Purification of D-alanine carboxypeptidase from Escherichia coli B on a penicillin-Sepharose column.

1. A soluble D-alanine carboxypeptidase from Escherichia coli strain B was purified on a p-aminobenzylpenicillin-Sepharose column. This one-step chromatography followed by an (NH4)2SO4 precipitation yielded an enzyme purified 1200-fold and some of its properties are reported. 2. The pure D-alanine carboxypeptidase was devoid of D-alanine carboxypeptidase II activity and migrated as a single protein band on analytical disc gel electrophoresis. 3. Triton X-100 in the purification procedure is an absolute requirement for obtaining a stable enzyme. 4. The enzymic activity of D-alanine carboxypeptidase was greatly affected in solution of high salt concentrations and varied somewhat with the nature of the cation tested.

Biosynthesis of ergotamine by Claviceps purpurea (Fr.) Tul.

High-yielding strains of Claviceps purpurea (Fr.) Tul, grown on a defined medium, have been used for a study of the biosynthesis of the peptide ergot alkaloid, ergotamine. l-[U-(14)C]tryptophan, dl-[2-(14)C]mevalonic acid lactone, sodium [2-(14)C]acetate, sodium [(14)C]formate and the methyl group of l-[methyl-(14)C]methionine were efficiently incorporated into the peptide alkaloids and specifically labelled the ergoline moiety of ergotamine. These results are the same as previously found for the biosynthesis of other ergot alkaloids. Time-course incubation experiments demonstrated that l-[U-(14)C]phenylalanine, l-[U-(14)C]proline and l-[U-(14)C]alanine were incorporated into the peptide ergot alkaloids. Chemical degradation of the radioactive alkaloid derived from additional precursor incubation experiments showed that phenylalanine and proline function as the most efficient precursors, and specifically label the constitutive side-chain phenylalanyl and prolyl moieties of the alkaloid. The evidence obtained from l-[U-(14)C]alanine-incorporation experiments was inconclusive. However, degradation of ergotamine isolated after incubation with dl-[1-(14)C]alanine, showed that the carboxyl group of the labelled amino acid was specifically incorporated into the alpha-hydroxy-alpha-amino acid residue of the alkaloid. This, in conjunction with the l-[U-(14)C]alanine-incorporation results, showed conclusively that all three carbon atoms of alanine were incorporated as a biosynthetic unit into the alpha-hydroxy-alpha-amino acid moiety of ergotamine.

Protein synthesis by perfused hearts from normal and insulin-deficient rats. Effect of insulin in the presence of glucose and after depletion of glucose, glucose 6-phosphate and glycogen.

In the absence of glucose, insulin stimulated the incorporation of (14)C-labelled amino acids into protein by perfused rat hearts that had been previously substantially depleted of endogenous glucose, glucose 6-phosphate and glycogen by substrate-free perfusion. This stimulation was also demonstrated in hearts perfused with buffer containing 2-deoxy-d-glucose, an inhibitor of glucose utilization. It is concluded that insulin exerts an effect on protein synthesis independent of its action on glucose metabolism. Streptozotocin-induced diabetes was found to have no effect either on (14)C-labelled amino acid incorporation by the perfused heart or on the polyribosome profile and amino acid-incorporating activity of polyribosomes prepared from the non-perfused hearts of these insulin-deficient rats, which show marked abnormalities in glucose metabolism. Protein synthesis was not diminished in the perfused hearts from rats treated with anti-insulin antiserum. The significance of these findings is discussed in relation to the reported effects of insulin deficiency on protein synthesis in skeletal muscle.

The role of glucose in the survival and 'recovery' of the anoxic isolated perfused rat heart.

Studies with the isolated perfused working rat heart were carried out to investigate factors that may enable the heart to recover after periods of anoxia. It was found that the presence of glucose in the perfusion fluid during anoxia was essential for complete post-anoxic recovery and the presence of a high concentration of K(+) increased not only the rate of recovery but also the final extent of recovery. In an attempt to clarify the roles played by glucose and K(+) in aiding the survival and recovery of the anoxic myocardium the concentrations of parameters associated with energy liberation and anaerobic glycolysis (ATP, ADP, AMP, P(i), creatine phosphate, glycogen and lactate) were measured in the presence and absence of glucose during the anoxic phase. Determinations of these parameters were carried out during the working aerobic control period, the anoxic period (K(+) arrest) and the recovery period. The results demonstrated that glucose acted as an energy source during anoxia and thus maintained myocardial concentrations of high-energy phosphates, particularly ATP. These studies have also shown a direct relationship between the ability of the heart to recover and the concentration of myocardial ATP at the time of reoxygenation.