Purification and characterization of Streptomyces griseus metalloendopeptidases I and II.

Two metalloendopeptidases, designated as Streptomyces griseus metalloendopeptidases I and II (SGMPI and SGMPII), were isolated from a commercial Pronase P by a method including affinity chromatography on carbobenzoxy-L-alaninyl-triethylenetetraminyl-Sepharose (Z-Ala-T-Sepharose). The two enzymes differed from each other in behavior on ion-exchange chromatography but showed the same amino-terminal sequence at least up to the 20th residue. Their molecular weights were both estimated to be 37,000 by SDS-polyacrylamide gel electrophoresis. Elemental and amino acid composition analyses indicated that both of them contained about 1 g atom of zinc and one cystine residue per mol of protein. Cleavage specificities of the two enzymes toward synthetic peptide-substrates were very similar to those observed with thermolysin. EDTA, o-phenanthroline, and phosphoramidon strongly inhibited these enzymes, while typical serine-protease inhibitors and cysteine-protease inhibitors had no effect. The findings clearly indicate that SGMPI and SGMPII can be classified into the family of zinc-endopeptidases. It was unexpectedly found, however, that these metalloendopeptidases were strongly inhibited by protein serine-protease inhibitors produced by Streptomycetes, such as Streptomyces subtilisin inhibitor (SSI), alkaline protease inhibitor-2c' (API-2c'), and plasminostreptin (PS).     

Interleukin-1 enhances the response of osteoblasts to platelet-derived growth factor through the alpha receptor-specific up-regulation.

Both platelet-derived growth factor (PDGF) and interleukin-1 (IL-1) are produced by activated macrophages and are thought to contribute to bone remodeling, but their precise roles remain to be clarified. The interaction between PDGF and IL-1 was, therefore, studied in normal osteoblast-like cells (MC3T3-E1). The expression of alpha- and beta-PDGF receptors on MC3T3-E1 cells was detected by RNA blot analysis and confirmed by immunoblot analysis. PDGF-induced chemotactic as well as mitogenic activities were synergistically enhanced by either IL-1 alpha or IL-1 beta (40 units/ml) pretreatment in serum-free medium, although IL-1 alone did not show any detectable chemotactic activities. This biological enhancement by IL-1 was accompanied by a selective increase of alpha-PDGF receptor expression, following the augmentation of alpha receptor autophosphorylation and inositol phosphate hydrolysis induced by PDGF-AA. These findings suggest that PDGF and IL-1 are jointly involved in the bone-remodeling microenvironment as local coupling factors.     

Light and electron microscopic immunocytochemistry on the localization of 3 beta-hydroxysteroid dehydrogenase/isomerase in the bovine adrenal cortical cells

The localization of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3 beta-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3 beta-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3 beta-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.     

Conformation change of effector-region residues in antiparallel beta-sheet of human c-Ha-ras protein on GDP----GTP gamma S exchange: a two-dimensional NMR study

The conformations of a truncated human c-Ha-ras gene product [ras(1-171) protein] in the GDP-bound form and in the GTP gamma S-bound form were compared by two-dimensional nuclear Overhauser effect spectroscopy (NOESY). As for the GDP-bound ras(1-171) protein, three NOESY cross peaks were observed in the region of 4.5-6.0 ppm, indicating a regular antiparallel beta-sheet structure. On the ligand exchange from GDP to GTP gamma S, one of the three NOESY cross peaks disappeared and the other two cross peaks were appreciably shifted. By analysis of the effects of specific deuteration of leucine residues and the homonuclear Hartmann-Hahn spectroscopy, the antiparallel beta-sheet was found to consist of residues 38-44 and residues 51-57. The conformations around Ser-39 and Leu-56 are of the regular antiparallel beta-strand type in the GDP-bound state, and largely distorted in the GTP gamma S-bound state, which is probably related to the conformational activation of the effector region of ras proteins by ligand exchange from GDP to GTP gamma S.     

Age-related decrease of urinary excretion of human epidermal growth factor (hEGF)

Epidermal growth factor (EGF) has been first isolated from the submandibular glands of the male mouse and recently from human urine. Despite its potent mitogenic effect in a variety of tissues, the physiological functions of EGF in human still remain undetermined. In order to study the effect of age on urinary human EGF (hEGF), we have evaluated urinary excretion of hEGF in normal subjects over a wide range of age (20–79 yr.) using homologous radioimmunoassay (RIA) for hEGF. Urinary excretion of hEGF expressed as a function of creatinine significantly decreased with increasing age, age, while females excreted significantly more hEGF than males. These data suggest that urinary excretion of hEGF decreases with age in normal subjects which may be due to reduced synthesis and/or secretion of hEGF.