Carlsberg Research Communications最新文献

A trypsin inhibitor with a Km of 5 x 10(-5) M has been isolated from kohlrabi (Brassica napus var. rapifera). Subtilisin DY is inhibited only weakly and chymotrypsin not at all. The inhibitor is closely related to napin as determined by amino acid sequence analysis which also showed the inhibitor to be polymorphous. The inhibitor has been further characterized by means of molecular weight determination using SDS gel-electrophoresis and by amino acid analysis, fluorimetry as well as circular dichroism. A simplified method for purification of napins is given.

Glutamate-1-semialdehyde aminotransferase (E.C. 5.4.3.8) was purified from barley and the cyanobacteria Synechococcus PCC 6301. The purification procedure involved serial affinity chromatography and preparative polyacrylamide gel electrophoresis under non-denaturing conditions. The aminotransferase of these two organisms showed different mobilities in non-denaturing gels. In SDS-PAGE the enzyme from both organisms migrated as a single protein with an apparent molecular weight of 46.000 Da. An antibody against the barley enzyme cross-reacted with the cyanobacterial aminotransferase. This antibody also recognized a 17 kDa peptide cleaved from the barley protein with cyanogen bromide. Amino acid sequences of the NH2-termini revealed significant homology between the eucaryotic and cyanobacterial enzyme.

A 39 kD protein has been extracted from barley flour with 0.1 M monothioglycerol at pH 5.0 and purified by (NH4)2SO4-precipitation, anion exchange and molecular sieve chromatography. It is an N-terminally blocked, non-glycosylated, single-chain protein present in at least two molecular forms of isoelectric points 5.18 and 5.22. The amino acid composition and partial sequence analysis reveal a relationship to barley endosperm Z protein which belongs to the serpin superfamily. The 39 kD protein inhibits alpha-chymotrypsin while little or no effect could be demonstrated on trypsin, subtilisin, proteinase K, S. aureus V8 protease, thermolysin or two malt thiol endoproteinases. The 39 kD protein is immunochemically related to the major protein component in beer.

The primary structure of malt carboxypeptidase III has been determined. The enzyme is a single N-terminally blocked polypeptide chain containing 411 amino acid residues. The sequence of these amino acid residues was deduced from analysis of fragments of the polypeptide chain obtained by chemical cleavages with either cyanogen bromide or hydroxylamine and by enzymatic cleavages with either trypsin, S. aureus V8 protease or proteinase A from yeast. A glycosylated asparagine was found in position 71. The determined sequence was 97% homologous with the amino acid sequence derived from the nucleotide sequence of a gene coding for a wheat protein postulated to be a carboxypeptidase. The malt carboxypeptidase III sequence showed 34% homology with the amino acid sequence of the single-chain carboxypeptidase Y, and about 25% homology with the combined A- and B-chains of malt carboxypeptidase I and II as well as wheat carboxypeptidase II.

DNA segments encoding signal peptides from mouse alpha-amylase, yeast acid phosphatase, and yeast invertase were fused in frame to a barley (1-3,1-4)-beta-glucanase cDNA gene and expressed in yeast cells under the control of the phosphoglycerate kinase gene promoter. Pure beta-glucanase is obtained by gel filtration of concentrated yeast cell supernatant. It was shown that the glucanase pre-protein was specifically processed and the mature protein efficiently secreted when the yeast invertase signal sequence directed secretion.

The complete amino acid sequence of acetolactate decarboxylase (EC 4.1.1.5) from Bacillus brevis has been determined by sequencing of the intact enzyme and of peptides obtained by cleavage with cyanogen bromide, Staphylococcus aureus V8 protease and trypsin, respectively. Determination of the C-terminal part was made by treatment with carboxypeptidases Y and M II. The enzyme has a molecular weight of 29,093 and consists of 260 amino acid residues arranged in a single peptide chain without disulphide bonds.

Carboxypeptidase Y is a serine carboxypeptidase assumed to contain a catalytic triad similar to the serine endopeptidases. On the basis of the homology between various serine carboxypeptidases His-397 is suspected to be part of the catalytic triad. To test this it was exchanged with Ala and Arg by site-directed mutagenesis of the cloned PRC1 gene. The catalytic efficiency of the mutant enzymes were reduced by a factor of 2 X 10(4) and 7 X 10(2), respectively, confirming the key role of His-397 in catalysis. Treatment of Ala-397-CPD-Y with Hg++ or CNBr, hence modifying Cys-341 located in the vicinity of the active site abolished the residual activity of the enzyme, indicating an additional involvement of this residue in catalysis.

The primary structure and physical chemical properties were determined of a nuclease expressed and secreted by Escherichia coli. The plasmid p403-SD2 carried a DNA sequence isolated from Serratia marcescens encoding the enzyme. During cultivation of the E. coli cells, 85% of the enzyme was released to the growth medium. The enzyme was purified and exhibited a single band with a molecular weight about 30,600 daltons on SDS-PAGE similar to nuclease isolated from S. marcescens. The amino acid composition and the amino acid sequence determined directly confirmed the primary structure of 245 amino acids predicted from the DNA sequence, and, in addition, the two disulfide bridges were assigned. Several physical chemical properties were examined. The ability of the enzyme to cross the outer membrane is proposed to depend upon the formation of the proper structures during the folding process.

Treatment of glucoamylase G2 with large excesses of different group specific reagents resulted in modification of 25% of the histidyl, 15% of the tyrosyl, 20-40% of the arginyl, 30-50% of the lysyl and none of the methionyl residues. The modified groups were not critical since the various derivatives possessed from 50% to 100% residual enzymatic activity and retained the thermostability. Carboxamidomethylation occurred specifically at His254 with essentially no change of the kinetic parameters for hydrolysis of maltose and starch. Removal of the two N-linked sugar units by endoglycosidase H was similarly without effect on activity, thermostability and chemical reactivity of the histidyl residues. H(+)-titration revealed that glucoamylase G2 carries a lower net charge throughout the pH-range 3-11 than predicted from its amino acid composition.

High molecular weight DNA released from isolated protoplasts was digested with rare-cutting restriction enzymes and separated by pulsed field gel electrophoresis. The average size of undigested DNA was above 1500 kbp. Digests made with NotI, SfiL, Mlul and SalI was hybridized to a probe, common to all genes of the Hor2 locus encoding B-hordein polypeptides, and this revealed the maximum size of the locus to be 360 kbp. Two probes, specific for individual B-hordein genes, enabled the identification of two fragment classes in the locus, each containing an equal number of B-hordein genes. Double digests allowed ordering of sites and construction of a map covering 650 kbp around the Hor2 locus. No evidence for physical linkage of the two fragment classes was obtained. The possible assignment of the two classes of hybridizing fragments to the B1- and B3-hordein subgroups is discussed.