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Biomedical subjects

F Ahmad

Publications and source records attributed to F Ahmad.

At least 181 records · Page 10Linked to original sources
PubMed1978

Purification and subunit structure of rat mammary gland acetyl coenzyme A carboxylase.

1. Acetyl coenzyme A carboxylase from lactating rat mammary gland has been purified to apparent homogeneity. 2. The purified enzyme has the following characteristics: (a) its specific activity approaches 15 units/mg of protein, (b) the sedimentation constants of the protomeric and polymeric forms of the enzyme are 12 to 13 S and greater than or equal to 40 S, respectively, (c) the polymeric form of the enzyme shows filamentous structures in the electron microscope, and (d) the polypeptide(s) arising from its dissociation reveals a single major component of Mr = 240,000 to 260,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 3. The enzyme contains 1 mol of biotin and approximately 6 mol of phosphate/240,000 g of protein.

Acetyl-CoA Carboxylase↗
PubMed1978

Solubilization and partial characterization of a phosphoprotein phosphatase from human myelin.

The phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) solubilized from human central nervous system myelin has been shown to possess a comparatively high degree of specificity towards myelin basic protein, a constituent of the membrane and most likely its natural substrate, rather than the mixed histones. The enzyme has a pH optimum of 7.5. Hydrolysis of both the substrates is stimulated by dithiothreitol and is almost completely dependent upon the presence of divalent metal ions. The maximum rate of dephosphorylation of basic protein is attained in the presence of 125 micrometer Mn2+ whereas a much higher concentration of Mg2+ (50--100 mM) is required for the optimal dephosphorylation of histones. The dephosphorylation of basic protein was also stimulated by Triton X-100 (0.15%, v/v) and was shown to result from a 3-fold increase in the V of the reaction catalyzed by the phosphatase. The apparent Km values for basic protein and histones were unaffected by the presence of Triton X-100 and were found to be approx. 1 and approx. 160 micrometer, respectively. Under optimal conditions of assay, the phosphatase cleaved approx. 32 and approx. 0.7 nmol of orthophosphate.min-1.mg-1 of protein from basic protein and histones, respectively.

Corpus Callosum↗
PubMed1978

Thermodynamics of the denaturation of pepsinogen by urea.

The denaturation of swine pepsinogen has been studied as a function of urea concentration, pH, and temperature. The unfolding of the protein by urea has been found to be fully reversible under different conditions of pH, temperature, and denaturant concentration. Kinetic experiments have shown that the transition shows two-state behavior at 25 degrees C in the pH range 6-8 covered in this study. Analysis of the equilibrium data obtained at 25 degrees C according to Tanford (Tanford, C. (1970), Adv. Protein Chem. 24, 1) and Pace (Pace, N.C. (1975), Crit. Rev. Biochem. 3, 1) leads to the conclusion that the free energy of stabilization of native pepsinogen, relative to the denatured state, under physiological conditions, is only 6-12 kcal mol-1. The temperature dependence of the equilibrium constant for the unfolding of pepsinogen by urea in the range 20-50 degrees C at pH 8.0 can be described by assigning the following values of thermodynamic parameters for the denaturation at 25 degrees C: deltaH=31.5 kcal mol-1; deltaS=105 cal deg-1 mol-1; and deltaCp=5215 cal deg-1 mol-1.

Animals↗
PubMed1978

The denaturation of covalently inhibited swine pepsin.

Studies are reported on the denaturation of freshly prepared, intact swine pepsin, which was inactivated by reaction with diazoacetylglycine ethyl ester, to prevent autolysis. Denaturation about pH 6 was found to involve a small expansion of the molecular domain with some loss of organized secondary structure. On the other hand, increasing concentrations of guanidine hydrochloride induced cooperative transitions in both the native and alkali denatured forms to give a cross-linked random coil. No conditions could be found in which these reactions were reversible. Removal of denaturing conditions usually resulted in aggregation and precipitation of protein. From these studies, it would seem that the active conformation is largely predetermined in the zymogen.

Animals↗
PubMed1978

Inorganic salt denaturants stabilize ribonuclease against denaturation by urea.

The isothermal denaturation of ribonuclease A by mixed denaturant systems was investigated at 25 degrees C. It was observed that low concentrations of lithium chloride stabilize the protein against denaturation by urea, even though the salt itself is a denaturant. This study also provides, for the first time, the most convincing evidence that the lithium chloride denatured ribonuclease A contains some of the native secondary and tertiary structure.

