Mechanism of stimulation of respiration by purines: I. Inhibitor studies

Abstract

A previous publication (1) reported a striking stinmulation of the endogenous respiration of nitrogenstarved Chlorella cells by low concentrations of various purines and purine analogs. This stimulation has sinice been shown in a large variety of nitrogenstarved nmicroorganisms as well as in some higher plants (2).Because of the widespread occurrence of this effect an(l the possibility that a novel regulatory functiotn of intracellular purines might be involved, the nmodle of action of the purines was further investigatedl. One mleanis of gaining insight into the possible metabolic pathways which nmight be involved is by means of imietabolic inhibitors with known sites of action. Thus it miight be possible to locate the purine effect in reactions involving the cytochrome system, oxidative phosplhorylation, amino acid metabolism, or glvcolvsis, for example. if it could be shown that known specific inhibitors of these systems inhibited the purine effect. This paper discusses the results fronm such studies andl points to the involvement of flavoprotein enzymes and cellular lipids in the respiration wlhiclh is causedt by purines. Further direct evidence for the existence of an electron transport system Nwhichl is mle(liated by free purines and is active in the oxi(lation of storage fat will be presented in future papers (3,4 (13). Total radioactivity was leternlinedl by plating the BaCOŵhich was recovered from the center wells of the Warburg oxygen flasks. The center wells wvere rinsed three tinmes into a centrifuge tube with distilled water an(d the trapped C'40., precipitate(l by the addition of a few drops of 12 % BaCI.2. The precipitate was washed with methanol and plate(d as previously dlescribe(l (1). Duplicate flasks were used in all cases. All radioactivities were corrected for self absorption and are expressed for infinite thickness.In the tinme course experimielnts 5 mll cells were suspen(le(l with or without 6-mercaptopurine in 25 nl Frlenmileyer flasks and shaken at 250 C in the (lark. At given intervals flasks were removedl. 3 ml of the contents pipette(l into Warburg flasks and the respirometric expel-imiients con(lucte(l.Although results from (luplicate flasks in these experiments always agreed to within less than 10 % there was consi(derable dlay to (lay variation (1). As a result, all experiments were con(lucted at least at two separate occasions and the results thus obtained were average(l after adjustnment of the uninhibited controls to the same level (1). Furthermore