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1. A method for the isolation of a new enzyme, myosin light-chain phosphatase, from rabbit white skeletal muscle by using a Sepharose-phosphorylated myosin light-chain affinity column is described. 2. The enzyme migrated as a single component on electrophoresis in sodium dodecyl sulphate/polyacrylamide gel at pH7.0, with apparent mol.wt. 70000. 3. The enzyme was highly specific for the phosphorylated P-light chain of myosin, had pH optima at 6.5 and 8.0 and was not inhibited by NaF. 4. A Ca2+-sensitive 'ATPase' (adenosine triphosphatase) system consisting of myosin light-chain kinase, myosin light-chain phosphatase and the P-light chain is described. 5. Evidence is presented for a phosphoryl exchange between Pi, phosphorylated P-light chain and myosin light-chain phosphatase. 6. Heavy meromyosin prepared by chymotryptic digestion can be phosphorylated by myosin light-chain kinase. 7. The ATPase activities of myosin and heavy meromyosin, in the presence and absence of F-actin, were not significantly changed (+/- 10%) by phosphorylation of the P-light chain.

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1.It was possible to find a steady-state lactate concentration in a closed-circuit heart perfusion containing added glucose. Insulin but not growth hormone (4 pglml) increased the equilibrium concentration in perfusate by 200 Ole. Intracellular lactate also remained constant and was about twice that in perfusate: this distribution is difficult to explain whether ionic or undissociated lactate is the permeating species.2. Pyruvate accumulated linearly in perfusate, reaching a peak after 30 min perfusion and then declined non-linearly. Insulin increased the peak concentration and growth hormone slowed the decline after the peak. The intracellular pyruvate concentration stayed constant and, except in the first few minutes, was lower than the perfusate concentration.3. Pyruvate addition a t zero time could inhibit pyruvate accumulation but altered neither the non-linear decline, the intracellular pyruvate level nor the lactate equilibrium. The [NAD]/[NADH]ratio, after 10 min of pre-perfusion on open circuit, was 23. During 2 h of perfusion the ratio rose slowly and then declined. If either insulin or growth hormone were present in the medium, the ratio fell to about 11 during the 6rst few minutes of perfusion and remained low for 90 min. These values could not easily be correlated with either the varying extracellular, or the constant intracellular [lactate]/[pyruvate] ratios.5. The glucose and oxygen consumption of the hearts during perfusion was measured, and the change in glycogen concentration in the tissue. It was then possible to draw up a balance sheet which indicated that the control hearts oxidied little other than carbohydrate for the 60 min of observation, but both insulin and growth hormone-treated hearts oxidised significant quantities of fatty acid during the first 15 min. It appears that growth hormone, like insulin, has an immediate effect on heart metabolism. The addition of pyruvate a t zero time appeared to stimulate carbohydrate oxidation in hearts perfused with insulin, even though the hormone had been present in the pre-perfusion fluid.6. Consideration of the variation in the extracellular [lactate]/[pyruvate] ratio with length of perfusion, and its lack of relationship to the intracellular [lactate]/[pyruvate] ratio, together with similar reports of other workers, leads us to conclude that neither lactate nor pyruvate establish a concentration gradient across the heart cell membrane which is based on passive diffusion. The discrepancy is particularly marked with respect to pyruvate. Our results strongly suggest that the extracellular [lactate]/[pyruvate] ratio cannot be used as an index of the cytoplasmic redox potential in perfused heart. 7.A tracer study of pyruvate metabolism in the reproducible non-steady-state conditions described can offer an explanation for the behaviour of the observed metabolic parameters : this study is reported in the following papers.The experiments which are reported in this and succeeding papers were designed to produce data that could be used to test a new metho...

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1. The kinetic properties of the 2-oxoglutarate dehydrogenase system were investigated. To this end, initial-velocity studies were carried out by the method of Fromm [(1967) Biochim. Biophys. Acta 139, 221-2301. Reciprocal plots of the results did not agree with those expected for the Hexa Uni Ping Pong mechanism previously proposed for the system. 2. The measured initial velocities were fitted to initial-rate equations corresponding to several possible mechanisms by using a computer optimization technique. Statistical analyses performed on the results ofthe optimization studies indicated that one mechanism was a significantly better fit to the experimental data than the other mechanisms tested. This mechanism is one in which there is a random order of binding of NAD+ and CoA and release of succinyl-CoA, although the binding of 2-oxoglutarate and release of CO2 is still given a Ping Pong mechanism, which precedes the binding of the other substrates. These conclusions were supported by NADH-inhibition studies. 3. The usefulness of the method of fitting initial-rate data to rate equations and the applicability of the proposed enzymic mechanism to the enzyme complex are discussed.

1. Glutamine synthetase activity has been determined in extracts of rat cardiac and skeletal muscle and kidney, after treatment to ensure that the rate of synthesis was proportional to time of incubation and to amount of extract added. The activity was measured by two methods, with hydroxylamine as substrate. 2. No activity was detected in rat heart extract by either method. The activity in skeletal muscle was of the order of 20mumol of glutamylhydroxamate synthesized/h per g of tissue under optimum conditions. The activity in kidney extracts was 180mumol/h per g of tissue when measured as ferric hydroxamate. 3. The activity in both skeletal-muscle and kidney extracts was inhibited by P(i). The inhibition is competitive for the muscle enzyme, with a K(i) of 12mm. For the kidney enzyme the inhibition is non-competitive, and less marked. Possible enzyme mechanisms that would lead to these types of inhibition are discussed. 4. Several observations are reported that suggest that the enzymes from muscle and kidney are not identical. 5. Growth hormone, either in vivo or in vitro, did not affect the measured glutamine synthetase activity of tissue extracts.

Thirty-eight soil samples from a sondage excavated through a 6- metre stratified tell (Gomoiava, in central Yugoslavia) have been analysed for nine biophile elements: Mg, Sr, Zn, Cu, Ni, Mn, Cr, Pb and B. The site was occupied from 7000 BC to 500 AD (calendar years). The results have been collated with earlier estimates of P and Ca, made on a larger series of samples from the same site. The results are discussed in terms of useful information, to supplement that given by phosphate analysis, about stratification and the relationship of the settlement to its environment.It is concluded that leaching probably distorts the results for Ca and Mg. The results for Mn may help to distinguish phosphate accumulation due to man from that due to farm livestock. There is significant accumulation of Sr and B, both of which have potential value, although some of the analytical results for the latter are suspect. The results for Zn, Cu and Ni are highly correlated, with sharp peaks which couid provide precise information about individual strata when more experience of interpretation has been gained. Only Cr and Pb appear to provide little useful information at this site.