Biochimica et biophysica acta最新文献

The albumin from an Atlantic salmonid, the brown trout (Salmo trutta), is 1730 Da higher in molecular mass than the albumin from a Pacific salmonid, the chinook salmon (Oncorhynchus tshawytscha), at 65230 Da. Digestion with neuraminidase revealed that purified brown trout albumin contained sialic acid while chinook salmon albumin did not. Concanavalin A-sepharose affinity chromatography was used to purify a glycopeptide from a total tryptic digest of brown trout albumin. The mass of this glycopeptide (3815 Da) was determined by mass spectrometry, and the sequence largely confirmed by N-terminal sequencing. The identified sequence of IAHCCNQSYSM-, contains an Asn-Gln-Ser glycosylation site and is identical to residues 475-485 derived from the cDNA of the albumin from the Atlantic salmon, the closest relative of the brown trout. Glycosylation of albumin is very unusual, and has not been identified in either reptilian or mammalian albumins. The finding of a glycoalbumin in salmonids, ancient members of the teleost fish subclass, coupled with evidence of albumin glycosylation in the oldest vertebrates, agnathans, as well as amphibians, suggests that albumin was originally a glycoprotein, but lost this modification sometime between the divergence of amphibians and reptiles.

Rat phosphatidylinositol transfer protein (PITP) is a 32 kDa protein containing 271 amino acids. It is involved in a number of cell functions including secretion and cell signaling. To further characterize structure/activity relationships of PITP, two C-terminal truncated derivatives, PITP(1-259) and PITP(1-253), were produced in Escherichia coli and purified to homogeneity. PITP(1-259) had transfer activity equal to 30-40% to that of native PITP in transfer of either phosphatidylcholine (PC) or phosphatidylinositol (PI) when transfer was measured using 95/5 mol% PC/PI donor and acceptor vesicles; PITP(1-253) had only slight transfer activity, even under the most favorable assay conditions. Thus, amino acids 254-258 are critical for transfer activity. The transfer activity of PITP(1-259) was strongly dependent on the composition of the donor and acceptor vesicles. With 100 mol% PC donor and acceptor vesicles, PITP(1-259) transfer activity ranged from 70 to 100% to that of PITP. The presence of 2 mol% phosphatidic acid (PA) in either donor or acceptor vesicles reduced transfer activity to between 10 and 20% that of full-length PITP under the same conditions. If both donor and acceptor contained 2% PA, PITP(1-259) was essentially inactive, though the activity of PITP was not affected significantly under these conditions. PITP(1-253) and PITP(1-259) bind much more avidly to vesicles than does PITP, and this enhanced binding reflects increased electrostatic interactions. Thus, the C-terminal residues modulate the affinity of PITP for vesicles and the efficiency of phospholipid transfer.

In the present study, the efficacy of a lipophilic Al complex, aluminum acetylacetonate, as a stimulator of Fe2+-initiated lipid peroxidation in phospholipid liposomes was examined, and results were compared with those from the liposomes treated with AlCl3. The extent of lipid peroxidation was assessed by the formation of thiobarbituric acid-reactive substances (TBARS). The results indicated that the stimulatory effect of Al complex on Fe2+-initiated lipid peroxidation in phosphatidylcholine liposomes was more effective than that of AlCl3 under the same conditions. The concentration dependence of Al complex on TBARS production showed that the concentration of the complex required to induce half-maximal stimulation of TBARS production was 43 microM. In contrast, the stimulatory effect of AlCl3 was not observed until the AlCl3 concentration is increased above 300 microM. In addition, it was found that there is a linear relationship between the TBARS values and the residual amounts of Fe2+ at an earlier stage (within 2 min after the addition of Fe2+) of the lipid peroxidation in PC liposomes with different concentrations of Al complex, suggesting that Fe2+ oxidation process is closely related to the stimulatory effect of Al complex. The stimulatory effect of Al complex upon the lipid peroxidation completely disappeared by treatment of Al complex-treated liposomes with Triton X-100. The results of fluorescence anisotropy measurements using 12-(9-anthroyloxy)stearic acid-labeled liposomes suggested that treatment of the liposomes with Al complex caused a decrease in their lipid fluidity. Furthermore, it was found that there is a correlation between the extents of the fluorescence anisotropy and the Fe2+ oxidation parameters in the liposomes with different concentrations of Al complex. From these results, it is suggested that the Al effect on Fe2+-initiated lipid peroxidation in the phospholipid liposomes is markedly enhanced by incorporation of Al complex into the liposomal membranes and that an acceleration of Fe2+ oxidation due to a strengthened packing between the acyl chains in the lipid layer may be one possible mechanism for the occurrence of a marked stimulatory effect of Al complex on Fe2+ initiated lipid peroxidation.

Determination of nucleotide sequence upstream to the neurotoxin binding protein (NBP) gene of type E Clostridium botulinum has revealed an open reading frame whose stop codon is only 18 bp apart from the start codon of the NBP gene. Amino acid sequence derived from the corresponding nucleotide sequence suggested the existence of the open reading frame as a 47.8 kDa protein (P-48). Protein data bank search revealed that the 47.8 kDa protein has 80% sequence identity to P-47 of type F C. botulinum. The gene organization of type E. Clostridium botulinum was predicted and compared to other types of C. botulinum. In type E C. botulinum, genes for the P-48, the neurotoxin binding protein and the neurotoxin form an operon which was similar to that of type F C. botulinum. However, type E C. botulinum has a P-18 gene instead of P-21 gene observed in type F C. botulinum, both located upstream to their respective P-48/P-47 gene.

The Leishmania cell surface virulence factor gp63 is a protease family that plays an important role in the survival of the parasite protozoon into the host macrophages. We have cloned and characterised the gp63 gene from L. infantum. The sequence analysis of the gene indicates the existence of a high degree of conservation with the other old world species L. major and L. donovani. The similarity is lower with new world species with the exception of L. chagasi which shows a strikingly high percentage of identity (99-100%). In L. infantum the gp63 gene expresses two polypeptides of 58 and 60 kDa, respectively, which show a similar proteolytic activity. The 60 kDa polypeptide is expressed during the whole life cycle of the promastigote form of the parasite with a moderate increase at the stationary phase of growth while the 58 kDa product, although slightly present in the logarithmic phase, notable increases its expression during the highly infectious stationary phase. RNA analysis showed that the presence in L. chagasi of these two polypeptides correlates with two RNA molecules and with the degree of parasite infectivity, whereas in the case of L. infantum a single 3 kb messenger RNA is detected through the whole promastigote life cycle. Our data indicate that in L. infantum, the differences in gene expression of the gp63 protease family according to parasite phase of growth seem to be due to a differential pattern of glycosilation of the polypeptides which correlates with the different infective forms of the promastigote form of the parasite.