The sequence of chemical reactions

A series of trehalose-based oligosaccharides were isolated from the cytoplasmic fraction of Mycobacterium smegmatis and purified by gel-filtration and paper chromatography and TLC. Their structures were determined by HPLC and GLC to determine sugar composition and ratios, MALDI-TOF MS to measure molecular mass, methylation analysis to determine linkages, 1H-NMR to obtain anomeric configurations of glycosidic linkages, and exoglycosidase digestions followed by TLC to determine sequences and anomeric configurations of the monosaccharides....

8p system191 01-06-2013 36 5   Download

The present work reports isolation and characterization of a highly glycosylated protein from bovine milk fat globule membranes, known as PAS III. Partial amino-acid sequencing of the purified protein allowed construction of degenerate oligonucleotide primers, enabling isolation of a full-length cDNA encoding a protein of 330 amino-acid residues. N-terminal amino-acid sequencing of derived peptides and the purified protein confirmed 76% of the sequence and demonstrated presence of a cleavable signal peptide of 23 residues, leaving a mature protein of 307 amino acids.

9p system191 01-06-2013 50 4   Download

Manganese lipoxygenase was isolated to homogeneity from the take-all fungus, Gaeumannomyces graminis. The C-terminal amino acids and several internal peptides were sequenced, and the information was used to obtain a cDNA probe by RT/PCR. Screening of a genomic library of G. graminis yielded a full-length clone of the Mn-Lipoxygenase gene. cDNA analysis showed that the gene spanned 2.6 kb and contained one intron (133 bp). Northern blot analyses indicated two transcripts (2.7 and 3.1 kb). The deduced amino-acid sequence of the Mn-Lipoxygenase precursor (618 amino acids, 67.

8p system191 01-06-2013 36 4   Download

An exo-1,3-b-glucanase has been isolated from cultural filtrate of Trichoderma viride AZ36. The N-terminal sequence of the purified enzyme (m ¼ 61 ^ 1 kDa) showed no significant homology to other known glucanases. The 1,3-b-glucanase displayed high activity against laminarins, curdlan, and 1,3-b-oligoglucosides, but acted slowly on 1,3-1,4-b-oligoglucosides. No significant activity was detected against high molecular mass 1,3-1,4-bglucans. The enzyme carried out hydrolysis with inversion of the anomeric configuration.

9p system191 01-06-2013 45 6   Download

We report the biochemical characterization of calhepatin, a calcium-binding protein of the S100 family, isolated from lungfish (Lepidosiren paradoxa) liver. The primary structure, determined by Edman degradation and MS/MS, shows that the sequence identities with the other members of the family are lower than those between S100 proteins from different species. Calhepatin is composed of 75 residues and has a molecular mass of 8670 Da. It is smaller than calbindin D9k (78 residues), the smallest S100 described so far....

9p research12 01-06-2013 44 4   Download

A systematic search for Escherichia coli proteins with the zinc-binding activity was performed using the assay of radioactive Zn(II) binding to total E. coli proteins fractionated by two methods of two-dimensional gel electrophoresis. A total of 30–40 radioactive spots were identified, of which 14 have been assigned from N-terminal sequencing.

11p research12 01-06-2013 35 3   Download

Two genes (CM-AAT1 and CM-AAT2) with strong sequence homology (87% identity at the protein level) putatively involved in the formation of aroma volatile esters have been isolated from Charentais melon fruit. They belong to a large and highly divergent family of multifunctional plant acyl-transferases and show at most 21% identity to the only other fruit acyl-transferase characterized so far in strawberry. RT-PCR studies indicated that both genes were specifically expressed in fruit at increasing rates in the early and mid phases of ripening.

8p research12 01-06-2013 49 4   Download

A technique has been developed to search a proteome database for new members of a functional class of membrane protein. It takes advantage of the highly conserved secondary structure of functionally related membrane proteins. Such proteins typically have the same number of transmembrane domains located at similar relative positions in their polypeptide sequence. This gives rise to a characteristic pattern of peaks in their hydropathy profiles.

7p research12 01-06-2013 32 4   Download

A eukaryotic-type protein serine/threonine kinase, PknA, was cloned from Mycobacterium tuberculosis strain H37Ra. Sequencing of the clone indicated 100% identity with the published pknA sequence of M. tuberculosis strain H37Rv. PknA fused to maltose-binding protein was expressed in Escherichia coli; it exhibited a molecular mass of % 97 kDa. The fusion protein was purified from the soluble fraction by affinity chromatography using amylose resin.

8p research12 01-06-2013 41 3   Download

To search for novel regulatory regions, we examined the features of chromatin structure in the rat B29/Ig-b gene and its flanking regions by determining DNase I hypersensitive sites (DHS) in plasmacytoma-derived Y3 cells. Six Y3 cellspecific DHS were detected at )8.6, promoter, +0.7, +4.4, +6.0, and +8.7 kb. The DHS at +4.4, +6.0, and +8.7 kb were present in the intergenic region between B29/Ig-b and growth hormone (GH) genes and were mapped inside conserved sequences in rat and humans.

