Carlsberg Research Communications最新文献

The nucleotide sequence of a 2065 base pair HindIII fragment, containing a gene (lambda hor1-14) belonging to the Hor1 locus in barley, has been determined. The fragment consists of 1044 bp of coding region interrupted by an amber codon at base 481, a 5' non-coding region of 428 bp and a 3' non-coding region with 593 bp. The deduced amino acid sequence of the mature protein (327 amino acids) is characterized by an octapeptide motif PQQPFPQQ which is repeated throughout the peptide chain between a unique 12 amino acid long NH2-terminal and an equally unique 10 amino acid long COOH-terminal end. The proline + glutamine content is 62% and the next three most abundant amino acids are leucine (9%), phenylalanine (8%) and isoleucine (3%). In the 5' non-coding region there is a TATA box at -98 bp from the start methionine. The 3' non-coding region has a polyadenylation signal 76 bp downstream from the TAA stop codon. The deduced amino acid sequences of the NH2- and COOH-terminals of lambda hor1-14 are very similar but not identical to those known from the Edman degradation and carboxypeptidase Y analysis of C-hordein polypeptides. The 3' coding and non-coding region of lambda hor1-14 is closely similar but different in detail to the known C-hordein cDNA clones. One polyadenylation signal is found in lambda hor1-14 whereas two are present in each of the three known C-hordein cDNAs. These differences and the amber codon interrupting the open reading frame indicate that this gene is silent.

It is demonstrated that site-directed mutagenesis successfully can be combined with chemical modification creating enzyme derivatives with altered properties. A methionyl residue located in the S1' binding site of carboxypeptidase Y was replaced by a cysteinyl residue and the mutant enzyme was isolated and modified with various alkylating and thioalkylating reagents. Treatment of the mutant carboxypeptidase Y with bulky reagents like phenacyl bromide and benzyl methanethiolsulfonate caused a drastic reduction in the activity towards substrates with bulky leaving groups in the P1' position, i.e. -OBzl, -Val-NH2 and amino acids (except -Gly-OH), while substrates with small groups in that position, i.e. -OMe and -NH2, were hydrolysed with increased rates. The presence of a positive charge, in addition to a bulky group, had a further adverse effect on the activity towards substrates with large leaving groups, whereas the activity towards those with small leaving groups remained unaffected by such a group. The derivatives obtained by modification of the mutant enzyme with benzyl methanethiolsulfonate and methyl methanethiolsulfonate were effective in deamidations of peptide amides and peptide synthesis reactions, respectively.

Glutamic acid 1-semialdehyde hydrochloride was synthesized and purified. Its prior structural characterization was extended and confirmed by 1H NMR spectroscopy and chemical analyses. In aqueous solution at pH 1 to 2 glutamic acid 1-semialdehyde exists in a stable hydrated form, but at pH 8.0 it has a half-life of 3 to 4 min. Spontaneous degradation of the material at pH 8.0 generated some undefined condensation products, but coincidentally a significant amount isomerized to 5-aminolevulinate. At pH 6.8 to 7.0, glutamate 1-semialdehyde is sufficiently stable to permit routine and reproducible assay for glutamate 1-semialdehyde aminotransferase activity. Only about 20% of the enzyme extracted from chloroplasts was sensitive to inactivation by gabaculine with no pretreatment. However, when the enzyme was exposed to 5-aminolevulinate, levulinate or 4,5-dioxovalerate in the absence of glutamate 1-semialdehyde, it was completely inactivated by gabaculine; 4,6-dioxoheptanoate had no effect on the enzyme. These results lead to the hypothesis that the aminotransferase exists in the chloroplast in a complex with pyridoxamine phosphate, which must be converted to the pyridoxal form before it can form a stable adduct with gabaculine. We propose that the enzyme catalyzes the conversion of glutamate 1-semialdehyde to 5-aminolevulinate via 4,5-diaminovalerate.

Carboxypeptidase S-1 from Penicillium janthinellum has been isolated by affinity chromatography and characterized. The enzyme activity is unusually stable in organic solvents, e.g. 80% methanol. The hydrolysis of peptide substrates is apparently dependent on three ionizable groups. One group, with pKa of 4.0-4.5, is a catalytically essential residue in its deprotonated form, and another group with a pKa of 6.5-7.0 functions in its protonated form, apparently as the binding site for the C-terminal carboxylate group of peptide substrates. The third group, with a pKa of 5.0-5.5, appears to be a carboxylic acid group located at the S1 binding site. Deprotonation of this group to form a negatively charged carboxylate group has an adverse effect on the hydrolysis of substrates with hydrophobic amino acid residues at the P1 position and a beneficial effect on the hydrolysis of substrates with the positively charged arginyl or lysyl residues at this position. The substrate preference of the enzyme is consequently pH dependent. At pH 6.0 (the optimum for ester hydrolysis) the enzyme is essentially specific for Bz-X-OMe substrates where X = Arg and Lys. Using amino acids and amino acid amides as nucleophiles carboxypeptidase S-1 efficiently catalyses the formation of peptide bonds by aminolysis of peptides (transpeptidation reactions) and peptide esters provided that the substrate contains a basic amino acid residue at the P1 position, e.g. Bz-Arg-OBu and Bz-Arg-Leu-OH. With several nucleophiles the fractions of aminolysis exceed those previously reported in similar studies with carboxypeptidase Y and malt carboxypeptidase II.

The polypeptides of the barley light-harvesting protein of photosystem I (LHCI) share certain epitopes. At least two of these common epitopes are present in chlorophyll a/b-protein 1 (Chla/b-P1 = CP29), as shown by cross-reacting monoclonal antibodies (14). These antibodies were employed for immunological identification of polypeptides translated in vitro in an mRNA-dependent cell-free rabbit reticulocyte lysate. The monoclonal antibody CMpLHCI:2 precipitated only one polypeptide of molecular weight 28 kD from in vitro translates primed with polyA+ RNA. No 28 kD precipitation band was found, if this antibody was mixed with a PSI-200 preparation before it was added to the translate. The translational capacity of the LHCI transcripts isolated from 12 hours greened barley was much higher than those isolated from 6 hours greened barley. Transcripts for LHCI polypeptides were also found among the polyA+ RNA of the mutant viridis-k23, which is devoid of LHCI polypeptides in its thylakoid membranes. The monoclonal antibody CMpCh1a/b-P1:1 precipitated a polypeptide of molecular weight 31 kD from in vitro translates primed with polyA+ RNA. Thus, the cross-reactivity the two antibodies show with the mature proteins is not found when the antibodies are reacted with the precursor proteins.

The psbE and psbF genes encoding the 9.4 and 4.4 kD apoproteins of cytochrome b-559 have been located in the chloroplast genome of barley. As in other plant species they are found adjacent to each other in the large single copy region of the chloroplast DNA. Both the nucleotide sequence and the deduced amino acid sequence for the two polypeptides are identical to that of wheat and more than 95% similar to those of spinach, tobacco and Oenothera. The region between the two genes spans 10 nucleotides (excluding the stopcodon) and contains a typical procaryotic ribosomal binding site. A dicistronic transcript is identified, but the presence of a ribosomal binding site between the two genes may allow independent translation.