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91.
Manju Basu Shu-Ai Weng Hongyu Tang Farhat Khan Federica Rossi Subhash Basu 《Glycoconjugate journal》1996,13(3):423-432
The galactosyltransferase, GalT-4, which catalyses the biosynthesisin vitro of neolactotetraosylceramide, nLcOse4Cer (Gal1-4GleNAc1-3Gal1-4Glc-Cer) from lactotriaosylceramide, LcOse3Cer (GlcNAc1-3Gal1-4Glc-Cer), and UDP-galactose has been purified 107 500-fold from a mineral oil induced mouse T-lyphoma P-1798, using affinity columns. The purified enzyme is partially stabilized in the presence of phospholipid liposomes. Two closely migrating protein bands of apparent molecular weights 56 kDa and 63 kDa were observed after sodium dodecyl sulfate polyacrylamide gel electrophoresis of highly purified mouse GalT-4. These two protein bands, when subjected to limited proteolysis, resulted in three peptides with identical mobilities indicating amino acid sequence identity between the proteins. Both protein bands from P-1798 gave a positive immunostain when tested with polyclonal antibody against bovine lactose synthetase (UDP-Gal:Glc 4-galactosyltransferase) following Western blot analysis on nitrocellulose paper. The enzyme has a pH optimum between 6.5 and 7.0 and like all other galactosyltransferases, GalT-4 has absolute requirements for divalent cation (Mn2+). TheK
m values for the substrate LcOse3Cer and donor UDP-galactose are 110 and 250 µm, respectively. Substrate competition studies with LcOse3Cer and either asialo-agalacto-1-acid glycoprotein orN-acetylglucosamine revealed that these reactions might be catalysed by the same protein. The only other glycolipid which showed acceptor activity toward the purified GalT-4 was iLcOse5Cer (GlcNAc1-1-3Gal1-4Lc3), the precursor for polylactosamine antigens. However, competition studies with these two active substrates using the most purified enzyme fraction, revealed that these two reactions might be catalysed by two different proteins since the experimental values were closer to the theoretical values calculated for two enzymes. Interestingly however, it seems that the GalT-4 from P-1798 has an absolute requirement for anN-acetylglucosamine residue in the substrate since the lyso-derivative (GlcNH21-3Gal1-4Glc-sphingosine) of the acceptor glycolipid LcOse3Cer is completely inactive as substrate while theK
m andV
max of the reacetylated substrate (GlcNac1-3Gal1-4Glc-acetylsphingosine) was comparable with LcOse3Cer. Autoradiography of the radioactive product formed by purified P-1798 GalT-4 confirmed the presence of nLcOse4Cer, as the product cochromatographed with authentic glycolipid. The monoclonal antibody IB-2, specific for nLcOse4Cer, also produced a positive immunostained band on TLC as well as giving a positive ELISA when tested with radioactive product obtained using a highly purified enzyme from mouse P-1798 T-lymphoma.Abbreviations EDTA
ethylenediamine tetraacetate
- ME
-mercaptoethanol
- PEG
polyethylene glycol
- PBS
phosphate buffered saline
- Suc
sucrose
- Mn2+
manganese
- Gal
galactose
- GlcNAc
N-acetylglucosamine
- UDP-Gal
Uridine diphosphate galactose
- Ab
antibody
- SDS
sodium dodecyl sulphate
- PAGE
polyacrylamide gel electrophoresis
- ECB
embryonic chicken brain
- Cer
ceramide
- nLc4 or NlcOse4Cer
Gal1-4GleNac1-3Gal1-4Glc-Cer, neoLactotetraosylceramide
- Lc3 or LcOse3Cer
GlcNac1-3Gal1-4Glc-Cer, lactotriaosylceramide
- iLc5
iLcOse5Cer, GlcNAc1-3nLcOse4Cer
- nLc6
nLcOse6Cer, Gal1-4iLcOse5Cer
- SA–Gal–1AGP
asialo-agalacto1-acid glycoprotein
- TLC
thin layer chromatography 相似文献
92.
