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Biomedical subjects

M Shin

Publications and source records attributed to M Shin.

At least 109 records · Page 6Linked to original sources
PubMed1989

Site-directed mutagenesis of human pancreatic secretory trypsin inhibitor.

Arg-42 or Lys-43 or Arg-44 of human pancreatic secretory trypsin inhibitor (PSTI) was replaced by Thr or Ser by site-directed mutagenesis, and the inactivation rates of the mutants after mixing with human trypsin were compared with that of the natural form. The inactivation rate decreased for one mutant (Arg-44----Ser), whereas no change was observed for another (Arg-42----Thr) and an increase was observed for a third (Lys-43----Thr). Kinetic studies on the interactions between human trypsin and synthetic peptides, comprising the regions of Phe39-Ser47 of the respective PSTI species, showed that human trypsin cleaved the Arg42-Lys43 bond preferentially to the Arg44-Gln45 bond. However, it is cleavage of the latter bond that is thought to cause inactivation of human PSTI. These results suggest that the Arg44-Gln45 bond of human PSTI is responsible for its inhibitory activity, and inactivation of human PSTI is probably caused by deletion of the dipeptide Lys43-Arg44.

Amino Acids↗
PubMed1989

Tryptophan metabolism by the isolated rat liver cells--effects of leucine and its metabolites.

1. The effects of leucine and its metabolites, such as alpha-ketoisocaproate and ketone bodies, on the metabolic fluxes of tryptophan were investigated in isolated rat liver cells using [benzene ring-U-14C]- or [methylene-14C]-tryptophan. 2. Tryptophan metabolized through the kynureninase flux decreased while that metabolized to acetyl CoA remained unchanged in the presence of leucine or its metabolites. Accordingly an amount of tryptophan metabolized through the quinolinate-NAD pathway, which was estimated by subtracting an amount of tryptophan metabolized via the acetyl CoA flux from that via the kynureninase flux, was decreased in the presence of leucine or its metabolites. 3. Less quinolinate accumulated during incubation with metabolites of leucine, however, the amount was still sufficient to saturate quinolinate phosphoribosyltransferase (EC 2.4.2.19). 4. Leucine and its metabolites added in vitro at 1 mM level did not inhibit quinolinate phosphoribosyltransferase activity in rat liver homogenate. 5. The results indicate that a decrease in NAD biosynthesis from tryptophan caused by metabolites of leucine in the isolated rat liver cells was neither due to insufficient supply of quinolinate nor to direct inhibitory effect on quinolinate phosphoribosyltransferase.

Acetoacetates↗
PubMed1988

Electron-microscopic studies on the threshold value of calcium concentration for the release of storage granules and the acceleration of their degradation in the rat parathyroid gland.

To determine both a threshold value of calcium concentration (CC) for the release of storage granules and that for the acceleration of degradation of these granules, the rat parathyroid glands were perfused in situ with HEPES-Ringer solutions containing different concentration of Ca2+ for 10 min. With perfusates containing 0.83-1.21 mM Ca2+ (equivalent to 8-11 mg/dl serum calcium), the number of type-I storage granules (large core) [NSG-I] and that of type-II storage granules (small core) [NSG-II] remained unchanged. With perfusates containing 0.83 mM Ca2+ (7.5 mg/dl) or less, however, both NSG-I and NSG-II decreased remarkably and the former was larger than the latter. On the contrary, with perfusates containing 1.27 mM Ca2+ (11.5 mg/dl) or more, NSG-II increased and the ratio of NSG-I to NSG-II was changed reversely. We concluded that a threshold value of CC required for the release of storage granules may be present between 0.88 and 0.83 mM Ca2+ (8 and 7.5 mg/dl) and that a threshold value of CC for accelerating the transformation of type-I granules into type-II, the degradation of storage granules, may be situated at about 1.27 mM Ca2+ (11.5 mg/dl). Additionally, it was suggested that both pro-secretory and storage granules are not only formed at the innermost Golgi cisterna but also at the trans-Golgi network.

