William B. Benjamin

Calcitonin gene‐related peptide acts presynaptically to increase quantal size and output at frog neuromuscular junctions

1 Calcitonin gene‐related peptide (CGRP) is found in dense‐cored vesicles in the motor nerve terminal. 2 Exogenous CGRP increased the size of the quanta. The increase in size reached a maximum after about 40 min. The lowest effective concentration of human CGRP (hCGRP) was 0.8 nM. The action of hCGRP was antagonized by (−)‐vesamicol, a drug that blocks active acetylcholine (ACh) uptake into synaptic vesicles, so it appears that hCGRP increases size by adding more ACh to the quanta. The action of hCGRP was antagonized by drugs that block the activation of protein kinase A (PKA). (In other preparations CGRP also activates PKA.) 3 The hCGRP effect was not blocked by fragment 8–37, an antagonist of one class of CGRP receptor. 4 hCGRP increases evoked quantal output and miniature endplate potential (MEPP) frequency, again by activating PKA. 5 CGRP release was measured by radioimmunoassay. Release was increased by depolarization with elevated K+, but the amounts released appear to be below those needed to affect quantal size or output. Moreover, although elevated K+ can increase quantal size it acts by a pathway that does not involve PKA. We suggest that the most likely target of endogenously released CGRP is the regulation of circulation of the muscle.

Phosphorylation of ATP‐citrate lyase by a cyclic AMP‐independent protein kinase from a rat liver

Insulin action in isolated adipocytes or hepatocytes incubated with 32Pi rapidly increases the amount of 32P radioactivity associated with a cytosolic phosphoprotein [l-4]. We isolated and purified this phosphoprotein from adipose and liver tissues and identified it as ATP-citrate lyase [5]. Concurrently, Alexander et al. [6] also found that in liver this phosphoprotein is ATP-citrate lyase. When ATP-citrate lyase is incubated with ATP, 2 mol phosphate/m01 enzyme are incorporated into histidine residues at the catalytic site (referred to as catalytic phosphate) which are acid labile [S ,7]. In addition, at least 2 mol acid-stable phosphate (referred to as structural phosphate), principally phosphoserine, have been found with each 1 mol enzyme [8]. Because structural site phosphorylation was affected by hormone action [ 1,2,5], we looked for a cyclic AMP-independent protein kinase in an insulin-sensitive tissue with specificity for phosphorylating the ATP-citrate lyase structural phosphate sites.

Insulin stimulates phosphorylation of a heat-stable protein in rat adipose tissue.

Insulin in rat adipose tissue acts to increase the phosphorylation about 2.5-fold of a low molecular weight protein in the cytosol designated phosphoprotein m. Isoproterenol had no effect on the phosphorylation of phosphoprotein m. Some of the properties of phosphoprotein m are: soluble in 1% trichloro acetic acid, heat-stable and has a molecular weight of 23,000 on polyacrylamide gels in the presence of sodium dodecyl sulfate. Phosphoserine and phosphothreonine are the phosphorylated amino acid residues of phosphoprotein m. The physical and chemical properties of phosphoprotein m are similar to those of previously described inhibitor and modulator proteins.

The effect of ecdysterone on nonihistone proteins in salivary gland chromosomes of Sciara coprophila

Synthesis and transport of proteins to the cell nucleus during puff induction was studied in S. coprophila. Changes in grain distribution along chromosomes (L-methionine [35S] incorporation into protein) were correlated with puffs induced by ecdysterone in vitro; A pattern of specific labelling at the sites of incipient puffs was noted within 2 h after the addition of the hormone, i.e. grains on the chromosomes were in clusters, characteristic for this time point and not seen in the controls (where only non-specific labeling was noted 0-4 h). Characteristic chromosomal puffs appeared between 3-4 h after the addition of ecdysterone. It was concluded that during ecdysterone-induced puff formation in salivary gland chromosomes, proteins which had been previously synthesized were selectively transported from the cytoplasm to specific sites on the chromosomes.

Chapter 22 Posttranscriptional Modifications of Nonhistone Proteins of Salivary Gland Cells of Sciara coprophila

Publisher Summary This chapter describes the posttranscriptional modifications of nonhistone proteins of salivary gland cells of Sciara coprophila . The techniques useful in studying nucleoprotein metabolism of polytene chromosomes are also discussed. The early appearance of nonhistone proteins in chromosome regions that will form puffs suggests that there may be a relationship between these proteins and the “activating” stimulus. The steroid molting hormone, ecdysone, induces puff formation only at specific stages of development. Either before or at the onset of puff formation there is a substantial accumulation of pre-existing protein in and around the developing puff. There is a pool of preexisting cytoplasmic proteins that, when acted upon either directly or indirectly by a specific hormone, allows recognition by this protein(s) of specific sites on the genome. The early appearance of nonhistone proteins may be part of the biochemical mechanism for the control of chromosomal loci designated to become activated by ecdysone. As the effects of endogenous ecdysone in Sciara coprophila are permanent, the transfer of cytoplasmic proteins to the nucleus may represent a mechanism for stable differentiation.