No abstract

Glucagon is a 29-amino acid polypeptide hormone synthesized by the A cells of the endocrine pancreas. Its primary site of action is the liver where it stimulates glycogenolysis, gluconeogenesis and ketogenesis. In mammals, biosynthetic studies have shown that glucagon is derived from a precursor of molecular weight (Mr) approximately 18,000 which is five to six times larger than glucagon. Glucagon-containing polypeptides and immunoreactants of various sizes have also been described from stomach, intestine, brain and salivary gland. Here, we have determined the structure of hamster pancreatic preproglucagon from the sequence of its cDNA. This 180-amino acid precursor contains the sequence of glucagon and two glucagon-like polypeptides arranged in tandem. The precursor also contains the sequences of several non-pancreatic glucagon-containing polypeptides which suggests that, in mammals, both pancreatic and non-pancreatic glucagon and glucagon-containing polypeptides may be derived from a common precursor by tissue-specific processing. We have tentatively identified each of the glucagon-like immunoreactants which have been described with respect to the sequence of proglucagon and have proposed a scheme for the processing of pancreatic proglucagon.

Fig.4 Southern blot analysis of PstI fragments of S. coelicolor plasmids. Lanes 1, pI1601; lanes 2, SCPl(410 kb); lanes 3, SCP1(440 kb); lanes 4, SCPl(470kb); lanes 5, pM138; lanes 6, pM146. Plasmids, SCPls(410, 440 and 470 kb), pM138 and pM146 were isolated from S. coelicolor A3(2), M138 and M146, respectively as in Fig. 1. These plasmids and pI1601 were digested with PstI and _ subjected to conventional agarose gel e1ec• trophoresis and Southern blot analysis with 32p. labelled pI1601 as a probe. 2 3 4 5 6

Saccharomyces cerevisiae cells were transformed with plasmids containing hybrid genes in which the sequence encoding mature human epidermal growth factor was joined to sequences encoding the leader region (preprosegment) of the precursor of the yeast mating pheromone afactor. These cells accurately process the hybrid protein and efficiently secrete authentic biologically active human epidermal growth factor into the medium. Previous studies on the expression of B-lactamase-proinsulin gene fusions in Escherichia coli showed that the /3-lactamase signal sequence is cleaved and proinsulin is secreted into the periplasm (4). We reasoned, therefore, that the leader sequences of secreted yeast proteins may allow more efficient processing and secretion of heterologous proteins by yeast.Most studies on secretion of yeast proteins have used enzymes such as invertase and acid phosphatase (2, 5, 6), which are secreted into the periplasmic space or cell wall.We have used the peptide mating pheromone a-factor, which is efficiently secreted into the medium. a-Factor is a 13-residue peptide, secreted by cells of the a mating type, that acts on cells of the opposite a mating type to promote efficient conjugation between the two cell types leading to the formation of aa diploid cells (7). Studies on the sequence of the afactor structural gene (8) and on the synthesis and processing of the a-factor peptide (9-11) have shown that a-factor is synthesized as a precursor of 165 amino acids containing an 83-residue leader and four a-factor coding regions, each preceded by a short spacer peptide. The leader and spacer amino acids appear to contain the signals necessary for proteolytic processing and secretion. A recent study (12) shows that fusion of the leader region of the a-factor precursor to invertase, another secreted yeast protein, directs its export.To investigate whether the a-factor leader sequences are sufficient to allow efficient processing and secretion, we have constructed plasmids in which the genes coding for foreign secreted proteins have been fused to the yeast a-factor gene and expressed under the control of the a-factor promoter. Several different constructions were made in which the

A mutated protein of human Cu(II)2Zn(II)2 SOD in which residues Phe50 and Gly51 at the dimer interface were substituted by Glu's, thus producing a monomeric species, has been characterized by electronic absorption spectroscopy, EPR, relaxivity and 1H NMR techniques. Such substitutions and/or accompanying remodeling and exposure of the dimer interface to solvent, alter the geometry of the active site: increases in the axiality of the copper chromophore and the Cu-OH2 distance have been observed. The affinity of both metal binding sites for Co(II) is also altered. The observed NMR parameters of the Co(II) substituted derivative have been interpreted as a function of the decrease of rotational correlation time as a consequence of the lower molecular weight of the mutated protein. Sharper NMR signals are also obtained for the reduced diamagnetic enzyme. Results are consistent with an active site structure similar to that observed for the dimeric analog. Thr137Ile characterized elsewhere. An observed proportional decrease in enzymatic activity and affinity for the N3-anion suggests the importance of electrostatic forces during substrate docking and catalysis.