Substrate regulation of mitochondrial oxidative phosphorylation in hypercapnic rabbit muscle.

Endurance muscle performance is highly dependent on ATP production from mitochondrial oxidative phosphorylation. To study the role of the mitochondrial oxidative enzymes in muscle fatigue, we analyzed the relationship between the concentrations of substrates associated with ATP synthesis and the muscle performance of electrically stimulated rabbit muscle under CO2-induced acidosis. Two different conditions of pacing-induced muscle performance were produced in the gastrocnemius and soleus muscle groups in anesthetized rabbits by stimulating the sciatic nerve submaximally at two frequencies. Phosphorus nuclear magnetic resonance was used to measure ATP, phosphocreatine, and Pi and to provide data for a calculation of intracellular pH and free ADP. To induce acidosis, the animal was ventilated with 20% CO2. The administration of CO2 effectively reduced the intracellular pH from 6.9 to 6.7 and reduced the isometric tension-time integral (TTI) to below half the value measured in normocapnia at the low pacing frequency. A twofold increase in the pacing frequency resulted in a doubling of the TTI in normocapnia and a tripling of TTI in hypercapnia. The increases in TTI corresponded with increases in free ADP and Pi concentrations. Under the various conditions, all free ADP values were near the in vitro Michaelis-Menten constant (Km) of ADP. The Michaelis-Menten relationship of the oxidative phosphorylative enzymes was applied to the change in substrate concentrations with respect to TTI. From this relationship we observed that the in vivo Km of free ADP was 26 microM, which is close to the in nitro Km, and that Km and maximal reaction velocity did not change under hypercapnia and increased pacing frequency.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of mutant acetolactate synthases resistant to triazolopyrimidine sulfonanilide

Triazolopyrimidine sulfanilides are a class of highly active herbicides whose primary target is acetolactate synthase. Spontaneous mutants of tobacco (Nicotiana tabacum) (KS-43) and cotton (Gossypium hirsutum) (PS-3 and DO-2) resistant to triazolopyrimidine sulfonanilide were selected in tissue culture. Acetolactate synthase partially purified from the three mutants were 80- to 1000-fold less sensitive to inhibition by the compound compared with the corresponding wild-type enzyme. The mutants also varied in the cross-resistance pattern to other acetolactate synthase inhibiting herbicides in the sulfonylurea, imidazolinone, and pyrimidyl-oxy-benzoate chemical families. Thus, acetolactate synthase from KS-43, PS-3, and DO-2 cultures have different mutations. The affinities for pyruvate, thiamine pyrophosphate, as well as the activity of the mutant enzymes were found to be comparable to the corresponding wild-type enzymes. However, the enzyme from PS-3 was highly resistant to feedback inhibition by valine and leucine. In contrast, acetolactate synthase from KS-43 and DO-2 were inhibited by valine and leucine to nearly the same extent as the wild-type enzymes. Also, PS-3 cultures accumulated much higher levels of the branched chain amino acids compared to the wild-type cotton culture. The mutation in the PS-3 enzyme has therefore rendered it insensitive to feedback regulation by valine and leucine.     

A NEW APPROACH TO CORRECTION OF D-LOOP TRANSPOSITION OF THE GREAT ARTERIES

An operative procedure is described for repair of transposition of the great arteries in which the left ventricle is the "aortic" ventricle and the right ventricle is the "pulmonic" ventricle. The technical feasibility of this procedure is supported by its successful application in experimental animals. A modified procedure, which may be applicable in certain complex forms of transposition of the great arteries and other perplexing anomalies for which there are no known repairs, is discussed.     

Variations on stoichiometry of ribosomal proteins in Escherichia coli

Experiments are described in which the Stoichiometry of the ribosomal proteins before and after ribosome release from mRNA is compared. Polysomes labeled with ³H (or ¹⁴C) and run-off 70 S particles (Subramanian el al., 1969) labeled with ¹⁴C (or ³H) were separately isolated, mixed, and the ribosomal proteins extracted and fractionated by two-dimensional gel electrophoresis. The measurement of the isotopic ratios shows that 47 proteins out of the 53 investigated are present in the same amount in polysomes as in run-off ribosomes indicating that they remain with the ribosome during the release step. Proteins S₁, S₂, S₆, S₂₁, $\text{L}{}_{\mathrm{<mtext>7</mtext>}}\text{L}{}_{\mathrm{<mtext>12</mtext>}}$ (Wittmann et al., 1971), however, show higher amounts in polysomes than in run-off ribosomes. The significance of these results is discussed.     

The presence of essential arginine residues at the active sites of citrate lyase complex from Klebsiella aerogenes

The acyl-transferase and acyl-lyase activities of $\text{Klebsiella aerogenes}$ citrate lyase complex are inactivated by the arginine specific reagents phenylglyoxal and 2,3-butanedione, the former reagent being the more potent inhibitor. Citrate and (3$\text{S}$)-citryl-CoA protect the transferase activity, while acetyl-CoA markedly enhances the rate of the inactivation. (3$\text{S}$)-Citryl-CoA protects the lyase subunit in the complex from inactivation. The kinetics of inactivation suggest the involvement of a single arginine residue at each of the active sites of the transferase and of the lyase subunits.     

Three-dimensional structure of the complex of the Rhizopus chinensis carboxyl proteinase and pepstatin at 2.5-A resolution

An X-ray diffraction analysis has been carried out at 2.5-A resolution of the three-dimensional structure of the Rhizopus chinensis carboxyl proteinase complexed with pepstatin. The resulting model of the complex supports the hypothesis [Marciniszyn, J., Hartsuck, J.A., & Tang, J. (1976) J. Biol. Chem. 251, 7088-7094] that statine (3-hydroxy-4-amino-6-methylheptanoic acid) approaches an analogue of the transition state for catalysis. The way in which pepstatin binds to the enzyme can be extended to provide a model of substrate binding and a model of the transition-state complex. This in turn has led to a proposed mechanism of action based on general acid-base catalysis with no covalent intermediates. These predictions are in general agreement with kinetic studies using several carboxyl proteinases, which together with their sequence homology and their common three-dimensional structures suggest that this mechanism can be extrapolated to all carboxyl proteinases.