Carbon assimilation by Pseudomonas oxalaticus (OX 1). 2. Formate and carbon dioxide utilization by cell-free extracts of the organism grown on formate
1959; Portland Press; Volume: 72; Issue: 4 Linguagem: Inglês
10.1042/bj0720631
ISSN0306-3283
Autores Tópico(s)Enzyme Catalysis and Immobilization
ResumoIsotopic experiments performed on cultures of Pseudomonas oxalaticu8 (OX 1) growing on formate as sole carbon and energy source (Quayle & Keech, 1958, 1959) indicated the existence in the organism of a carbon dioxide-fixation cycle similar to that found in photosynthetic tissue (Bassham et al. 1954) and autotrophic bacteria (Santer & Vishniac, 1955;Trudinger, 1955).An investigation of the ability of cell-free extracts of P. oxaksticus (OX 1) to incorporate formate carbon and carbon dioxide into 3-phos- phoglyceric acid and certain C4 acids is reported in this paper. METHODS AND MATERIALSMaintenance and growth of the organism.The maintenance and growth of stock cultures of Pseudomona oxalaiwus (OX 1) have been described (Quayle & Keech, 1959).Large quantities of the organism were grown in 401. of medium inoculated with 800 ml. of a bacterial suspension in the logarithmic phase of growth.The growth vessel used was a modified stainless-steel container of a commercial washing machine (Hunt, Rogers & Hughes, 1959).Growth was maintained at 300 under conditions of vigorous aeration, formic acid (90%, w/v; A.R.) being added to keep the pH at 7-5-8-5.After 42 hr.the bacteria (31 g. wet wt.) were harvested in a Sharples centrifuge and stored at -100.Preparation of ceU-free extracts.The harvested cells (0-5-2-0 g. wet wt.) were suspended in 2 vol. of mm-2- amino-2-hydroxymethylpropane-1:3-diol (tris) buffer, pH 7-8, containing about 200 ,ug.each of crystalline ribo- nuclease and deoxyribonuclease (L.Light and Co. Ltd., Colnbrook, Bucks).Extracts were then prepared by orushing the frozen suspension in a Hughes press (Hughes, 1951), at -20°.The crushed material was homogenized with a glass homogenizer and the resulting extract was used for some experiments.More frequently, the supernatant fraction obtained from the homogenate by centrifuging at 15 000 g for 20 min. in a refrigerated angle-head centrifuge was used.This fraction always contained 12-20 mg. of protein/ml.Experiment8 with radioactiveJ;fiation.The standard-aasay mixture for measuring isotope incorporation into 3-phosphoglyceric acid (PGA) from [14C]bicarbonate or [14C]- formate contained: 5 1tmoles of tris buffer, pH 7-8, 2-5 - * Part 1. Quayle & Keech (1959).moles of MgCl,, 0-5 jsmole of ribulose 1:5-diphosphate,
Referência(s)