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Biomedical subjects

F Ahmad

Publications and source records attributed to F Ahmad.

At least 163 records · Page 9Linked to original sources
PubMed1982

Estimation of the free energy of stabilization of ribonuclease A, lysozyme, alpha-lactalbumin, and myoglobin.

The denaturation of ribonuclease A, lysozyme alpha-lactalbumin, and myoglobin by urea, guanidine hydrochloride, and guanidine thiocyanate has been followed with the use of difference spectral measurements. The free energy of stabilization (delta GH2OD) has been determined by the linear extrapolation of the free energy of denaturation to zero denaturant concentration. The values of delta GH2OD are 7.3 +/- 0.2, 8.9 +/- 0.1, 4.3 +/- 0.1;, and 7.9 +/- 0.2 kcal/mol at 25 degrees C for ribonuclease A (pH 7.0), lysozyme (pH 7.0), alpha-lactalbumin (pH 7.0), and myoglobin (pH 6.6), respectively. The dependence of the free energy of denaturation on concentration ranges from 0.88 to 2.08 kcal/mol.M for urea and from 1.27 to 4.22 kcal/mol.M for guanidine hydrochloride for four proteins. The ratio of this dependence in guanidine hydrochloride to that in urea may depend on the polarity of the amino acid residues in the unfolding unit.

Animals↗
PubMed1982

Irreversible inhibition of fatty acid synthase from rat mammary gland with S-(4-bromo-2,3-dioxobutyl)-CoA. Effect on the partial reactions, protection by substrates and stoichiometry studies.

Fatty acid synthase from lactating rat mammary gland is rapidly and irreversibly inhibited by S-(4-bromo-2,3-dioxobutyl)-CoA. Of the seven partial reactions catalysed by the enzyme, the inhibition of the overall catalytic activity is closely paralleled only by inhibition of the beta-oxoacyl synthase (condensing) partial reaction. Three partial reactions. Beta-oxoacyl reductase, beta-hydroxyacyl dehydratase and enoyl reductase, are inhibited to a modest degree. The three partial reactions known to involve an acyl-CoA/CoA-binding site, acetyl acyltransferase, malonyl acyltransferase and palmitoyl thioesterase, are not inhibited by S-(4-bromo-2,3-dioxobutyl)-CoA. The modification process does not cause the enzyme to dissociate into catalytically incompetent monomers. Stoichiometric studies suggest that approx. 6 mol of reagent are incorporated per mol of totally inhibited enzyme (dimer). The formation of acylated enzyme from either acetyl-CoA or malonyl-CoA protects the enzyme equally well against S-(4-bromo-2,3-dioxobutyl)-CoA. Also, pretreatment of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid), a thiol-specific reagent reported to block essential thiol groups in the condensing partial reaction, protects against inhibition by the reagent. On the other hand, the presence of up to 770 microM-S-acetonyl-CoA or dethio-CoA does not protect the enzyme from irreversible inhibition. Together, the results suggest that the primary inhibitory process is a bimolecular reaction resulting in alkylation of essential thiol groups in the condensing partial reaction: this process does not require the obligatory formation of a Michaelis-Menten complex of enzyme and reagent before the alkylation reaction.

Acyl Coenzyme A↗
PubMed1982

Rat mammary-gland fatty acid synthase. A simple purification procedure and stoicheiometry of CoA ester binding.

A simple procedure was devised which allows purification of rat lactating-mammary-gland fatty acid synthase to a high degree of purity, with recoveries of activity exceeding 50%. Over 50 mg of enzyme was isolated from 60 g of mammary tissue. The specific activity of the purified enzyme was about 2.5 mumol of NADPH oxidized/min per mg of protein at 37 degrees. The enzyme appeared homogeneous by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by immunodiffusion analysis. Each mol (Mr 480 000) of the enzyme bound 3 mol of acetyl and 3-4 mol of malonyl groups when the binding experiments were performed at 0 degrees for 30 s. The presence of NADPH did not influence the binding stoicheiometry for these acyl-CoA derivatives. Approx. 2 mol of taurine was found per mol of the performic acid-oxidized enzyme, suggesting that there were 2 mol of 4'-phosphopantetheine in the native enzyme. Rat mammary-gland fatty acid synthase required free CoA for activity.

Acyl Coenzyme A↗
PubMed1981

Detection of ligand-induced perturbations affecting the biotinyl group of mammalian acetyl-coenzyme A carboxylase by using biotin-binding antibodies.

