Subunit ratios of separated hybrid hexamers of Neurospora NADP-specific glutamate dehydrogenase containing complementing mutationally modified monomers
- 30 November 1978
- journal article
- research article
- Published by Portland Press Ltd. in Biochemical Journal
- Vol. 175 (3) , 1125-1133
- https://doi.org/10.1042/bj1751125
Abstract
The am1 and am3 mutational variants of the N. crassa NADP-specific glutamate dehydrogenase [Ec 1.4.1.4] show complementation activity in hybrid hexamers. A freeze-thaw hybridization method was used to construct hybrids from purified enzymes and the products were separated into species of different monomer ratio by affinity chromatography. Hexamers with am1: am3 ratios of 1:5, 2:4, 3:3, 4:2 and 5:1 were all recovered as resolved or partially resolved peaks in quantities approximating to a binomial distribution. Reassociation of monomers during the hybridization process was random, except for some differential loss of am3 protein by precipitation and an apparent absence of reassociated am1 homohexamers. Compelmentation activity was shown by hybrids of all 5 monomer ratios, owing to activation of am3 monomers by conformational constraints arising from the intrinsically inactive am1 monomers. The activating effect of such constraints was greatest in hexamers containing a single am1 monomer and least in the 5 am1:1 am3 species. When fully activated by L-glutamate all am3 were equivalent in intrinsic catalytic activity, irrespective of the number of am1 monomers per hexamer.This publication has 12 references indexed in Scilit:
- Affinity chromatography of the Neurospora NADP-specific glutamate dehydrogenase, its mutational variants and hybrid hexamersBiochemical Journal, 1977
- The molecular basis of an osmotically reparable mutant of Neurospora crassa producing unstable glutamate dehydrogenaseJournal of Molecular Biology, 1977
- Mutational amino acid replacements in Neurospora crassa NADP-specific glutamate dehydrogenaseJournal of Molecular Biology, 1976
- Protein ComplementationAnnual Review of Biochemistry, 1975
- Slow conformational changes of a Neurospora glutamate dehydrogenase studied by protein fluorescenceBiochemical Journal, 1974
- Amino-Acid Sequence of NADP-Specific Glutamate Dehydrogenase of Neurospora crassaProceedings of the National Academy of Sciences, 1974
- Nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase of Neurospora. I. Isolation, subunits, amino acid composition, sulfhydryl groups, and identification of a lysine residue reactive with pyridoxal phosphate and N-ethylmaleimide.1973
- Multiple active varieties of Neurospora glutamate dehydrogenase formed by hybridization between two inactive mutant proteins in vivo and in vitroJournal of Molecular Biology, 1966
- Proof of hybrid enzyme formation in a case of inter-allelic complementation in Neurospora crassaJournal of Molecular Biology, 1965
- Complementation at the am locus of Neurospora crassa: a reaction between different mutant forms of glutamate dehydrogenaseJournal of Molecular Biology, 1963