The Creatine Phosphoryltransfer Reaction in Iodoacetate-Poisoned Muscle

The iodoacetate-nitrogen-poisoned muscle offers the possibility of studying the stoichiometry of the single muscle twitch since metabolic resynthesis by glycolysis and oxidative phosphorylation are blocked, and there remains as an energy source only the creatine phosphoryltransfer system, creatine phosphate reacting with adenosinediphosphate to give the triphosphate and creatine. It is shown, preparatory to a determination of the amount of phosphocreatine split in a single twitch, that iodoacetate does not inhibit creatine phosphoryltransferase at concentrations which block glycolysis. An analysis is developed which assumes that the transferase maintains the creatine phosphoryl transfer reaction in equilibrium following contraction, and further that the creatine phosporyltransfer reaction and the myokinase reaction are isolated in muscle. On the basis of this analysis and the data obtained, an estimate of the equilibrium constant of the creatine phosphoryl reaction in muscle is obtained which agrees with values determined in vitro. Using the estimated equilibrium constant, and the concentrations of creatine, creatine phosphate, and adenosinetriphosphate found, a value for the concentration of free adenosinediphosphate is obtained which is considerably less than that found by direct chemical analysis.     

The Mechanochemistry of Muscular Contraction : I. The isometric twitch

The dependence of PC¹ and ATP¹ dephosphorylation on the number of isometric twitches in the iodoacetate-nitrogen-poisoned muscle has been examined. There is no net dephosphorylation of adenosinetriphosphate. PC dephosphorylation varies linearly with the number of twitches and produces equivalent amounts of C¹ and P_1i.¹ Iodoacetate concentrations which block the enzyme, creatine phosphokinase, render the muscle non-contractile. A value of 0.286 µmole/gm. for the amount of PC split per twitch is obtained which gives a value of -9.62 kcal./mole for the "physiological" heat of hydrolysis of PC in agreement with expectations based on thermochemical data. In a single maximal isometric twitch it is estimated that 2 to 3 PC molecules are dephosphorylated per myosin molecule, or 1 per actin molecule. The results support the view that under the conditions of these experiments PC dephosphorylation is the net energy yielding reaction. The in vivo stoichiometry of the mechano-chemistry of contraction revealed by these studies on the one hand, and the known stoichiometry of actin polymerization and its coupling to the creatine phosphokinase system on the other are strikingly similar and strongly suggest that the reversible polymerization of actin is involved in a major way in the contraction-relaxation-recovery cycle of muscle.     

The ISBsu2 mobile element is present in a plasmid of a soil strain and in the chromosomes of several other strains of Bacillus subtilis

Chromosomes of several Bacillus subtilis strains were shown to contain homologs of the ISBsu2 mobile genetic element, which was earlier revealed in a cryptic plasmid of a soil strain of B. subtilis.     

Potential sources of the 1995 Venezuelan equine encephalitis subtype IC epidemic

Venezuelan equine encephalitis viruses (VEEV) belonging to subtype IC have caused three (1962-1964, 1992-1993 and 1995) major equine epizootics and epidemics. Previous sequence analyses of a portion of the envelope glycoprotein gene demonstrated a high degree of conservation among isolates from the 1962-1964 and the 1995 outbreaks, as well as a 1983 interepizootic mosquito isolate from Panaquire, Venezuela. However, unlike subtype IAB VEEV that were used to prepare inactivated vaccines that probably initiated several outbreaks, subtype IC viruses have not been used for vaccine production and their conservation cannot be explained in this way. To characterize further subtype IC VEEV conservation and to evaluate potential sources of the 1995 outbreak, we sequenced the complete genomes of three isolates from the 1962-1964 outbreak, the 1983 Panaquire interepizootic isolate, and two isolates from 1995. The sequence of the Panaquire isolate, and that of virus isolated from a mouse brain antigen prepared from subtype IC strain P676 and used in the same laboratory, suggested that the Panaquire isolate represents a laboratory contaminant. Some authentic epizootic IC strains isolated 32 years apart showed a greater degree of sequence identity than did isolates from the same (1962-1964 or 1995) outbreak. If these viruses were circulating and replicating between 1964 and 1995, their rate of sequence evolution was at least 10-fold lower than that estimated during outbreaks or that of closely related enzootic VEEV strains that circulate continuously. Current understanding of alphavirus evolution is inconsistent with this conservation. This subtype IC VEEV conservation, combined with phylogenetic relationships, suggests the possibility that the 1995 outbreak was initiated by a laboratory strain.     

The ISBsu2 Mobile Element Is Present in a Plasmid of a Soil Strain and in the Chromosomes of Several Other Strains of Bacillus subtilis

Chromosomes of several Bacillus subtilis strains were shown to contain homologs of the ISBsu2 mobile genetic element, which was earlier revealed in a cryptic plasmid of a soil strain of B. subtilis.     

Ca-induced fusion of cardiolipin/phosphatidylcholine vesicles monitored by mixing of aqueous contents

The kinetics of Ca²⁺-induced fusion of large (0.1 μm) unilamellar cardiolipin/phosphatidylcholine (1:1) vesicles have been investigated by continuous monitoring of the mixing of the aqueous vesicle contents. In parallel, release of vesicle contents to the external medium has been followed. Initial fusion of the vesicles is non-leaky, release of vesicle contents being largely a secondary phenomenon. The minimal Ca²⁺ concentration required for fusion in this system is approx. 9 mM. At higher Ca²⁺ concentrations fusion is extremely fast, occurring on the time scale of seconds.