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M Hamada

Publications and source records attributed to M Hamada.

At least 649 records · Page 36Linked to original sources
PubMed1976

Effect of guanidine hydrochloride on the holo- and apo-enzymes of pig heart lipoamide dehydrogenase.

1. The effect of guanidine hydrochloride (GuHCl) on pig heart lipoamide dehydrogenase [NADH: lipoamide oxidoreductase, EC 1.6.4.3.] was investigated by means of enzymatic activity and optical measurements (CD, absorption, and fluorescence spectra). The activity of the enzyme decreased on increasing the concentration of GuHCl and the enzyme was completely inactivated in 2.0 M GuHCl. 2. The contents of alpha-helix, beta, and unordered forms in lipoamide dehydrogenase were estimated to be 34, 14, and 52%, respectively. On increasing the concentration of GuHCl, the content of alpha-helix in lipoamide dehydrogenase decreased, whereas the content of the beta form hardly changed. 3. The native lipoamide dehydrogenase showed absorption, CD, and fluorescence spectra characteristic of bound FAD in the visible region, suggesting hydrophobic interaction between the protein moiety and FAD chromophore. The absorption, CD, and fluorescence spectra of the enzyme in 2.0 M GuHCl were similar to those of free FAD in the buffer, suggesting the release of FAD from the protein moiety. 4. The protein fluorescence spectrum of lipoamide dehydrogenase had a maximum at 350 nm blue-shifted by 8 nm from that of tryptophan in aqueous solution. The maximum of the enzyme in 2.0 M GuHCl was red-shifted to 357 nm. This suggests exposure of tryptophan residues to a polar environment. The maximum, 352nm, of the apoenzyme shifted to 350 nm on addition of FAD. These results show that the conformation in the microenvironment of some tryptophan residues in lipoamide dehydrogenase is affected by the dissociation-association of FAD. 5. The contents of alpha-helix, beta, and unordered forms in the apoenzyme were estimated to be 35, 8, and 57%, respectively. These values are similar to those of the native holoenzyme. The alpha-helical structure in the apoenzyme molecule was more sensitive to GuHCl than that in the holoenzyme. FAD and two hydrophobic probes, 8-anilinonaphthalene-1-sulfonate (ANS) and 4 benzolamido-4'-aminostilbene-2,2'-disulfonate (MBAS), which can bind to the apoenzyme, stabilized the alpha-helical structure in the apoenzyme molecule.

Animals↗
PubMed1976

Interaction of hydrophobic probes with the apoenzyme of pig heart lipoamide dehydrogenase.

The interaction of hydrophobic probes, 8-anilinonaphthalene-1-sulfonate (ANS) and 4-benzoylamido-4'-aminostilbene-2, 2'-disulfonate (MBAS), with pig heart lipoamide dehydrogenase [NADH: lipoamide oxidoreductase, EC 1.6.4.3] was investigated. When ANS or MBAS was mixed with the apoenzyme of lipoamide dehydrogenase, the fluorescence quantum yield, of each dye was enhancedd markedly and the emission maxima concurrently shifted to the blue. The quantum yield, 0.038, of ANS bound to the apoenzyme, calculated from the corrected emission spectrum, was eight times higher than that in buffer solution, and the value, 0.0090, for bound MBAS was eighteen times higher than that in buffer solution. Moreover, the absortion bands of both ANS and MBAS shifted to the red upon binding with the apoenzyme. A general feature of the absorption spectra of these dyes observed on changing the solvent from polar to apolar was a red shift of the absorption bands. These results indicate that ANS or MBAS bound to the apoenzyme of lipoamide dehydrogenase is situated in a hydrophobic region of the apoenzyme molecule. It was found that 2 moles of each dye was bound per mole of the apoenzyme, which contains two polypeptide chains. The dissociation constants for the ANS- and MBAS-apoenzyme complexes were estimated to be 1.03X10(-5) and 1.54X10(-5) M, respectively. The enhanced fluorescence of both dyes bound to the apoenzyme decreased linearly upon adding FAD and disappeared at about 2 moles of FAD per mole of the apoenzyme. This suggests that both ANS and MBAS were displaced from their binding sites on the apoenzyme by FAD. The protein fluorescence spectrum of the apoenzyme had a maximum at 352 nm, which was blue-shifted by 6 nm from that of tryptophan in the buffer. Upon binding ANS or MBAS, the maximum of the protein fluorescence of the apoenzyme returned to 350 nm for the holoenzyme, and the fluorescence intensity decreased. Thus, the conformation around some tryptophan residues was affected by the binding of the dyes. When guanidine hydrochloride (GuHCl) was added to the ANS-apoenzyme complex solution, the enhanced fluorescence due to the bound ANS decreased and the emission maximum concurrently shifted to the red. Further, the maximum of the protein fluorescence of the apoenzyme shifted to the red, indicating the exposure of some tryptophan residues buried in an apolar region of the apoenzyme. Thus the binding of ANS to the apoenzyme was inhibited by protein denaturation due to GuHCL. In contrast, the holoenzyme of lipoamide dehydrogenase did not bind ANS or MBAS at all.

Anilino Naphthalenesulfonates↗
PubMed1976

Properties and subunit structure of pig heart pyruvate dehydrogenase.

Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (alpha and beta), with estimated molecular weights of 41,000 (alpha) and 36,000 (beta), respectively, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as alpha2beta2. The content of right-handed alpha-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively. The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5 X 10(-5) M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and alpha-ketobutyrate, but it also showed some activity with alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuribenzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.

Amino Acids↗
PubMed1976

Structure-activity relationships among negamycin analogs.

Various negamycin analogs were examined for (1) miscoding activity and (2) inhibition of the termination of protein synthesis. Since properties (1) and (2) do not correlate for the investigated compounds they may depend on different structural features of negamycin analogs. The results of biochemical and antimicrobial studies indicate that (a) the natural configuration of the carbon atom carrying the beta-amino group is essential, (b) the delta-hydroxyl group is unnecessary, and (c) the acylation of the epsilon-amino group causes loss of activity.

Anti-Bacterial Agents↗
PubMed1975

A kinetic study of the alpha-keto acid dehydrogenase complexes from pig heart mitochondria.

The kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease complexes from pig heart mitochondria were studied at pH 7.5 and 25 degrees. A three-site ping-pong mechanism for the actin of both complexes was proposed on the basis of the parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvate concentration for various levels of CoA and a level of NAD+ near its Michaelis constant value. Rate equations were derived from the proposed mechanism. Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reaction are: 2-oxoglutarate, 0.220 mM; CoA, 0.025 mM; NAD+, 0.050 mM. Those of the pyruvate dehydrogenase complex are: pyruvate, 0.015 mM; CoA, 0.021 mM; NAD+, 0.079 mM. Product inhibition studies showed that succinyl-CoA or acetyl-CoA was competitive with respect to CoA, and NADH was competitive with respect to NAD+ in both overall reactions, and that succinyl-CoA or acetyl-CoA and NADH were uncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for succinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These results are consistent with the proposed mechanisms.

Acetyl Coenzyme A↗