93.
The role played by glycogenolysis in the ischemic heart has been recently put into question because it is suspected that a slowing down of this process could be beneficial for the tolerance of the myocardium to ischemia. The role of the intracellular effectors that control the rate of glycogenolysis has therefore regained interest. We aimed to understand the role played by those intracellular effectors which are directly related to the energy balance of the heart. To this end, we review some of the previously published data on this subject and we present new data obtained from P-31 and C-13 NMR spectroscopic measurement on isolated rat heart. Two conditions of ischemia were studied: 15 min global no-flow and 25 min low-flow ischemia. The hearts were isolated either from control animals or from rats pre-treated with isoproterenol (5 mg.kg–1 b.w. i.p.) 1 h before the perfusion in order to C-13 label glycogen stores. Our main results are as follows: (1) the biochemically determined glycogenolysis rate during the early phase of ischemia (up to 10–15 min) was larger in no-flow ischemia than in low-flow conditions for both groups, (2) direct measurement of the glycogenolysis rate, as determined by C-13 NMR, after labelling of the glycogen pool in the hearts from isoproterenol-treated rats, confirms the estimations from the biochemical data, (3) glycogenolysis was slower in the hearts from pre-treated animals than in control hearts for both conditions of ischemia, (4) the total activity of glycogen phosphorylase (a + b) increased, by 50%, after 5 min no-flow ischemia, whereas it decreased by 42% after the same time of low-flow ischemia. However, the ratio phosphorylase a/a + b was not altered, whatever the conditions, (5) the concentration of inorganic phosphate (Pi) increased sharply during the first minutes of ischemia, to values above 8–10 mM, under all conditions studied. The rate of increase was larger during no-flow ischemia than during low-flow ischemia. The concentration of Pi was thereafter higher in controls than in the hearts from isoproterenol-treated animals.The calculated cytosolic concentration of free 5 AMP increased sharply at the onset of ischemia, reaching in a few minutes values above 30 M in controls and significantly lower values, around 15 M, in the hearts from isoproterenol-treated rats. (6) The hearts from isoproterenol-treated rats displayed a reduced intracellular acidosis, when compared to controls, under both conditions of ischemia.We conclude that the intracellular effectors, mainly free AMP, play an essential role in the control of glycogenolysis via allosteric control of phosphorylase b activity. The alteration in the concentration of free Pi, the substrate of both forms of phosphorylase, can also be considered as determinant in the control of the rate of glycogenolysis.The attenuation of ischemia-induced intracellular acidosis in the hearts from isoproterenol-treated rats could be a consequence of a reduced glycogenolytic rate and is likely to be related to a better resumption of the mechanical function on reperfusion. 相似文献
94.
Selenium,zinc, and thyroid hormones in healthy subjects 总被引:3,自引:0,他引:3
Oliviero Olivieri Domenico Girelli Anna Maria Stanzial Luigi Rossi Antonella Bassi Roberto Corrocher 《Biological trace element research》1996,51(1):31-41
Iodothyronine 5′ deiodinase, which is mainly responsible for peripheral T3 production, has recently been demonstrated to be a selenium (Se)-containing enzyme. The structure of nuclear thyroid hormone
receptors contains Zinc (Zn) ions, crucial for the functional properties of the protein. In the elderly, reduced peripheral
conversion of T4 to T3 with a lower T3/T4 ratio and overt hypothyroidism are frequently observed. We measured serum Se and RBC GSH-Px (as indices of Se status), circulating
and RBC Zinc (as indices of Zn status), thyroid hormones and TSH in 109 healthy euthyroid subjects (52 women, 57 men), carefully
selected to avoid abnormally low thyroid hormone levels induced by acute or chronic diseases or calorie restriction. The subjects
were subdivided into three age groups. To avoid under- or malnutrition conditions, dietary records were obtained for a sample
of 24 subjects, randomly selected and representative of the whole population for age and sex. Low T3/T4 ratios and reduced Se and RBC GSH-Px activity were observed only in the older group. A highly significant linear correlation
between the T3/T4 ratio and indices of Se status was observed in the older group of subjects (r=0.54;p<0.002, for Se;r=0.50;p<0.002, for RBC GSH-Px). Indices of Zn status did not correlate with thyroid hormones, but RBC Zn was decreased in older as
compared with younger subjects. We concluded that reduced peripheral T4 conversion is related to impaired Se status in the elderly. 相似文献
95.
