Harvey R. Kaslow

Defect of Receptor–Cyclase Coupling Protein in Pseudohypoparathyroidism

Hormone-sensitive adenylate cyclase contains a recently discovered protein component that is required for stimulation of cyclic AMP synthesis by hormones and guanine nucleotides; the component presumably couples the membrane receptor to the cyclase. We studied this protein (termed N) in erythrocyte membranes of patients with pseudohypoparathyroidism, using assays of the proteins biochemical activity and of its susceptibility to radiolabeling in the presence of [32P]NAD and cholera toxin. By both assays, the proteins activity was reduced by 40 to 50 per cent in erythrocytes of five of 10 patients with Type I pseudohypoparathyroidism as compared with those of normal and hypoparathyroid subjects and one patient with Type II pseudohypoparathyroidism. If activity of the N protein is reduced in other tissues, this deficiency could cause the resistance of target organs in pseudohypoparathyroidism to parathyroid hormone and other hormones that work via cyclic AMP. Erythrocytes of five patients with Type I pseudohypoparathyroidism, all in one family, showed no defect in activity of the N protein; the biochemical defect of this family remains undefined.

A regulatory component of adenylate cyclase is located on the inner surface of human erythrocyte membranes

Summary Human erythrocyte membranes contain the guanine nucleotide regulatory component of adenylate cyclase termed N, which can be assayed by in vitro complementation of adenylate cyclase in membranes of the cyc − variant of the S49 lymphoma cell. We used inside-out (IO) and rightside-out (RO) membrane vesicles to determine the membrane orientation of N: Trypsin inactivates N in IO but not in RO vesicles. Treatment with cholera toxin and NAD + activates N in IO but not in RO vesicles. These experiments provide strong evidence that N is located on the inner surface of the human erythrocyte membrane.

Apparent phosphorylation of glycogen synthase in mammalian cells lacking cyclic AMP-dependent protein kinase

The capacity of liver extracts to synthesize glycogen utilizing UDP-glucose as the glucose donor was first reported in [ 11. Glycogen synthase (uridine diphosphate glucose: glycogen 4_cr-glucosyl transferase, EC 2.4.1 .l l), the enzyme catalyzing this reaction, is a key enzyme in the regulationof glycogen synthesis in vivo [2,3]. Hormones promote the interconversion of synthase between activated dephosphorylated and deactivated phosphorylated forms [4,5]. Purified cyclic AMP-dependent protein kinase phosphorylated and deactivated purified glycogen synthase [6]. Thus it has been assumed that regulation of glycogen synthase involved interconversion between two kinetic forms: a D (or b) form, with activity largely dependent on the presence of the allosteric activator, glucose-6-P; and an I (or a) form, with activity essentially independent of glucose-6-P. This assumption led to the widespread use of the -/+ glucose-6-P activity ratio as a measure of the activation state of glycogen synthase. However, the regulation of glycogen synthase is more complex (reviewed [3,7,8]). These reports in& cate: (9

Defect of Receptor-Cyclase Coupling Protein in Pseudohypoparathyroidism

Hormone-sensitive adenylate cyclase contains a recently discovered protein component that is required for stimulation of cyclic AMP synthesis by hormones and guanine nucleotides; the component presumably couples the membrane receptor to the cyclase. We studied this protein (termed N) in erythrocyte membranes of patients with pseudohypoparathyroidism, using assays of the proteins biochemical activity and of its susceptibility to radiolabeling in the presence of [32P]NAD and cholera toxin. By both assays, the proteins activity was reduced by 40 to 50 per cent in erythrocytes of five of 10 patients with Type I pseudohypoparathyroidism as compared with those of normal and hypoparathyroid subjects and one patient with Type II pseudohypoparathyroidism. If activity of the N protein is reduced in other tissues, this deficiency could cause the resistance of target organs in pseudohypoparathyroidism to parathyroid hormone and other hormones that work via cyclic AMP. Erythrocytes of five patients with Type I pseudohypoparathyroidism, all in one family, showed no defect in activity of the N protein; the biochemical defect of this family remains undefined.