In mammals, lipoate-activating enzyme (LAE) catalyzes the activation of lipoate to lipoyl-nucleoside monophosphate. The lipoyl moiety is then transferred to the specific lysine residue of lipoate-dependent enzymes by the action of lipoyltransferase. We purified LAE from bovine liver mitochondria to apparent homogeneity. LAE activated lipoate with GTP at a 1000-fold higher rate than with ATP. The reaction absolutely required lipoate, GTP, and Mg 2؉ ion, and the reaction product was lipoyl-GMP. LAE activated both (R)-and (S)-lipoate to the respective lipoyl-GMP, although a preference for (R)-lipoate was observed. Similarly, lipoyltransferase equally transferred both the (R)-and (S)-lipoyl moieties from the respectively activated lipoates to apoH-protein. Interestingly, however, only H-protein carrying (R)-lipoate was active in the glycine cleavage reaction. cDNA clones encoding a precursor LAE with a mitochondrial presequence were isolated. The predicted amino acid sequence of LAE is identical with that of xenobiotic-metabolizing/medium-chain fatty acid:CoA ligase-III, but an amino acid substitution due to a single nucleotide polymorphism was found. These results indicate that the medium-chain acyl-CoA synthetase in mitochondria has a novel function, the activation of lipoate with GTP.

The control of malate metabolism and stimulation of 1-sinapolyglucose: L-malate sinapoyltransferase (SMT) activity in radish (Raphanus sativus L. var. sativus) cotyledons has been studied. The light-induced and nitrate-dependent activity of SMT catalyzes the formation of O-sinapoly-L-malate via 1-O-sinapoyl-β-D-glucose. When dark-grown radish seedlings, cultivated in quartz sand with nutrient solution containing NO 3 (-) as the sole N source, were treated with light, SMT activity increased concomitantly with free malate in the cotyledons. This light effect was suppressed in seedlings grown in a culture medium which contained in addition to NO 3 (-) also NH 4 (+) . However, treatment with methionine sulfoximine neutralized this ammonium effect, resulting again in both rapid accumulation of malate and rapid increase in SMT activity. When seedlings grown on NO 3 (-) nitrogen were subsequently supplied with NH 4 (+) nitrogen, the accumulated level of L-malate rapidly dropped and the SMT increase ceased. The enzyme activity decreased later on, reaching the low activity level of plants which were grown permanently on NO 3 (-) /NH 4 (+) -nitrogen. An external supply (vacuum infiltration) of malate to excised cotyledons and intact seedings, grown on NO 3 (-) /NH 4 (+) -nitrogen medium, specifically promoted a dose-dependent increase in the activity of SMT. In summary these results provide evidence indicating that the SMT activity in cotyledons of Raphanus sativus might be related to the metabolism of malic acid.

Smith & Ackers (Proc. Natl. Acad. Sci. 82,5347 -5351 (1985)) introduced a three-state model for hemoglobin action. The kinetics of interconversion between such states should be measurable at appropriate levels of 02-binding. For triligated hemoglobin the interconversion of two states has been measured by Zhang et a1 (Biophys. J. 58,333 -340 (1990)). If hemoglobin action involves three allosteric states, then the additional interconversion, though likely acting faster than the previously measured interconversion, should also be measurable by appropriate experimental design. We use numerical simulation to study the feasibility of observing such interconversions. We simulate the kinetics of the reaction of hemoglobin with molecular oxygen, in which rapid mixing is followed by a very fast temperature jump (see G. Czerlinski, J.Theor. Biol. 154, 119 -126 (1992)). Values for rate constants are used when known, otherwise estimated. The simulation is performed by numerical solution of a system of differential equations reflecting the kinetics of the three-state model of Smith & Ackers (I.c.). We consider three models and determine under what experimental conditions one may distinguish between these models: (1) The model of Ackers with only one path to and from each bisubstituted species.(2) The full model of Ackers. (3) A mechanism-altering change in the value of one interconversion constant.