No abstract

Rat aortic rings cultured for 24 h in protein-free medium showed a significant reduction in the contractile response to potassium (-60%) and to phorbol 12,13-dibutyrate (-65%). The addition of plasma to the medium attenuated the loss in responsiveness, and the supplementation of the plasma-containing medium with all trans-retinoic acid (RA) returned the response to potassium (85%) and phorbol ester (135%) to near normal or supramaximal compared with fresh tissue. Furthermore, the combined additions of plasma and RA resulted in significant preservation of the contractile response (75%) for at least 3 days in organ culture. Removal of the endothelium before organ culture eliminated the enhancement of contraction by plasma and RA. However, the removal of the endothelium from tissues that had been cultured with an intact endothelium or the blockade of nitric oxide synthesis in these tissues before contractile measurements had no effect on the contractile response. Finally, the incubation of tissues with phorbol ester to downregulate protein kinase C resulted in a marked attenuation (-75%) of the contractile response compared with control tissues in culture. The results suggest that circulating factors may be necessary for the maintenance of contractile function of aortic smooth muscle. Based on the opposing actions of RA and phorbol ester, the long-term regulation of contractile function may involve protein kinase C activity.

No abstract

The isopropylmalate isomerase (EC 4.2.1.33) of Neurospora crassa is a globular protein consisting of a single polypeptide chain with a molecular weight of about 90,000. The isomerase cannot easily be freed of a contaminating protease which cleaves the enzyme into two major fragments, one of approximately 56,000 and the other 37,000 daltons. This suggests that the folded polypeptide chain may contain some hinge point or loop exposed on the surface which makes it susceptible to proteolytic attack. Most of the isomerase activity extracted from the wild-type strain is in monomer form. However, a small fraction of the activity in crude extracts is found in multimeric aggregates, and the active isomerase extracted from complementing leu-2 heterokaryons consists entirely of dimers and higher multimers. These observations suggest that, though active as a monomer, a significant fraction of the normal enzyme might be organized in multimeric form within the cell.

The capacity to synthetize isopropylmalate isomerase (EC 4.2.1.33) by Neurospora crassa increased during induction in the presence of cycloheximide but was inhibited by proflavine and other inhibitors of RNA synthesis. Turnover of the enzyme once formed appeared negligible, but the message (measured as enzyme-forming capacity) had a half-life of 4 to 8 min. A comparison of the kinetics of induction in the wild type and a newly isolated alpha-isopropylmalate-permeable strain suggested strongly that feedback control by leucine of alpha-isopropylmalate production can adequately serve as the primary physiological regulator of endogenous inducer concentration. Genetic data are presented which implicate the involvement of two unlinked genes, ipm-1 and ipm-2, in determining permeation of alpha-isopropylmalate.