ENZYMATIC OXIDATION OF Α-Ketoglutarate AND COUPLED PHOSPHORYLATION

Kaufman, Seymour; Gilvarg, Charles; Cori, Osvaldo; Ochoa, Severo

doi:10.1016/s0021-9258(19)52356-0

Cited by 168 publications

(12 citation statements)

References 23 publications

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“…140-fold purification achieved with KG dehydrogenase (Kaufman et at., 1953;Hiroshima et al, 1967). Electrophoresis of the DEAE-cellulose eluate (after dialysis of 2 ml of a 7% enzyme solution vs. barbiturate buffer, pH 8.6; 225 V; 3 mA) showed, after 1 hr, a trailing shoulder (on an otherwise single symmetrical peak) which represented about 5 % of the total protein.…”

Section: Resultsmentioning

confidence: 99%

Carboligase activity of α-ketoglutarate dehydrogenase

Schlossberg¹,

Bloom²,

Richert³

et al. 1970

Biochemistry

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Section: Resultsmentioning

confidence: 99%

Carboligase activity of α-ketoglutarate dehydrogenase

Schlossberg¹,

Bloom²,

Richert³

et al. 1970

Biochemistry

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“…Succinyl-CoA synthetase was isolated from E. coli as described previously (Bowman & Nishimura, 1975). The enzyme was assayed by the method of Kaufman et al (1953), as modified by Grinnell and Nishimura (1969). The method depends on the continuous conversion of succinyl-CoA in the presence of hydroxylamine to succinyl hydroxamate, which is detected as its ferric complex (Lipmann & Tuttle, 1945).…”

Section: Methodsmentioning

confidence: 99%

Chemical modification of tryptophan residues in Escherichia coli succinyl-CoA synthetase. Effect on structure and enzyme activity

Ybarra¹,

Prasad²,

Nishimura³

1986

Biochemistry

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Succinyl-CoA synthetase of Escherichia coli is an alpha 2 beta 2 protein containing active sites at the interfaces between alpha- and beta-subunits. The alpha-subunit contains a histidine residue that is phosphorylated during the reaction. The beta-subunit binds coenzyme A and probably succinate [see Nishimura, J. S. (1986) Adv. Enzymol. Relat. Areas Mol. Biol. 58, 141-172]. Chemical modification studies have been conducted in order to more clearly define functions of each subunit. Tryptophan residues of the enzyme were modified by treatment with N-bromosuccinimide at pH 7. There was a linear relationship between loss of enzyme activity and tryptophan modified. At one tryptophan residue modified per beta-subunit, 100% of the enzyme activity was lost. In this enzyme sample, one methionine residue in each alpha- and beta-subunit was oxidized to methionine sulfoxide, although loss of enzyme activity could not be related in a linear manner to the formation of this residue. Subunits were prepared from enzyme that was inactivated 50% by N-bromosuccinimide with 0.5 tryptophan modified per beta-subunit but with insignificant modification of methionine residues in either subunit. Small decreases in the tyrosine and histidine content were observed in the alpha-subunit but not in the beta-subunit. In this case, modified beta-subunit when mixed with unmodified alpha-subunit gave a population of molecules that was 50% as active as the refolded, unmodified control but was only slightly changed with respect to phosphorylation capacity and unchanged with respect to rate of phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…The enzyme was purified from E. coli as previously described (Bowman & Nishimura, 1975). Enzyme activity was assayed either by the method of Cha (1969) or by the hydroxamate assay (Kaufman et al, 1953) as modified by Grinnell & Nishimura (1969a). The enzyme as prepared here had a specific activity of 590 units/mg (Kaufman et al, 1953).…”

Section: Methodsmentioning

confidence: 99%