Biotin-binding antibodies were raised in rabbits by injecting biotin-bovine serum albumin conjugate. Neither the protomer nor the polymer of rat mammary-gland acetyl-CoA carboxylase formed precipitin bands with the anti-biotin. By virtue of its ability to bind biotin (apparent binding constant for free biotin about 1mum), the anti-biotin inhibited the carboxylase activity under certain conditions. This property of the antibody was employed to detect the ligand-induced changes affecting the biotinyl group in different conformational states of mammalian carboxylase. Depending on the ligand present, the biotinyl group in the protomeric form was either accessible or inaccessible to the antibody. The biotinyl group of the protomer generated by a relatively high concentration of NaCl (0.5m) reacted with the antibody, and the antibody-carboxylase complex could not be converted into active enzyme by citrate. Further experiments showed that citrate failed to induce polymerization in this protomer-antibody complex and that anti-biotin could be displaced rapidly from this complex with excess of biotin. The resulting protomer was converted into the polymeric state on citrate addition, with parallel regain of enzyme activity. In the presence of ADP+Mg(2+), ATP+Mg(2+) or ATP+Mg(2+)+HCO(3) (-), however, the enzyme remained as a protomer, but its configuration was such that the biotinyl group was essentially inaccessible to the antibody. Likewise, the biotinyl group of the different polymeric forms of the carboxylase (s approximately 30-45S) engendered by phosphate, malonyl-CoA, acetyl-CoA or citrate remained essentially inaccessible, since their activity was minimally affected by the anti-biotin. In the presence of 0.15m-NaCl, the phosphate-induced polymer reverted to a approximately 19S form with concomitant appearance of anti-biotin-sensitivity, whereas the other polymeric forms remained unaffected under similar experimental conditions.

Acetyl-CoA Carboxylase↗
PubMed1981

Acyl coenzyme A carboxylase of Propionibacterium shermanii: detection and properties.

An acyl coenzyme A (CoA) carboxylase, which catalyzes the adenosine triphosphate-dependent fixation of CO2 into acetyl-, propionyl-, and butyryl-CoA, was detected in fractionated cell extracts of Propionibacterium shermanii. Catalytic activity was inhibited by avidin but was unaffected by avidin pretreated with excess biotin. The carboxylase levels detected were relatively small and were related to cellular growth. Maximal carboxylase activity was detected in cells grown for about 96 h. Thereafter, the activity declined rapidly. Optimal CO2 fixation occurred at pH 7.5. Other parameters of the assay system were optimized, and the apparent Km values for substrates were determined. The end product of the reaction (with acetyl-CoA as the substrate) was identified as malonyl-CoA. The stoichiometry of the reaction was such that, for every mole of acetyl-CoA and adenosine triphosphate consumed, 1 mol each of malonyl-CoA, adenosine diphosphate, and orthophosphate was formed. These data provide the first evidence for the presence of another biotin-containing enzyme, an acyl-CoA carboxylase, in these bacteria in addition to the well-characterized methylmalonyl-CoA carboxyltransferase.

Acetyl Coenzyme A↗
PubMed1981

Stability of acetylcholinesterase in guanidine hydrochloride solution.

The irreversible unfolding of acetylcholinesterase (acetylcholine hydrolyase, EC 3.1.1.7) by guanidine hydrochloride was studied by difference spectral, circular dichroic, and enzyme activity measurements. At pH 7.0 and in 1.1 M denaturant solution, a conformational state in which enzyme is completely inactive was detected. It is identical to the native enzyme as far as sedimentation coefficient and molecular weight are concerned, but differs from the native molecule by a slight loss in secondary structure and by a small perturbation of aromatic residues. Acetylcholinesterase in concentrated guanidine hydrochloride solution containing beta-mercaptoethanol dissociates and exists as a random coil of molecular weight 68 000.

Acetylcholinesterase↗
PubMed1980

Immunofluorescent localization of acetyl CoA carboxylase, fatty acid synthetase and pyruvate carboxylase during the adipocyte conversion of 3T3 fibroblasts.

Three enzymes involved in the conversion of 3T3-L2 fibroblasts into fat cells, acetyl CoA carboxylase (ACC), fatty acid synthetase (FAS) and pyruvate carboxylase (PC) have been localized by immunofluorescence techniques. The method enables the identification of cells undergoing the conversion while they are still fibroblastic in appearance, often before the obvious appearance of fat droplets. Specific fluorescence for each enzyme can be seen in "clones" of cells derived from single cells, which may undergo an event during logarithmic growth, which programs the cells to differentiate subsequent to confluence of and addition of induction medium.

Acetyl-CoA Carboxylase↗
PubMed1980

125I-insulin receptor binding to cord blood erythrocytes of varying gestational age and comparison with adult values.

The measurement of 125I-insulin binding to erythrocytes from 34 samples of cord blood (% B/T = 11.2 +/- 3.8) showed statistically significant differences when compared to adult erythrocytes (% B/T = 6.6 +/- 1.7). Preterm erythrocytes (% B/T = 14.7 +/- 3.4) bound significantly more insulin when compared to term erythrocytes (% B/T = 10.1 +/- 3.2). The negative correlation (r = -0.613; P = 0.001) between insulin binding and gestational age indicates that the trend during the final trimester of gestation is toward the lower binding observed for adult cells. The major differences in binding between adult and term cord blood erythrocytes may be primarily attributed to differences in the number of binding sites per cell. Additionally, there are small differences in both affinity and number of sites per cell in preterm as compared to term and adult erythrocytes.

Adult↗
PubMed1980

Comparison of conventional and immunofluorescent techniques for the detection of Entamoeba histolytica in rectal biopsies.

