Isolation of H-protein loaded with methylamine as a transient species in glycine decarboxylase reactions.
A three-step protocol was devised to purify H-protein, which can be readily released as a soluble protein from pea mitochondria. After the final step of purification (anion-exchange chromatography) the native enzyme was eluted as two distinct peaks at 250 and 350 mM-KCl if the lysis buffer contained glycine. Each from exhibited an identical Mr of 15000 on SDS/PAGE and they were not distinguishable by PAGE under non-denaturating conditions. Both forms catalysed the rapid fixation of [14C]bicarbonate to the carboxy group atom of glycine during the exchange reaction, whereas the reversible exchange of electrons between NADH and lipoamide bound to the H-protein in the presence of 5,5'-dithiobis-(2-nitrobenzoic acid) was seen only with the form eluted at 350 mM-KCl. During the early steps of H-protein isolation, when P- and H-protein react together in the presence of glycine, the methylamine intermediate bound to the lipoamide of the H-protein accumulates in the medium at the expense of oxidized H-protein. Under these conditions the methylamine intermediate, which is a rather stable structure, was easily separated from the oxidized H-protein on ion-exchange chromatography. The methylamine bound to the lipoamide of the H-protein prevented the reversible exchange of electrons between NADH and lipoamide. High concentrations of glycine were required for the loading of H-protein with methylamine catalysed by a large excess of P-protein.
Neuburger
M
M
Département de Biologie Moléculaire et Structurale, Centre d'Etudes Nucléaires et Université Joseph Fourier, Grenoble, France.
Jourdain
A
A
Douce
R
R
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D007887:Fabaceae; D050961:Glycine Decarboxylase Complex H-Protein; D050841:Glycine Dehydrogenase (Decarboxylating); D008744:Methylamines / Q000378:metabolism*; D008928:Mitochondria / Q000737:chemistry*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D010944:Plants / Q000648:ultrastructure*; D010946:Plants, Medicinal; D011189:Potassium Chloride
D002352:Carrier Proteins; D050961:Glycine Decarboxylase Complex H-Protein; D008744:Methylamines; D009243:NAD; D011189:Potassium Chloride; D004228:Dithionitrobenzoic Acid; C027451:methylamine; D000594:Amino Acid Oxidoreductases; D050841:Glycine Dehydrogenase (Decarboxylating)