No abstract

The kinetics of the hydrolysis of adenosine triphosphate (ATP) by the muscle protein myosin have received considerable attention in recent years, and several reviews have been devoted to this subject (Szent-Gyiirgyi, '55; Morales et al., '55; Buchthal et al., '56). The BTP-ase activity of myosin is modified by pressure (Laidler and Beardell, '55)' by calcium, magnesium and nucleotides (Blum, '55), and by calcium, pH and temperature (Green and Mommaerts, '54; Ouellet et al., '52). The effects of pressure, temperature and ions on ATP-ase activity have been interpreted in terms of a simple Michaelis-Menten reaction scheme (Laidler and Beardell, '55 ; Green and Mommaerts, '54)' although other studies of the behavior of myosin have implicated a series of reactions (Mommaerts and Hanson, '56; Morales et al., '55; Watanabe et al., '53). I n fact, Spicer ('55) has suggested that reversible denaturations may explain gelation phenomena in solutions of myosin B. Our interest in the problem arose from the observation that pressure reduces the isometric twitch tension of intact muscle at lower temperatures and increases it at higher (Cattell and Edwards, '28). This has been ascribed to the effect of presl This investigation was aided by grants from the Dazian Foundation for Medical Research,

It is now generally agreed that the S shape of the curve for the oxygenation of mammalian hemoglobin (fraction oxygenated ~s. oxygen pressure) virtually implies interaction between the four heroes attached to each hemoglobin molecule. In other words, a hemoglobin molecule will react more readily with oxygen after some of its heroes have been oxygenated (1-3). The intramolecular mechanism of this interaction is still very obscure.The problem is often attacked by consideration of the effects of changes in pH (1) or by consideration of reactions of the iron atoms of the molecule (4), although Riggs (5) has studied the effect of compounds known to react with sulfhydryl groups of the protein. Compounds that react with nitrogenous groups of the protein might also be expected to affect the combination of oxygen with hemoglobin, and the study of such reactions should provide useful information. In the present experiments, formaldehyde was chosen as a reagent because its reactions with amino acids have been explored and their equilibrium constants have been determined (6). Unfortunately, formaldehyde reacts with many functional groups of the protein moiety of hemoglobin, and the results permit no simple interpretation. However, the experiments indicate that the interaction involves nitrogen-containing groups able to combine with formaldehyde.Methods 11emogtotrin.--Concentrated ceils from freshly drawn, heparinized human blood were washed three times in isotonic saline solution, then laked for 90 to 120 minutes by the addition of an equal volume of water and 0.4 volume of toluene. After the resulting solution was centrifuged to remove debris, enough cold potassium phosphate buffer was added to make the final phosphate concentration 0.4 M. Then the solution was centrifuged again to clarify it for optical measurements before it was introduced into the tonometer. The hemoglobin was deoxygenated by successive evacuations of the tonometer, interspersed with washings with nitrogen. All steps in the preparation were carried out at 2-5°C.Measurements.--The methods of measurement have been described previously (2).