Animals↗
PubMed1977

The denaturation of beta-lactoglobulin-A at pH 2.

The denaturation of beta-lactoglobulin-A by heat and guanidine hydrochloride at pH 2 has been investigated. The effect of ethylene glycol on the thermal denaturation at this pH has also been studied. The conditions of the experiments have been chosen so as to eliminate complications arising out of disulfide interchange, changes in the degree of association of the protein during denaturation, and intermolecular aggregation. The physical parameters characterizing the denatured states of the protein which are produced by heat and guanidine hydrochloride have been determined. The thermodynamic parameters for these transitions have been estimated using a two-state hypothesis in each case. Both the physical and thermodynamic parameters indicate that the heat-denatured state of beta-lactoglobulin-A retains about 15-20% of residual structure which is destroyed on adding guanidine hydrochloride.

Circular Dichroism↗
PubMed1976

Reversible unfolding of the major fraction of ovalbumin by guanidine hydrochloride.

The guanidine hydrochloride induced unfolding of the major fraction of ovalbumin (i.e. A1 which contains two phosphate groups and constitutes about 77% of the total protein) was investigated systematically by difference spectran and viscosity measurements. As judged by the intrinsic viscosity (3.9 ml/g), the native protein conformation is compact and globular. Difference spectral results showed extensive disruption of the native structure by guanidine hydrochloride with and without 0.1 M beta-mercaptoethanol were 31.1 and 27.0 ml/g. These and optical rotation results indicated that the denatured protein existed in a cross-linked random coil conformation in 6 M guanidine hydrochloride alone. Strikingly, in contrast to whole ovalbumin, the denaturation of its A1 fraction by guanidine hydrochloride was fully reversible and obeyed first-order kinetic law under different experimental condit ions of pH, temperature, and the denaturant concentration. The monotonic variation of deltaH for the unfolding of ovalbumin A1 by guanidine hydrochloride with temperature, the coincidence of the two transition curves obtained by measuring two independent properties (namely reduced viscosity and difference in light absorption at 288 nm (or 293 nm) as a function of the denaturant concentration, and finally the adherence of the unfolding as well as refolding reactions to first-order kinetic law suggested that the transition of ovalbumin. A1 can reasonably be approximated by a two-state mode. Analysis of the equilibrium data obtained at pH 7.0 and 25 degrees C according to Aune and Tanford (Aune, K.C.,and Tanford, C. (1969), Biochemistry 8, 4586) showed that 12 additional binding sites for the denaturant with an association constant of 1.12 were freshly exposed by the unfolding process and that the native protein was marginally more stable (approximately 6 kcal/mol) than its unfolded form even under native condition. The temperature dependence of the equilibrium constant for the unfolding of ovalbumin A1 by guanidine hydrochloride which was studied in the range 10-60 degrees C at pH 7.0 can be described by assigning the following values of the thermodynamic parameters for the unfolding process: deltaH = 52 kcal/mol at 25 degrees C; deltaS = 153 cal deg-1 mol-1 at 25 degrees C; and delta Cp = 2700 +/- 400 cal deg-1 mol-1.

Calorimetry↗
PubMed1975

Isolation of peptides from the carboxyl carrier subunit of transcarboxylase. Role of the non-biotinyl peptide in assembly.