10p research12 01-06-2013 29 4   Download

A gene having high sequence homology (45–49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer....

8p research12 01-06-2013 27 4   Download

Mitogen activated protein kinase kinase (MKK) 3 and 6 are the main p38 mitogen-activated protein kinase activators in mammals. In the present study, three Atlantic salmon MKK6 orthologs were identified. The deduced amino acid sequences of the salmon MKK6 proteins were highly similar to mammalian MKK6 sequences, and they were ubiquitously expressed. All three were shown to be upstream activators of salmon p38.

16p research12 29-04-2013 27 3   Download

Arecombinant streptococcalC5apeptidasewas expressed in Escherichia coliand its catalytic properties and thermal sta-bility were subjected to examination. It was shown that the NH2 -terminal region ofC5a peptidase (Asn32–Asp79/ Lys90) forms the pro-sequence segment. Upon maturation the propeptide is hydrolyzed either via an autocatalytic intramolecular cleavage or by exogenous protease strepto-pain. At pH 7.4 the enzyme exhibited maximum activity in the narrow range oftemperatures between 40 and 43
C....

13p research12 29-04-2013 46 3   Download

The RuvA, RuvB andRuvCproteins ofEscherichia coliact together to process Holliday junctions formed during recombination and DNA repair. RuvA has a well-de®ned DNA binding surface that is sculptured speci®cally to accommodate a Holliday junction and allow subsequent loading of RuvB and RuvC. A negatively charged pin pro-jecting from the centre limits binding of linear duplexDNA. The amino-acid sequences forming the pin are highly con-served.

9p research12 29-04-2013 39 3   Download

The disaccharide galactosea1,3galactose (theaGal epitope) is the major xenoantigen responsible for the hyperacute vascular rejection occurring in pig-to-primates organ trans-plantation. The synthesis of theaGal epitope is catalyzed by the enzyme a1,3-galactosyltransferase (a1,3GalT). To be able to control porcinea1,3GalT gene expression speci®c-ally, we have analyzed the upstreamportionof thea1,3GalT gene, and identi®ed the regulatory sequences.

10p research12 29-04-2013 33 2   Download

In addition to kallikrein hK3, a serine protease generally reported as PSA (prostate-speci®c antigen), at least two other enzymes in human seminal plasma also cleave syn-thetic peptidyl substrates derived from the sequence of human semenogelins. We have identi®ed one of these as prostatic acid phosphatase (PAP), a major component of prostatic ¯uid whose physiological function is unclear.

6p research12 29-04-2013 31 4   Download

Propionate CoA-transferase fromClostridium propionicum hasbeenpuri®edand thegene encoding the enzymehasbeen cloned and sequenced. The enzyme was rapidly and irre-versibly inactivated by sodium borohydride or hydroxyl-amine in the presence of propionyl-CoA. The reduction of the thiol ester between a catalytic site glutamate and CoA with borohydride and the cleavage by hydroxylamine were used to introduce a site-speci®c label, which was followed by MALDI-TOF-MS. This allowed the identi®cation of glutamate 324 at the active site. ...

9p research12 29-04-2013 30 4   Download

b1,4-N-Acetylglucosaminyltransferase III (GnT-III) plays a regulatory role in the biosynthesis of N-glycans, and it has been suggested that its product, a bisecting GlcNAc, is involved in a variety of biological events as well as in regu-lating the biosynthesis of the oligosaccharides. In this study, it was found, on the basis of sequence homology, that GnT-III contains a small region that is signi®cantly homologous to both snail b1,4GlcNAc transferase andb1,4Gal trans-ferase-1.

9p research12 29-04-2013 30 3   Download

Weak neurotoxins from snake venom are small proteins with five disulfide bonds, which have been shown to be poor bindersof nicotinicacetylcholine receptors.We report on the cloning and sequencing of four cDNAs encoding weak neurotoxins fromNaja sputatrixvenomglands. The protein encoded by one of them, Wntx-5, has been synthesized by solid-phase synthesis and characterized.

10p research12 23-04-2013 43 4   Download

Two novel toxins, Lqh6 andLqh7, isolated from the venom of the scorpionLeiurus quinquestriatus hebraeus,have in their sequence amolecular signature (8Q/KPE10) associated with a recently defined group ofa-toxins that target Na channels, namely thea-like toxins [reviewed inGordon, D., Savarin, P.,Gurevitz,M.&Zinn-Justin, S. (1998)J. Toxicol. Toxin Rev.17, 131–159]. Lqh6 and Lqh7 are highly toxic to insects and mice, and inhibit the binding of a-toxins to cockroach neuronal membranes.

14p research12 23-04-2013 37 1   Download