Two comparative studies of six staining techniques in detection of Entamoeba histolytica in rectal biopsies were performed. The techniques evaluated were direct and indirect fluorescent antibody technique, and four conventional stains: PAS, H & E, Trichrome, and PTAH). On ethanol-fixed tissue, the direct fluorescent antibody technique was the most sensitive, detecting amoebas in 84% of rectal biopsies from 19 aspirate-positive dysentery patients. PAS staining was specific, almost as sensitive, and amoebas could be rapidly detected. The other three conventional methods, however, detected amoebas in only 53--58% of the aspirate-positive patients and required considerable efforts to find the amoebas. The indirect fluorescent antibody technique performed on ethanol-fixed tissues was comparable to the direct technique. The same technique used on formalin-fixed tissues had high background staining, but amoebas could still be detected more efficiently than with conventional staining. Following 20 hr or more treatment with metronidazole, no patient had amoebas detectable in their rectal biopsies by any technique used. Patients with amoebic dysentery and necrotic ulcers on protoscopy had amoebas detected more frequently in rectal biopsy than did patients with mild disease as determined by proctoscopy. Entamoeba histolytica in rectal biopsies can be detected most easily in ethanol-fixed tissue stained with either a fluorescent antibody or with PAS. If amoebas are not detected in the rectal biopsy, amoebic colitis cannot be excluded.

Adult↗
PubMed1979

Increase in fatty acid synthetase content of 3T3-L cells undergoing spontaneous and chemically induced differentiation to adipocytes.

3T3-L fibroblasts differentiate into adipose cells when maintained in a non-growing state. The specific activity of fatty acid synthetase of differentiated cells was 25--30-fold higher than that present in 3T3-L fibroblasts or in 3T3-C2 cells that possess an extremely low incidence of differentiation to adipocytes. The results of immunochemical analysis indicate that the increased specific activity of fatty acid synthetase in the differentiated cells is due to an increase in the cellular content of this enzyme. The rate of conversion of adipose cells was accelerated by brief exposure of confluent non-growing cultures of 3T3-L cells to 3-isobutyl-1-methylxanthine. This was accompanied by an increase in the specificity activity of fatty acid synthetase, which was also shown to be due to an increase in the cellular content of this enzyme. The continuous presence of 3-isobutyl-1-methylxanthine in the culture medium was not required to elicit the morphological and biochemical changes in 3T3-L cells that occurred many days after the removal of the inducer but earlier than the onset of spontanous differentiation.

1-Methyl-3-isobutylxanthine↗
PubMed1979

The pH dependence of the reversible unfolding of ovalbumin A1 by guanidine hydrochloride.

The pH dependence of the reversible guanidine hydrochloride denaturation of the major fraction of ovalbumin (ovalbumin A1) was studied by a viscometric method in the pH range 1-7, at 25 degrees C and at six different denaturant concentrations (1.5-2.6 M). At any denaturant concentrationa reduction in pH favoured the transition from the native to the denatured state. The latter was essentially 'structureless', as revealed by the fact that the reduced viscosity of the acid and guanidine hydrochloride denatured state of ovalbumin A1 (obtained at different denaturant concentrations in acidic solutions) was measured (at a protein concentration of 3.8 mg/ml) to be 29.2 ml/g which is identical to that found in 6 M guanidine hydrochloride wherein the protein behaves as a cross-linked random coil. A quantitative analysis of the results on the pH dependence of the equilibrium constant for the denaturation process showed that on denaturation the intrinsic pK of two carboxyl groups in ovalbumin A1 went up from 3.1 in the native state to 4.4 in the denatured state of the protein.

Guanidines↗
PubMed1979

Characterization of a stable intermediate in the unfolding of diazoacetylglycine ethyl ester--pepsin by urea.

The irreversible unfolding of covalently inhibited swine pepsin by urea was studied by spectrophotometric and viscosity measurements. At pH 4.5 and 25 degrees C in 8 M urea, a stable intermediate form of the protein was detected. It differed from the native protein by a slight loss of secondary structure and an increased intrinsic viscosity ([pi] = 7.5 mL g-1), indicating the intermediate to have an increased molecular volume or to be more asymmetric in shape. The protein was transformed into a random coil form by increases of temperature and pH. Comparison with other results suggested that at pH 6 pepsin is less stable than its inactive precursor, pepsinogen, by about 3 Kcal mol-1 (1 cal = 4.187 J).

Azo Compounds↗
PubMed1978

Spectrophotometric titration of phenolic groups of pepsin.

The ionization of tyrosine residues in diazotized pepsin under various solvent conditions was studied. All tyrosyl residues of the protein titrated normally with a pK of 10.02 in 6 M guanidine hydrochloride solution. On the other hand, two stages in the phenolic group titration curve were observed for the inactivated protein in the absence of guanidine hydrochloride; only about 10 tyrosine residues ionized reversibly up to pH 11, above which titration was irreversible. The irreversible titration zone corresponds to the pH range 11--13 in which unfolding, leading to the random coil state, was shown to occur by circular dichroism and viscosity measurements. The number of tyrosine residues exposed in the native and alkali-denatured (pH 7.5) states of diazotized protein were also studied by solvent perturbation techniques; 10 and 12 groups are exposed in the native and denatured states, respectively.

Animals↗