Production of recombinant salmon calcitonin by in vitro amidation of an Escherichia coli produced precursor peptide.

Salmon calcitonin (sCT) is a 32 amino acid peptide hormone that requires C-terminal amidation for full biological activity. We have produced salmon calcitonin by in vitro amidation of an E. coli produced precursor peptide. Glycine-extended sCT, the substrate for amidation, was produced in recombinant E. coli as part of a fusion with glutathione-S-transferase. The microbially produced soluble fusion protein was purified to near homogeneity by affinity chromatography. Following S-sulfonation of the fusion protein, the glycine-extended peptide was cleaved from the fusion by cyanogen bromide. The S-sulfonated peptide was recovered and enzymatically converted to the amidated peptide in a reaction with recombinant peptidylglycine alpha-amidating enzyme (alpha-AE) secreted from Chinese hamster ovary (CHO) cells. After reformation of the intramolecular disulfide bond, the sCT was purified with a step yield of 60%. The ease and speed of this recombinant process, as well as its potential for scale-up, make it adaptable to production demands for calcitonin, a proven useful agent for the treatment of post-menopausal osteoporosis. Moreover, the relaxed specificity of the recombinant alpha-AE for the penultimate amino acid which is amidated allows the basic process to be applied to the production of other amidated peptides.

The effect of adenylate cyclase inhibitor (ACI) on guanylate cyclase, phosphodiesterase and other enzymes in heart.

The effect of an inhibitor of adenylate cyclase (ACI) was measured on some enzymes associated with cyclic nucleotide-regulated metabolism. Soluble guanylate cyclase was inhibited; both soluble and particulate cyclic GMP-phosphodiesterases were stimulated. Cyclic AMP phosphodiesterases were unaffected. In contrast, the activities of Na, K-ATPase, protein kinase, phosphorylase kinase, glycogen synthetase and a number of glycosidases were not altered by equipotent amounts of the inhibitor. It is concluded that this substance acts as a modulator of both cyclic AMP and cyclic GMP metabolism in heart and other tissues.

Antigenic differences in (Na+, K+)-ATPase preparations isolated from various organs and species.

Antisera to purified (Na+, K+)-ATPase raised in rabbits and in sheep were purified by an absorption procedure employing purified canine kidney (Na+, K+)-ATPase. The antibodies were fractionated into two components, one which inhibited catalytic activity, and a second which inhibited ouabain binding. Under certain conditions, the fraction that inhibited ouabain binding also inhibited catalytic activity, and the effectiveness of both was dependent to some extent on the ligands present in the incubation medium. Thus, both antibody fractions appeared to detect conformations of the enzyme that depended upon ligand-induced perturbations. When the antibody raised against catalytic activity was incubated with erythrocyte membrane fragments, an inhibition of the (Na+, K+)-ATPase occurred, but only minimal or no effect on potassium influx or on digoxin-induced inhibition of potassium flux in intact erythrocytes was noted. In a similar experiment, however, the antibody against ouabain binding significantly inhibited potassium influx, suggesting specificity in terms of the macromolecular surfaces of the pump which were exposed to the external medium. We concluded that there may be organ and species differences among (Na+, K+)-ATPase preparations. Antibodies prepared in rabbits and sheep were fractionated by absorption to dog brain enzyme. Both the antibody fraction which bound to the brain enzyme and that which did not bind inhibited the dog kidney (Na+, K+)-ATPase, but only the former inhibited dog brain (Na+, K+)-ATPase. When the two fractions were recombined, inhibition was restored to the extent of the unfractionated antibody.