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X39 622nd MEETING. LEICESTER Novel nucleotide diversity of succinate thiokinase T I M O T H Y M. J E N K I N S and P. D. J . WEITZMAN Dcptirtmont of' Biochcwiistrj. Universi!,. of Buth. C l r w r t o n Down. Buth BA2 7 A Y . U .K. observed in the presence of both A D P and I D P a t saturating levels (Table IA). Interestingly. although G D P is not utilized by either enzyme, it appears to inhibit the two STKs differentially (Table IA). The I-STK shows a greater degree of inhibition by G D P than does the A-STK, and this may be due to the structural similarity between IDP and G D P . In addition. the two STKs exhibit different pH profiles and have different K , values for their respective nucleotides: K , (IDP) = 26.7 f 2.1 P M and K , (ADP) = 89.4 11.9~~. The relative specific activities of the two STKs in B. niogutrrium were found to vary depending upon the stage of growth and also upon the growth medium. O n nutrient broth a 10-fold variation in the ratio of A-STK/I-STK occurred between early-logarithmic and late-stationary growth phases, and between different growth media at the same stage of growth (Table 1 B). Along with the evidence presented in Table IA, these results strongly suggest the presence of two distinct nucleotide-specific STKs, the relative proportions of which differ according to physiological conditions. The marked elevation of I-STK activity during growth on succinate may reflect increased formation of succinyl-CoA from succinate under these conditions. The situation in Gram-positive bacteria may therefore resemble that in animal tissues, in which it has been suggested that A-STK may function in the energetic role of the citric acid cycle, whereas the second STK may operate in the opposite metabolic direction, converting succinate to succinylCoA for biosynthesis (Jenkins & Weitzman, 1986). Investigations in progress are aimed at further exploration o f this possibility. The nucleotide specificity of succinate thiokinase (STK) from a variety of sources has been studied (Palmer & Wedding. 1966; McClellan & Ottaway, 1980; Weitzman & Jaskowska-Hodges, 1982). Gram-negative bacteria can utilize both adenine and guanine nucleotides (ADP/ATP and G D P G T P ) on a single 'large' (tetrameric) enzyme. In contrast, the utilization of these nucleotides by animal STKs occurs on two distinct 'small' (dimeric) enzymes (Weitzman ct t d . . 1986). Gram-positive bacteria were previously thought to contain a single 'small' (dimeric) STK specific for ADP/ATP. However. this communication presents evidence for the existence of a second STK activity in Gram-positive bacteria which is specific for inosine nucleotides (IDP/ITP). In animal tissues, where both A-STK and G-STK activities are present. I-STK activity can be detected, but in tissues with apparently only A-STK. no I-STK is measurable. Similarly. in Gram-negative bacterial STK, inosine nucleotides may replace their guanine counterparts with similar kinetic characteristics. In Gram-positive bacteria, where n o ti-STK occurs, a n STK specific for inosine nucleotides has been found and this I-STK appears distinct from the A-STK. Bacteria were grown in nutrient broth a t 30°C or 37°C. Btic~illus nicJgutrrium was also grown in minimal salts medium, with 25 mM-succinate, glucose or glutamate as the sole carbon source, to detect any changes in levels of A-STK and I-STK activities. STK activity was assayed polarographically (Weitzman & Jaskowska-Hodges, 1982). Hp.1.c. analysis of all the nucleotides confirmed their purity and hence ruled out the possibility of the detection of activity with a minor impurity. I-STK was detected in the following Gram-positive bacteria: B. mc>gaterium, Bacil1u.s suhtilis, Sttiph!.loc.oc.c,u.s uurws, Arthrohucter simples and Kurthiu ~ o p f i iPartial . additivity which has previously been used as a criterion for distinct enzymes (Weitzman et ul., 1986) was Jenkins, T. M. & Weitzman. P. D. J. (19x6) FEBS L e i / . 205. 215 218 McClellan. J . A. & Ottaway. J. H. (1980) Comp. B i o h m . P l ~ ~ i o l . 678, 697 6x4 Palmer. J. M . & Wedding. R. T. (1966) Biochini. B i ~ p h x . Actu ~. 113. 167 174 WcitLman. 1'. D. J . & Jaskowska-Hodges, H. (19x2) FEBS Lrir. 143. 237 240 Wcit~man.P. D. J . . Jenkins. T. M.. Else. A. J . & Holt. R. A. (1986) F'ERS L r i i . 199. 57 60 Abbreviations used: STK. succinate thiokinase; Hp.1.c.. high pressure liquid chromatography; NDP. nucleoside diphosphate. Rcceived I April 19x7 Table I . Succinate thiokinase activities ,from Bacillus mcguterium A Nutrient broth Late-stationary +ADP +IDP +GDP 74.7 16.12 0.0 +(ADP + IDP) +(ADP 80. I + GDP) 58.2 /n ' Inhibition +(IDP 22.2 + GDP) 7.27 B A-STK I-STK Ratio A-STKLSTK Nutrient broth Early-logarithmic Mid-logarithmic Late-stationary 31.0 73.3 48.74 0.35 4.8 5.56 xx.57 15.27 8.77 25 mM-Succinate Late-stationary 41.09 6.75 6.1 25 mM-Glucose Late-stationary 57.9 I .04 55.67 25 mM-Glutamate Late-stationary 28.06 0.426 65.87 Growth media (NDP Vol. I5 = 0.5mM; all activity values are in nmol/min per mg protein.) Yn Inhibition 54.8