably acts as an active site specific reagent for the catalytic subunit.

Affinity Labels↗
PubMed1985

Inactivation of guanosine cyclic 3',5'-monophosphate dependent protein kinase from bovine lung by o-phthalaldehyde.

Guanosine cyclic 3',5'-monophosphate (cGMP) dependent protein kinase is inactivated by o-phthalaldehyde. The loss of phosphotransferase activity following treatment with o-phthalaldehyde was rapid, and the second-order rate constant at 25 degrees C and pH 7.3 was 35 M-1 s-1. The inactivation reaction did not follow saturation kinetics. The cGMP-dependent protein kinase was protected from inactivation by its substrates, MgATP and Ser-peptide. Fluorescence excitation and emission spectroscopic data showed that an isoindole derivative was formed following the reaction between cGMP-dependent protein kinase and o-phthalaldehyde. Four moles of isoindole per mole of the cGMP-dependent protein kinase dimer was formed following complete inactivation by o-phthalaldehyde. In the absence of cGMP, the protein kinase lost only 50% of its cGMP binding activity while there was almost a complete loss of its phosphotransferase activity. Studies in the presence of 20 microM cGMP, however, showed that about 2 mol of isoindole groups per mole of the protein kinase dimer was formed following complete inactivation by o-phthalaldehyde. The second-order rate constant for inactivation of cGMP-dependent protein kinase by o-phthalaldehyde in the presence of 20 microM cGMP was 40 M-1 s-1. Fluorescence measurements of samples containing inactivated, iodoacetamide-modified, or 5'-[p-(fluorosulfonyl)benzoyl]adenosine-modified, cGMP-dependent protein kinase and o-phthalaldehyde showed that the intensity of fluorescence in each case was about 50% of that obtained from unmodified, active cGMP-dependent protein kinase and o-phthalaldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehydes↗
PubMed1985

Interaction of guanosine cyclic 3',5'-phosphate dependent protein kinase with lin-benzoadenine nucleotides.

Using the activated cGMP-dependent protein kinase in the presence of the phosphorylatable peptide [[Ala34]histone H2B-(29-35)], we found that lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) was a competitive inhibitor of the enzyme with respect to ATP with a Ki (22 microM) similar to the Kd (20 microM) determined by fluorescence polarization titrations. The Kd for lin-benzo-ADP determined in the absence of the phosphorylatable peptide, however, was only 12 microM. ADP bound with lower affinity (Ki = 169 microM; Kd = 114 microM). With [Ala34]histone H2B-(29-35) as phosphoryl acceptor, the Km for lin-benzo-ATP was 29 microM, and that for ATP was 32 microM. The Vmax with lin-benzo-ATP, however, was only 0.06% of that with ATP as substrate [0.00623 +/- 0.00035 vs. 11.1 +/- 0.17 mumol (min.mg)-1]. Binding of lin-benzo-ADP to the kinase was dependent upon a divalent cation. Fluorescence polarization revealed that Mg2+, Mn2+, Co2+, Ni2+, Ca2+, Sr2+, and Ba2+ supported nucleotide binding to the enzyme; Ca2+, Sr2+, and Ba2+, however, did not support any measurable phosphotransferase activity. The rank order of metal ion effectiveness in mediating phosphotransferase activity was Mg2+ greater than Ni2+ greater than Co2+ greater than Mn2+. Although these results were similar to those observed with the cAMP-dependent protein kinase [Hartl, F. T., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 2347], major differences in the Vmax with lin-benzo-ATP as substrate and the effect of peptide substrates on nucleotide (both lin-benzo-ADP and ADP) binding were observed.

Animals↗
PubMed1985

A parallel study of enalapril and captopril and 1 year of experience with enalapril treatment in moderate-to-severe essential hypertension.

Angiotensin converting enzyme (ACE) inhibitors, enalapril (5 to 20 mg twice daily) or captopril (25 to 100 mg thrice daily) and matching ACE inhibitor placebos were given to 32 moderate-to-severe essential hypertension patients who were already on 50 mg hydrochlorothiazide daily. Alpha-methyldopa (250 to 500 mg twice daily) was given to 16 patients following 6 weeks of ACE inhibitor therapy. Both enalapril and captopril significantly (p less than 0.05) lowered the supine and upright blood pressures (BPs) (acutely and long-term) without significant reflex heart rate changes. The BPs of enalapril patients were, however, significantly lower (supine diastolic p less than 0.03, supine systolic p less than 0.05, and upright diastolic p less than 0.04) than those of captopril patients when compared by repeated measures of analysis of variance. Eleven enalapril patients have been followed for 1 year with continued BP control. Skin rash occurred in one captopril patient and reversible renal insufficiency developed in two enalapril patients during the first 16 weeks. It is concluded that (although both ACE inhibitors lowered BP, enalapril was more effective than captopril and twice-daily enalapril was well tolerated during 52 weeks of treatment.

Blood Pressure↗
PubMed1984

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: nucleotide binding to the chemically modified catalytic subunit.