BET3 encodes a novel hydrophilic protein that acts in conjunction with yeast SNAREs. 总被引:2,自引:1,他引:1
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G Rossi K Kolstad S Stone F Palluault S Ferro-Novick 《Molecular biology of the cell》1995,6(12):1769-1780
Here we report the identification of BET3, a new member of a group of interacting genes whose products have been implicated in the targeting and fusion of endoplasmic reticulum (ER) to Golgi transport vesicles with their acceptor compartment. A temperature-sensitive mutant in bet3-1 was isolated in a synthetic lethal screen designed to identify new genes whose products may interact with BET1, a type II integral membrane protein that is required for ER to Golgi transport. At 37 degrees C, bet3-1 fails to transport invertase, alpha-factor, and carboxypeptidase Y from the ER to the Golgi complex. As a consequence, this mutant accumulates dilated ER and small vesicles. The SNARE complex, a docking/fusion complex, fails to form in this mutant. Furthermore, BET3 encodes an essential 22-kDa hydrophilic protein that is conserved in evolution, which is not a component of this complex. These findings support the hypothesis that Bet3p may act before the assembly of the SNARE complex. 相似文献
96.
Abstract. The Argentine Chaco is a mosaic of grassland and open forest habitats maintained by natural disturbance activities such as fire. Since the introduction of domestic livestock and other human activities, the balance of this mosaic has been significantly altered, both in plant species and structural composition. This study focuses on the impact of such changes on the diversity of ground-dwelling arthropods within semi-arid Chaco forest. Quantitative measures of habitat structure and arthropod diversity were taken in forest areas previously subjected to grazing, logging and ploughing. Results indicated that arthropod diversity was smaller on sites with reduced structural complexity, with marked changes in arthropod family composition. The habitat components relating to plant architectural and vertical diversity were particularly influential on arthropod diversity. The guild size ratio of predatory to non-predatory arthropods also differed significantly between habitats suggesting a change in the resource base available to some groups. The latter suggests a shift in the functional organisation of the forest ecosystem which could have important repercussions for the diversity of other trophic levels. 相似文献
97.
Marcelo V. Sousa Michael Richardson Wagner Fontes Lauro Morhy 《Journal of Protein Chemistry》1994,13(8):659-667
Enterolobin, a 55-kDa cytolytic, inflammatory, and insecticidal protein isolated from seeds of the Brazilian treeEnterolobium contortisiliquum (Leguminosae-Mimosoideae) has been further purified and partially sequenced by using both manual and automated methods. A computational search of enterolobin partial amino acid sequence against the PIR database revealed possible sequence similarities with aerolysins, cytolytic proteins fromAeromonas species. An alignment of enterolobin partial sequence to the amino acid sequences ofA. hydrophila andA. sobria aerolysins showed several similar regions with many residue identites. The seed protein enterolobin and the bacterial aerolysins may be homologous proteins despite the distant phylogenetic relationship. 相似文献
98.
99.
Lucia Potenza Antonella Amicucci Ismaela Rossi Francesco Palma Roberta De Bellis Paola Cardoni Vilberto Stocchi 《Biotechnology Techniques》1994,8(2):93-98
Summary Different species of truffle were studied in order to identify species-specific markers. The isolation of two Tuber magnatum Pico markers is reported. One of these could be used as a probe in dot blot hybridization, allowing the development of a rapid test able to identify Tuber magnatum species. 相似文献
100.