A systematic study of the variables affecting the in vitro reassembly of yeast glyceraldehyde 3-phosphate dehydrogenase from urea-dissociated subunits led to the following optimum reversal conditions: (1) pH 6.9, 0.05 m imidazole buffer; (2) 0.3 m KCI; (3) 0.05 mg/ml of protein concentration; (4) 27 mM nicotinamide–adenine dinucleotide; (5) 27 mM glutathione; and (6) temperature: dilution at 0°, followed by incubation at 16° for 1 hr. The refolding of yeast D-glyceraldehyde 3-phosphate dehydrogenase exhibits an almost absolute requirement for nicotinamide-adenine dinucleotide. In 0.15 m KCI, to approximate in vivo conditions, the recoveries of activity relative to the native enzyme control are 3 and 82% respectively; with 0.8 m KCI, it is 93%. The profile of activity recovery shows a low activity plateau at nicotinamide-adenine dinucleotide concentrations below 10-6 M, an ionic strength dependent transition centered in the region 0.6-2.2 mM, and a high activity plateau above 27 mM. High salt (0.8 m KCI) substitutes for nicotinamide-adenine dinucleotide, yielding 68% recovery. The 3-acetylpyridine nicotinamide-adenine dinucleotide analog serves as a weak coenzyme and enhances the refolding whereas nicotinamideadenine dinucleotide phosphate does neither; therefore, a free adenosyl 2′-hydroxyl appears to be critical, but the nicotinamide-adenine dinucleotide pyridineamide probably is not. The in vitro reassembly is fast (half-time = 7 min at 16°) and gives complete recovery (93 %) suggesting that it approximates the in vivo folding process. The in vitro requirement for nicotinamide-adenine dinucleotide for folding of yeast dglyceraldehyde 3-phosphate dehydrogenase emphasizes that the amino acid sequence alone may not be adequate to provide the correct folding of nascent or completed polypeptides in vivo and that nicotinamide-adenine dinucleotide may accomplish this and thereby control the rate of enzyme synthesis. Two models are described for translational control by control of folding. The procedures used here gave activity recoveries greater than 50 % with six different enzymes. © 1969, American Chemical Society. All rights reserved.
