Determination of the primary structure of Paim II, an alpha-amylase inhibitor from Streptomyces coruchorushii, by high-performance tandem mass spectrometry

This study indicates one of the advantages of tandem mass spectrometry; the primary structures of proteins with little structural difference can be determined by using tandem mass spectrometry without prior purification of each component. The primary structure of Paim II, a protein alpha-amylase inhibitor from Streptomyces coruchorushii, was determined by using tandem mass spectrometry. Paim II consists of two component proteins with ragged N-terminus, and was sequenced on the basis of the structure of Paim I, an analogous alpha-amylase inhibitor from the same natural origin.     

Effects of proteinase inhibitors on preimplantation embryos in the rat

Proteinase inhibitors of microbial origin were injected into the uterine horns of mated rats at 14:00 h on Day 5 of pregnancy (spermatozoa in vaginal smear = Day 1), and 5 or 6 h later the embryos were flushed from the horns and examined. Chymostatin and alpha-MAPI, inhibitors of chymotrypsin-like serine proteinase and thiol proteinases, as well as thiolstatin, an inhibitor of thiol proteinases, significantly inhibited embryo growth. The inhibitory activity of alpha-MAPI on embryonic growth was distinctly greater than that of thiolstatin, although the ID50 values of the two inhibitors to papain are similar. Antipain and leupeptin which are inhibitors of trypsin-like and thiol proteinases, and talopeptin, an inhibitor of metal proteinases, significantly interrupted the removal of the zona pellucida from expanding blastocysts. These results suggest that (1) a chymotrypsin-like proteinase seems to be important to the growth of the embryo, (2) a thiol proteinase may participate in embryonic growth, and (3) a trypsin-like proteinase and a metal proteinase are likely to participate in zonalysis.     

A protein containing the cystic fibrosis antigen is an inhibitor of protein kinases

To investigate myeloid cell maturation, we established a panel of monoclonal antibodies that recognize myeloid cell nuclear antigens. One of these monoclonal antibodies was used to purify a specific protein complex (PC) from a human spleen. This PC, which is present at high levels in peripheral blood monocytes and granulocytes, contains a protein that is the cystic fibrosis (CF) antigen. The purified PC was shown to inhibit the activity of casein kinase I and II but not cAMP-dependent protein kinase, protein kinase C, v-abl tyrosine kinase, or insulin receptor tyrosine kinase. The observed Ki values for casein kinases I and II purified from several sources were 1 microM or less. Furthermore, the addition of the purified PC to a nuclear extract from human cells was able to prevent protein kinase-mediated stimulation of RNA polymerase activity. The unique inhibitory character of the PC and its elevated levels in monocytes and granulocytes and of the CF antigen in CF patients implies that this complex may be associated with myeloid cell functions and perhaps with the cause or consequence of the clinical manifestations of CF.     

A protein complex expressed during terminal differentiation of monomyelocytic cells is an inhibitor of cell growth

A protein complex (PC) composed of the MRP8 and MRP14 proteins has previously been shown to be a specific inhibitor of casein kinase I and II. This PC is expressed during the late stages of terminal differentiation induced in human promyelocytic HL-60 leukemia cells by 1 alpha,25-dihydroxyvitamin D3 and in human monocytic THP-1 leukemia cells by phorbol 12-myristate 13-acetate. This expression is associated with terminal cell differentiation because incubation of HL-60 cells with an agent or condition that causes suppression of growth but not induction of differentiation does not result in expression of the PC. At concentrations of 5-15 nM, the purified PC inhibited the growth of HL-60 cells and THP-1 cells, as well as other cell types belonging to different cell lineages. This growth inhibition was preceded by a reduction in [32P]phosphate incorporation and, at the higher PC concentrations, was associated with a reduction in [3H]thymidine, [3H]uridine, and [32S]methionine incorporation. The specific expression pattern and growth-inhibitory character of the PC suggests that the complex may have a role in suppressing cell growth during monomyelocytic terminal differentiation induced by specific chemical stimuli and during physiological and pathological events associated with monomyelocytic cell functions.     

The states of tyrosyl and tryptophyl residues in a protein proteinase inhibitor (Streptomyces subtilisin inhibitor

The states of tyrosyl and tryptophyl residues of a dimeric protein proteinase inhibitor, Streptomyces subtilisin inhibitor (Sato, S & Murao, S. (1973), Agric. Biol. Chem. 37, 1067) were studies by solvent perturbation difference spectroscopy with methanol, ethylene glycol, polyethylene glycol, and deuterium oxide as perturbants, and by spectrophotometric titration at alkaline pH. It appeared that all three tyrosyl residues per monomer of the inhibitor were exposed on the surface of the molecule, and their apparent pK values were estimated separately to be 9.58, 11.10, and 12.42. The single tryptophyl residue per monomer of the inhibitor appeared to be partially buried in the protein molecule.     

Nucleotide sequences of serine tRNAs from Bacillus subtilis.

Three B. subitilis serine tRNAs were sequenced including modified nucleosides. All the serine tRNAs contained 1-methyl-adenosine in the D-loop. As other characteristic modified nucleosides, 5-methoxyuridine was found in the first letter of the anticodon in the tRNA(UGA).