Animals↗
PubMed1988

Isolation and purification of mature bovine adrenocortical ferredoxin with an elongated carboxyl end.

Mature bovine adrenocortical ferredoxin (adreno-ferredoxin) was extracted from fresh adrenal glands at pH 9.0. Extraction and purification at this alkaline pH protected the mature adreno-ferredoxin molecule from proteolytic degradation. The mature adreno-ferredoxin was extensively purified by a rapid procedure including two kinds of column chromatography, hydrophobic and ion exchange. The purified adreno-ferredoxin was homogeneous on the basis of two HPLC analyses, hydrophobic and ion exchange, and had the highest purity so far reported. Then it was digested by trypsin and the carboxyl-terminal peptide was isolated from the tryptic digest by a novel column chromatographic method using a cation-exchange HPLC column, TSK-gel SP-5PW. The carboxyl-terminal amino acid was isoleucine, so the adreno-ferredoxin had 127 amino acid residues, the longest polypeptide so far determined chemically for bovine adreno-ferredoxin. Only Glu-128 was lacking within the carboxyl-terminal elongated peptide that was found by nucleotide sequencing of the adreno-ferredoxin gene. There was no evidence obtained on whether the deletion of Glu-128 was due to so-called carboxyl-terminal processing or to proteolytic degradation during storage and purification.

Adrenal Cortex↗
PubMed1987

Evidence from high-pressure liquid chromatography for the existence of two ferredoxins in plants.

Crude ferredoxin preparations were obtained from blue-green algae, green algae, ferns, and higher plants. We analyzed the preparations by high-pressure liquid chromatography using two different types of columns, a hydrophobic phenyl-5PW column and an ion-exchange DEAE-5PW column. Two ferredoxins were detected in all plants analyzed. The ferredoxins from some plants were separated by use of both columns and those from others were separated by one of the two columns. Thus, there were three possible ways in which pairs of ferredoxins from a single species of plant could be separated. We suspect that there are two different ferredoxins in most if not all species of plants.

Chlorella↗
PubMed1987

Base sequence-specific interactions of operator DNA fragments with the lambda-cro repressor coupled with changes in their conformations.

The mechanism of interaction of the operator DNA with the lambda-cro repressor protein was investigated using proton n.m.r. and photo CIDNP. Three kinds of DNA duplexes, the lambda-OR3 17-mer, phi80-OR2 19-mer and CRP binding site 22-mer, were prepared, and all of their imino proton resonances of the complexes with lambda-cro were assigned to individual base pairs. By monitoring the assigned signals of the DNA fragments and lambda-cro, it was found that in the complex of lambda-cro with lambda-OR3, two subunits of the cro dimer bind to the right and left halves of the OR3, respectively, and the bidentate binding induces a structural distortion in the middle of the 17-mer. lambda-cro itself also undergoes a conformational change including loosening of the dimeric form. In the complex of lambda-cro with phi 80-OR2, which has a 6-bp sequence common to that of lambda-OR3, one subunit of the cro dimer seems to bind specifically to the common part. However, there is only a slight conformational change in the cro dimer. In the mixture of the CRP binding site 22-mer and lambda-cro, soft contact without any conformational change was observed between them.

Base Sequence↗
PubMed1987

Production of recombinant human pancreatic secretory trypsin inhibitor by Escherichia coli.

A synthetic gene for human pancreatic secretory trypsin inhibitor (PSTI) was fused to the coding sequence for the amino-terminal 135 amino acid residues of human interferon-gamma (IFN-gamma) by interposing a methionine codon sequence, and the resulting hybrid gene was efficiently expressed in Escherichia coli cells. Recombinant human PSTI (rHu-PSTI) was separated from the IFN-gamma/PSTI fused protein by cleavage at the methionine residue with cyanogen bromide. Finally, rHu-PSTI was purified by affinity chromatography on a bovine trypsin-CH-Sepharose 4B column. The amino acid composition, partial amino-terminal sequence, disulfide formation, human trypsin inhibitory activity, and immunoreactivity against rabbit anti-human PSTI serum of rHu-PSTI corresponded to those of the natural form.