5'-[p-(Fluorosulfonyl)benzoyl]adenosine (FSBA) inactivates the catalytic subunit of the adenosine cyclic 3',5'-monophosphate dependent protein kinase isolated from bovine cardiac muscle by covalent modification of lysine-71, whereas 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) react with cysteines-199 and -343 to inactivate the enzyme. All three of these reagents have been postulated to modify residues at or near the active site of the catalytic subunit. ATP (2 mM) in the presence of excess Mg2+ (10 mM) protects the enzyme against inactivation by these reagents. AMP did not afford any protection, but adenosine slightly decreased the rate of inactivation. The specific effects of covalent modification of lysine-71 and cysteines-199 and -343 on nucleotide binding were characterized by fluorescence-polarization titrations with lin-benzoadenine nucleotides as fluorescent ligands. lin-Benzoadenosine is a competitive inhibitor of the catalytic subunit with respect to ATP with a Ki (38 microM) similar to the Ki for adenosine (35 microM). This value agrees well with the Kd (32 microM) for adenosine determined by fluorescence-polarization titrations. lin-Benzoadenosine 5'-diphosphate (lin-benzo-ADP) has been shown to be a competitive inhibitor with respect to ATP [Hartl, F. T., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 2347], and lin-benzoadenosine 5'-triphosphate (lin-benzo-ATP) is a substrate for the phosphotransferase activity of the protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Chloro-7-nitrobenzofurazan↗
PubMed1983

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: a new fluorescence displacement titration technique for characterizing the nucleotide binding site on the catalytic subunit.

We determined the dissociation constant (Kd) of a series of nucleotides for the bovine skeletal muscle type II catalytic subunit by displacing lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) with increasing concentrations of competing nucleotide. The Kd of each nucleotide was calculated from the decreases in the fluorescence polarization of lin-benzo-ADP that accompany its displacement from the catalytic subunit. We found that modifications of the adenine moiety reduce nucleotide affinity for the enzyme. The effect was most pronounced with modifications at position 6 of the base. Replacement of the 3'-hydroxyl group of ribose with a hydrogen increased the affinity of the nucleotide; addition of phosphate to the 2'- or 3'-hydroxyl groups, on the other hand, decreased nucleotide affinity. MgATP and MgADP exhibited Kd's of about 10 microM. AMP, which contains a negatively charged alpha-phosphate, bound with reduced affinity (643 microM). Adenosine, which lacks a charged alpha-phosphate, bound with a higher affinity (32 microM). To learn more about the nature of the alpha-phosphate binding site, a series of uncharged and positively charged derivatives of the 5'-position on the ribose moiety was prepared. The uncharged derivatives bound with much greater affinity than the negatively charged AMP. The Kd's for 5'-tosyladenosine and 5'-iodo-5'-deoxyadenosine were 30 and 32 microM, respectively. Like the negatively charged AMP, positively charged derivatives also bound less tenaciously than the neutral species. The positively charged 5'-amino-5'deoxyadenosine, for example, exhibited a 15-fold higher Kd (506 microM) than the neutral congenors.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1982

Site-specific interaction of ATPase-pumped protons with photosystem II in chloroplast thylakoid membranes.

The chloroplast thylakoid ATPase proton pump-driven H+ accumulation in the dark was compared to the light-dependent proton pump driven by either photosystem II or I, in regard to the effects of the resultant acidity on chemical modification reactions. The assays used to detect the acidity effects were: (a)the incorporation of [3H]-acetic anhydride into membrane protein -NH2 groups, and (b) the effect of a certain level of that chemical modification on inhibition of photosystem II water oxidation activity. Based on labeling data with [3H]-acetic anhydride, 20-30 nmol.(mg chl)-1 of -NH3+ groups appear to be metastable in the dark in untreated membranes. The term metastable is used because proton leak-inducing treatments in the dark lead to about 20-30 nmol . (mg chl)-1 increase in acetic anhydride labeling probably due to reaction with the -NH2 form of amine groups. Addition of low levels of uncoupler or a brief thermal treatment caused a loss of protons from the membrane equivalent to the increase in acetic anhydride derivatization. The increase in acetic anhydride derivatization caused inhibition of water oxidation activity. Using thermally sensitized membranes, photosystem II but not photosystem I electron transport (each giving a steady-state proton accumulation of about 50 nmol H+ . (mg chl)-1 restored the lower level of acetic anhydride reactivity as in previous results (Baker et al., 1981). In dark-maintained, thermally treated membranes, ATPase activity, i.e., the proton pump associated with it, also restored the lower level of acetic anhydride labeling, and again acetic anhydride no longer inhibited water oxidation. Because photosystem I activity did not elicit this type of response to acetic anhydride, there appears to be a pathway for ATPase pumped protons which allows them to reach a restricted domain, perhaps intramembrane, common with the photosystem II water oxidation mechanism and unavailable to protons pumped by photosystem I. The membrane structure(s) which determines this site specificity is not yet understood.

Adenosine Triphosphatases↗