Transcarboxylase is made up of a central hexameric subunit (S20,W similar 12 S), three peripheral dimeric metallo subunits (S20,W similar to 5 S), and six biotinyl carboxyl carrier subunits (S20,W similar to 1.3 S). The results presented here show that the carboxyl carrier subunit is required for assembly of the 12S and 5S subunits into the oligomer. However, only a portion of the subunit is required for assembly. On treatment of transcarboxylase briefly with trypsin at pH 6.3 extremely susceptible peptide bonds of the carboxyl carrier protein are cleaved releasing biotinyl peptides of about similar to 66 and similar to 40 residues. The resulting trypsinized transcarboxylase, though enzymatically inactive, remains essentially intact as judged by its hydrodynamic and molecular sieving properties. The modified enzyme can be dissociated at pH 8 to the central 12S subunit and peripheral 5S subunit to which the residual portion(s) of the cleaved carboxyl carrier protein is still attached. These components can then be separated by molecular sieving. The residual portion of the carboxyl carrier protein (non-biotinyl peptide) can then be isolated by dissociation of the 5S subunit complex at pH 9 and by chromatography over Bio-Gel A-1.5m. The isolated non-biotinyl peptide has been shown to contain the combining domain of the 1.3SE carboxyl carrier protein since it causes combination of the 12S and 5S subunits. Active enzyme is formed by combination of the intact carboxyl carrier protein and the 12S and 5S subunits and an inactive oligomer of similar size is formed if the non-biotinyl peptide is used in place of the carboxyl carrier protein. The similar to 66- and similar to 40-residue biotinyl peptides, which are released by the trypsin treatment, apparently occur on an exposed portion of the enzyme. This portion of the carboxyl carrier protein apparently serves to place the biotinyl group adjacent to the two substrate sites of the enzyme, one of which is on the peripheral subunit and the other on the central subunit. Thus the carboxyl carrier protein has two functions: one portion holds the 12S and 5S subunits in juxtaposition and the other portion orients the biotinyl group adjacent to the substrate sites so that it may function as a carboxyl carrier between the sites.

Acetyltransferases↗
PubMed1975

Evidence that the two partial reactions of transcarboxylation are catalyzed by two dissimilar subunits of transcarboxylase.

The results presented here show that isolated subunits of transcarboxylase specifically catalyze the two partial reactions of transcarboxylation as shown in eq 1-3. The 12S central subunit is active in the transcarboxylation with methylmalonyl-CoA but inactive with oxalacetate and the peripheral metallo 5S subunit is active in the transcarboxylation with oxalacetate but inactive with methylmalonyl-CoA. These subunits, likewise, are specific for the reverse partial reactions; the central subunit catalyzing transfer from the carboxylated biotinyl group to propionyl-CoA to yield methylmalonyl-CoA and the peripheral subunit to pyruvate to yield oxalacetate. Thus, the central subunit contains the sites for the CoA esters (methylmalonyl-CoA and propionyl-CoA) and the peripheral metallo subunits for the keto acids (oxalacetate and pyruvate). In the overall reaction the biotinyl carboxyl carrier protein acts as a shuttle to carry the carboxyl groups between the two subunits. Biotin and certain biotin analogs are inactive in these partial reactions but the similar to 40- or similar to 66-residue biotinyl peptides, which are derived from the carboxyl carrier protein, are active. Transcarboxylase can be reconstituted from its isolated subunits and a comparison was made of the rate of the overall reaction when the subunits were assembled, as in the intact enzyme, with that obtained when the reaction was catalyzed by the nonassembled subunits. In the latter case, since the biotinyl carboxyl carrier subunit must diffuse from one subunit to the other, the overall reaction is much slower than with the assembled subunits. The reaction with trypsinized transcarboxylase from which the similar to 66-residue and similar to 40-residue biotinyl peptides have been stripped, likewise, was slow even though the biotinyl peptides were added to the reconstitution mixture. The 12SH and 5SE subunits remain assembled after trypsin treatment but the biotinyl peptides apparently do not combine firmly or properly with the trypsinized enzyme and the biotinyl group apparently must oscillate as a carboxyl carrier between the two sites on the subunits by diffusion.

Acetyltransferases↗
PubMed1975

Intrinsic viscosity of ovomucoid in random coil conformation.

Ovomucoid is denatured by concentrated solutions of guanidine hydrochloride. The intrinsic viscosities of the glycoprotein in 6 M guanidine hydrochloride in the absence and presence of beta-mercaptoethanol were found to be 8.1 and 16.0 ml/g, respectively. Ovomucoid with disulphide bonds reduced exists in linear random coil conformation. However, the intrinsic viscosity of the randomly coiled protein was less than that predicted from the empirical equations describing the molecular weight dependence of intrinsic viscosities of random coil proteins in 6 M guanidine hydrochloride. On excluding the carbohydrate content of the protein, which is theoretically justified, the calculated intrinsic viscosity interestingly became closer to the measured one. The temperature dependence of the intrinsic viscosity of ovomucoid in linear random coil conformation was studied in the temperature range, 25-55 degrees. The features of the intrinsic viscosity-temperature profile are not comparable with those exhibited by other linear random coil proteins in 6 M guanidine hydrochloride.

Chemical Phenomena↗