Amino Acid Sequence↗
PubMed1987

Freeze-fracture replica studies of effects of ACTH treatment and hypophysectomy on the cell surface of the rat adrenal inner cortex.

The morphological change of the cell surface in the inner zone of the adrenal cortex was studied in intact, hypophysectomized, and ACTH-treated hypophysectomized rats, using freeze-fracture replicas. In both intact and hypophysectomized ACTH-treated animals, the P fracture face of the plasma membranes of the cortical cells facing capillaries shows groups of characteristic finger-shaped microvilli, which arise horizontally from the edges of a small cavity on the cell surface to form various configurations ranging from incompletely organized, flower bud-like to complicated, arboraceous ones. In the non-treated hypophysectomized animals, however, the microvilli in such groups decrease in number and shorten in length but increase in width, looking like a blade of a prickly-pear cactus or an irregular-shaped plate. On the other hand, presumptive exocytotic and endocytotic specializations of intra-membraneous and experimental animals.

Adrenal Cortex↗
PubMed1986

Heterogeneity of adrenocortical ferredoxin.

Bovine adrenocortical ferredoxin (adreno-ferredoxin) was purified from adrenocortical mitochondria by an improved method that included hydrophobic chromatography on Toyopearl gels. The purified ferredoxin was electrophoretically homogeneous. It was further separated into five fractions by hydrophobic chromatography on a TSK-gel phenyl-5PW column with a high-pressure liquid chromatography system. The properties of the three main fractions were examined. The fractions had identical absorption spectra and almost the same activity in an NADPH-cytochrome c reducing system. Their amino-terminal sequences all corresponded to the reported sequence, but the carboxyl-terminal residues were glycine or serine, not alanine as reported. These results indicate that these adreno-ferredoxins had additional amino acid residues at the carboxyl end. It seems that adreno-ferredoxin extracted from mitochondria undergoes proteolytic attack during purification to become heterogeneous.

Adrenal Cortex↗
PubMed1986

Separation and identification of two native forms of spinach ferredoxin by hydrophobic chromatography.

Hydrophobic chromatography on a TSK-gel Phenyl-5PW column separated highly purified spinach ferredoxin into two distinct molecular species in their native forms. The two ferredoxins showed almost the same absorption spectra in spite of a difference in amino acid composition. Both ferredoxins were active in the NADPH-cytochrome c reducing system, and no significant difference was observed between their activities. The new separation method was also applied to ferredoxins highly purified from wheat plants and barley. Interestingly, all ferredoxin preparations so far examined contained two molecular species of ferredoxin.

Amino Acids↗
PubMed1986

Dog and rat pancreatic phospholipases A2: complete amino acid sequences deduced from complementary DNAs.

We performed molecular cloning of the cDNAs that encode dog and rat pancreatic phospholipases A2 to predict the primary structures of these enzymes. The deduced amino acid sequences exhibited a highly conservative feature which is common to a group of pancreatic phospholipases A2 from various animal species. Furthermore, the structures of the signal sequences of dog and rat pancreatic phospholipases A2 were predicted, although the assignment of the positions cleaved post-translationally is only tentative at the present time.

Amino Acid Sequence↗
PubMed1986

An NMR study on the structure of OR3 in the lambda cro-OR3 complex.

1H-NMR spectra of lambda-OR3 17mer in the complex with lambda-cro has been analysed and the change in its conformation on the complexation has been deduced. The changed conformation is retained until the duplex melts above 55 degrees C. Increase in heat stability of the cro dimer was observed. The result was compared with those of other DNA fragments phi 80-OR2 19 mer and the CRP binding site 22mer mixed with lambda-cro.

Amino Acid Sequence↗