Structure of the horseradish peroxidase isozyme C genes

We have isolated, cloned and characterized three cDNAs and two genomic DNAs corresponding to the mRNAs and genes for the horseradish (Armoracia rusticana) peroxidase isoenzyme C (HPR C). The amino acid sequence of HRP C1, deduced from the nucleotide sequence of one of the cDNA clone, pSK1, contained the same primary sequence as that of the purified enzyme established by Welinder [FEBS Lett. 72, 19-23 (1976)] with additional sequences at the N and C terminal. All three inserts in the cDNA clones, pSK1, pSK2 and pSK3, coded the same size of peptide (308 amino acid residues) if these are processed in the same way, and the amino acid sequence were homologous to each other by 91-94%. Functional amino acids, including His40, His170, Tyr185 and Arg183 and S-S-bond-forming Cys, were conserved in the three isozymes, but a few N-glycosylation sites were not the same. Two HRP C isoenzyme genomic genes, prxC1 and prxC2, were tandem on the chromosomal DNA and each gene consisted of four exons and three introns. The positions in the exons interrupted by introns were the same in two genes. We observed a putative promoter sequence 5' upstream and a poly(A) signal 3' downstream in both genes. The gene product of prxC1 might be processed with a signal sequence of 30 amino acid residues at the N terminus and a peptide consisting of 15 amino acid residues at the C terminus.     

Molecular cloning of complementary DNA encoding one of the human pancreatic protease E isozymes

We have cloned a DNA from a human pancreatic cDNA library using a cloned rat pancreatic elastase 1 cDNA as a probe, and determined its nucleotide sequence. This cDNA contains a coding region of 810 nucleotides which encodes a 270-amino-acid protein. The deduced amino acid sequence shows less than 60% homologies with rat and porcine pancreatic elastase 1, although its substrate binding region is homologous with those of the above elastases 1. When this deduced amino acid sequence was compared with known amino acid sequences of pancreatic proteases other than elastases, it was found to contain an amino acid sequence which was highly homologous with the N-terminal amino acid sequence of porcine pancreatic protease E. We also purified human pancreatic protease E isozymes from human pancreatic juice, and determined their N-terminal amino acid sequences. One of the isozymes does not hydrolyze elastin but does hydrolyze a synthetic substrate. Endoglycosidase F digests glycoside bonds of the isozyme. These results suggest that the cDNA cloned by us corresponded to one of the human protease E isozymes.     

Differentiation of oocyte- and somatic-type 5S rRNAs in animals

In some amphibians and bony fishes, oocyte- and somatic-type 5S rRNA genes are expressed differently in oocytes and somatic cells. In order to determine at what stage of animal evolution this differential expression system appeared and how it is regulated, the sequences of oocyte and somatic 5S rRNAs from three invertebrates (sea urchin, sea hare, and silkworm) and two vertebrates (lamprey and chick) were analyzed. It was found that the oocyte 5S rRNA from lamprey consists of two components, while its somatic 5S rRNA consists of only one. In other animals, such differential expression of 5S rRNA in oocytes and somatic cells was not seen. A phylogenetic tree of 63 animal 5S rRNAs was constructed by means of the parsimony method, and the evolution of oocyte and somatic-type 5S rRNAs was discussed.     

Efficient synthesis and secretion of a thermophilic alpha-amylase by protein-producing Bacillus brevis 47 carrying the Bacillus stearothermophilus amylase gene.

Bacillus subtilis and Bacillus brevis 47-5, carrying the Bacillus stearothermophilus alpha-amylase gene on pUB110 (pBAM101), synthesized the same alpha-amylase as the donor strain as determined by the enzyme's thermal stability and NH2-terminal amino acid sequence. Regardless of the host, the 34-amino acid signal peptide of the enzyme was processed at exactly the same site between two alanine residues. B. brevis 47-5(pBAM101) secreted the enzyme most efficiently of the hosts examined, 100, 15, and 5 times more than B. stearothermophilus, Escherichia coli HB101(pH1301), and B. subtilis 1A289(pBAM101), respectively. The efficient secretion of the enzyme in B. brevis 47-5(pBAM101) was suggested to be due to the unique properties of the cell wall of this organism.     

A new type of glycogen storage disease caused by deficiency of cardiac phosphorylase kinase

A five-month-old Japanese boy was found to have marked glycogen accumulation only in the heart. A survey of enzymes revealed normal activities of phosphorylase, cyclic AMP-dependent protein kinase, acid maltase and amylo-1,6-glucosidase. However, the heart had capacity of activating neither rabbit muscle phosphorylase b nor endogenous phosphorylase b, which was converted to active form only when supplemented rabbit muscle phosphorylase kinase. In contrast to the heart, activities of phosphorylase kinase were found within normal levels in other organ tissues so far tested. These findings indicate that the present case of the cardiac glycogenosis is caused by deficiency of cardiac phosphorylase kinase.     

Effect of 1,25-dihydroxyvitamin D3 on pancreatic B and D cell function

To investigate the effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on pancreatic B and D cell function in normal rats, 1 microgram of 1,25(OH)2D3 was administered intravenously 20 hours before the experiment. The plasma 1,25(OH)2D3 and calcium concentrations were significantly elevated, and plasma insulin levels also increased in 1,25(OH)2D3-administered rats compared with controls. Glucose-induced insulin and somatostatin release from the isolated pancreas perfused with lower calcium, however, was the same between the 1,25(OH)2D3-administered group and the controls. On the other hand, when the isolated pancreas was perfused with higher calcium, the glucose-induced insulin release was significantly increased in the 1,25(OH)2D3-administered group, while no significant difference in somatostatin release was observed in any group. These results suggest that the sensitivity of pancreatic B cells to glucose perfused with more calcium may increase when 1,25(OH)2D3 has been previously administered. In addition, 1,25(OH)2D3 does not seem to affect the somatostatin release from the pancreatic D cells.     

Effects of prostaglandin E2 and D2 on gastric somatostatin and gastrin secretion

The effects of PGE2 and PGD2 on gastric somatostatin and gastrin releases were investigated using the isolated perfused rat stomach. In the presence of 5.5 mM glucose, the infusion of PGE2 elicited a significant augmentation in somatostatin release, but suppressed gastrin secretion from the perfusate. On the other hand, PGD2 did not affect somatostatin release, although the gastrin secretion decreased significantly, the same as after PGE2 infusion. These results suggest that PGE2 and PGD2 may be important in the regulation of gastric endocrine function, but that PGD2 does not affect gastric somatostatin secretion.     

How do inositol phosphates regulate calcium signaling?

Activation of a variety of cell surface receptors results in the phospholipase C-catalyzed hydrolysis of the minor plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate, with concomitant formation of inositol 1,4,5-trisphosphate and diacylglycerol. There is strong evidence that inositol 1,4,5-trisphosphate stimulates Ca2+ release from intracellular stores. The Ca2+-releasing actions of inositol 1,4,5-trisphosphate are terminated by its metabolism through two distinct pathways. Inositol 1,4,5-trisphosphate is dephosphorylated by a 5-phosphatase to inositol 1,4-bisphosphate; alternatively, inositol 1,4,5-trisphosphate can also be phosphorylated to inositol 1,3,4,5-tetrakisphosphate by a 3-kinase. Although the mechanism of Ca2+ mobilization is understood, the precise mechanisms involved in Ca2+ entry are not known; the proposal that inositol 1,4,5-trisphosphate secondarily elicits Ca2+ entry by emptying an intracellular Ca2+ pool is considered.