Biomedical subjects

J Perrier

Publications and source records attributed to J Perrier.

At least 19 recordsLinked to original sources

PubMed2000

The rat kidney acylase I, characterization and molecular cloning. Differences with other acylases I.

The soluble acylase I from rat kidney was purified to homogeneity using a five-step procedure. As the resulting protein was found to have a relative molecular mass of 125 kDa based on size-exclusion chromatography and 44 kDa based on SDS/PAGE, the native protein was taken to consist of three subunits. The amino-acid sequence of a peptide resulting from limited proteolysis of the polypeptide chain with proteinase K, which was determined by microsequencing (RHEFHALRAGFALDEGLA), was found to be very similar to the corresponding sequence of porcine kidney acylase I. However, as N-furyl-acryloyl-L-methionine, a synthetic substrate for porcine acylases, was not hydrolyzed by the rat enzyme, it was suggested that the polypeptide chain might differ in other respects from those of the other acylases I. A full length cDNA coding for the rat kidney acylase I was therefore isolated and found to contain a 1224-bp open reading frame encoding a protein consisting of 408 amino-acid residues, which corresponded to a calculated molecular mass of 45 847 Da per subunit. The deduced amino-acid sequence showed 93.6% and 87.2% identity with that of the human liver and porcine kidney, respectively.

Amidohydrolases↗

PubMed1999

Cloning, sequencing and further characterization of acylpeptide hydrolase from porcine intestinal mucosa.

Acylpeptide hydrolase was purified to homogeneity from porcine intestinal mucosa using a seven-step procedure including ammonium sulfate precipitation, gel filtration as well as anion exchange and affinity chromatography. The specific activity of the enzyme reached 105000 nmol/mg protein per min and the purification was as high as 5500-fold. This tetrameric enzyme is composed of four apparently identical subunits, the molecular mass of which was estimated to be 75 kDa, based on the results of amino acid analysis and gel electrophoresis performed under denaturing conditions. It is likely that the NH(2)-terminal residue may be acetylated, while serine was found to be the COOH-terminal residue. The hydrolytic activity of the enzyme toward N-acetyl-L-alanine p-nitroanilide at the optimum pH value was increased twofold in the presence of the chloride anion. The K(m) value calculated from the kinetics of the hydrolysis of acetylalanyl peptides was found to be 0.7+/-0.1 mM, whereas the V(max) values decreased from 200 to 50 nmol/min per microgram of enzyme, depending on the peptidic chain lengths. The V(max) value of the synthetic substrate (250 nmol/min per microgram of enzyme) was 25-500% higher than those of the acetylalanyl peptides, depending on the peptide chain length, although the enzyme affinity was slightly lower (1.8 mM as compared with 0.7 mM). In line with data on other animal species and on various tissues, the enzyme seemed likely to be a serine protease, since it was readily inhibited by diisopropyl fluorophosphate and diethyl pyrocarbonate. A 2377-nucleotide long cDNA coding for the enzyme was isolated from pig small intestine. The deduced amino acid sequence consisted of 731 residues and showed a single different amino acid with that of the porcine liver APH, except the N-terminal amino acid which is still probably lacking.

Amino Acid Sequence↗

PubMed1998

The human pancreatic alpha-amylase isoforms: isolation, structural studies and kinetics of inhibition by acarbose.

A rapid method is proposed for isolating the two main components of human pancreatic alpha-amylase (HPA I and HPA II). The isoelectric point of HPA I (7.2), the main component, was determined using an isoelectrofocusing method and found to differ from that of HPA II (6. 6). The molecular mass of HPA I (55862+/-5 Da) and that of HPA II (55786+/-5 Da) were determined by performing mass spectrometry and found to be quite similar to that of the protein moiety calculated from the amino acid sequence (55788 Da), which indicates that the human amylase is not glycosylated. The structure of both HPA I and HPA II was further investigated by performing limited proteolysis. Two fragments with an apparent molecular mass of 41 kDa and 14 kDa were obtained by digesting the isoforms with proteinase K and subtilisin, whereas digestion with papain yielded two cleaved fragments with molecular masses of 38 kDa and 17 kDa. Proteinase K and subtilisin susceptible bonds are located in the L8 loop (A domain), while the papain cut which occurs in the presence of the calcium chelator EDTA is in the L3 loop (B domain). The kinetics of the inhibition of HPA I and HPA II by acarbose, a drug used to treat diabetes and obesity, were studied using an amylose substrate. The Lineweaver-Burk primary plots of HPA I and HPA II, which did not differ significantly, indicated that the inhibition was of the mixed non-competitive type. The secondary plots gave parabolic curves. All in all, these data provide evidence that two acarbose molecules bind to HPA. In conclusion, apart from the pI, no significant differences were observed between HPA I and HPA II as regards either their molecular mass and limited proteolysis or their kinetic behavior. As was to be expected in view of the high degree of structural identity previously found to exist between human and porcine pancreatic amylases, the present data show that the inhibitory effects of acarbose on the kinetic behavior of these two amylases are quite comparable. In particular, the process of amylose hydrolysis catalyzed by HPA as well as by PPA in both cases requires two carbohydrate binding sites in addition to the catalytic site.

Acarbose↗

PubMed1998

Isolation of a methanogenic bacterium, Methanosarcina sp. strain FR, for its ability to degrade high concentration of perchloroethylene.

Tetrachloroethylene (PCE) is a toxic compound essentially used as a degreasing and dry-cleaning solvent. A methanogenic and sulfate-reducing consortium that dechlorinates and mineralizes high concentrations of PCE was derived from anaerobically digested sludge obtained from a waste water treatment plant (Bourg-en-Bresse, France). A methanogenic bacterium, strain FR, was isolated from this acclimated consortium. On the basis of morphological and physiological characteristics, strain FR was classified in the genus of Methanosarcina. Phylogeny analysis with the 16S rRNA gene sequence revealed that strain FR is highly related to Methanosarcina mazei and Methanosarcina frisia (99.6 and 99.5% identity, respectively). High concentrations (50-87 microM) of PCE were completely dechlorinated by strain FR cultures at the rate of 76 nM-mg protein(-1).day(-1). PCE dechlorination produced a nonidentified compound. The tracer experiments with [13C]PCE revealed that the product was nonchlorinated. Dechlorination of PCE to trichloroethylene was still active in the presence of boiled cell extract of the strain FR. However, no further dechlorination was observed. This result suggests that a cofactor rather than an enzymatic system is responsible for the first dechlorination of PCE. Dechlorination-active fractions purified from cell extracts on a XAD-4 column revealed the presence of F(420), F(430), and cobamides cofactors. This is the first report of the isolation of a methanogenic bacterium with the ability to dechlorinate high concentrations of PCE to a nonchlorinated product.

Cell-Free System↗

PubMed1996