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121.
The sugar binding activity of MR60, a mannose-specific shuttling lectin, requires a dimeric state 总被引:1,自引:0,他引:1
MR60 is an intracellular membrane protein which has been shown to act as a
mannoside specific lectin and to be identical to ERGIC-53, a protein
characteristic of the endoplasmic reticulum-Golgi apparatus- intermediate
compartment, acting as a shuttle. According to its primary sequence, this
MR60/ERGIC-53 protein contains a luminal domain including the carbohydrate
recognition domain, a stem, a transmembrane segment and a cytosolic domain.
The endogenous MR60/ERGIC-53 protein is spontaneously oligomeric, (dimers
and hexamers). In this paper, we study the relationship between the
oligomerization state and the sugar binding capacity by using recombinant
proteins. The expression of the recombinant proteins was evidenced by
immunocytochemistry and by immunoprecipitation followed by SDS-PAGE
analysis. The full size recombinant protein binds mannosides and is
oligomeric, up to the hexameric form. Two truncated proteins lacking the
transmembrane and the cytosolic domains were prepared and characterized. A
long one, containing the cysteine 466 close to the C-terminal end of the
recombinant protein but lacking the cysteine 475, close to the C- terminal
end of the native protein, does bind mannosides and forms dimers but no
higher oligomeric forms. A shorter one, lacking both the cysteines 466 and
475, does not bind mannosides and does not form dimers or higher polymers.
The two cysteines in the carbohydrate recognition domain (C190 and C230)
are not involved in the stabilization of oligomers. In conclusion, this
study shows that the luminal moiety of MR60/ERGIC-53 contains a device
allowing both its oligomeric pattern and its sugar binding capability.
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122.
Pig heart short chain L-3-hydroxyacyl-CoA dehydrogenase revisited: sequence analysis and crystal structure determination.
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J. J. Barycki L. K. O''''Brien J. J. Birktoft A. W. Strauss L. J. Banaszak 《Protein science : a publication of the Protein Society》1999,8(10):2010-2018
Short chain L-3-hydroxyacyl CoA dehydrogenase (SCHAD) is a soluble dimeric enzyme critical for oxidative metabolism of fatty acids. Its primary sequence has been reported to be conserved across numerous tissues and species with the notable exception of the pig heart homologue. Preliminary efforts to solve the crystal structure of the dimeric pig heart SCHAD suggested the unprecedented occurrence of three enzyme subunits within the asymmetric unit, a phenomenon that was thought to have hampered refinement of the initial chain tracing. The recently solved crystal coordinates of human heart SCHAD facilitated a molecular replacement solution to the pig heart SCHAD data. Refinement of the model, in conjunction with the nucleotide sequence for pig heart SCHAD determined in this paper, has demonstrated that the previously published pig heart SCHAD sequence was incorrect. Presented here are the corrected amino acid sequence and the high resolution crystal structure determined for pig heart SCHAD complexed with its NAD+ cofactor (2.8 A; R(cryst) = 22.4%, R(free) = 28.8%). In addition, the peculiar phenomenon of a dimeric enzyme crystallizing with three subunits contained in the asymmetric unit is described. 相似文献
123.
Argininosuccinate lyase deficiency: evidence for heterogeneous structural gene mutations by immunoblotting. 总被引:1,自引:0,他引:1
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Argininosuccinate lyase (AS lyase) deficiency is an inborn error of the urea cycle with extensive clinical and genetic heterogeneity. We investigated the biochemical basis of the enzyme defect and the genetic heterogeneity in this disorder using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting of fibroblast extracts. The AS lyase monomer in control fibroblasts was present in two bands of approximately 51 and approximately 49 Kd. Each of 28 mutant strains had some cross-reactive material (CRM) of the lower (approximately 49 Kd) MW, in quantities ranging from trace to substantial levels. The approximately 51 Kd band was found in only six mutants with near-normal amounts of AS lyase CRM or high residual enzyme activity. The residual AS lyase enzyme activity in a mutant did not necessarily reflect the amount of the 49-51 Kd monomer in that strain. In contrast, there was a strong general correlation between the quantity of 49-51 Kd CRM in a mutant and the frequency of complementation by that mutant. In addition to the CRM of normal molecular weight (MW) (49-51 Kd), the majority of mutants (but not controls) had significant CRM present in one to five bands of MW less than 49 Kd. The immunoprecipitation of at least one of these low MW bands was inhibited by purified human AS lyase. Mutants indistinguishable by clinical, enzymatic, or complementation analysis have been shown to be heterogeneous in their content of AS lyase CRM, greatly extending the number of distinct mutant alleles identified at this locus. These data demonstrate that multiple unique mutations in the structural gene coding for the monomer cause AS lyase deficiency and that the AS lyase monomers made by these mutants may be unstable. Integration of these findings with enzymatic and complementation data has indicated the functional domain of the AS lyase monomer likely to be altered in certain mutants. 相似文献
124.
New mutation and prenatal diagnosis in ornithine transcarbamylase deficiency. 总被引:12,自引:5,他引:7
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R L Nussbaum B A Boggs A L Beaudet S Doyle J L Potter W E O''''Brien 《American journal of human genetics》1986,38(2):149-158
Ornithine transcarbamylase (OTC) (E.C.2.1.3.3) is an X-linked hepatic enzyme in the urea cycle necessary for ammonia detoxification. Deficiency of OTC results in neonatal hyperammonemia, coma, and death in childhood. Because fibroblasts do not express OTC, prenatal diagnosis in the past has required fetal liver biopsy. Using a complementary DNA (cDNA) for OTC for Southern blot analysis of genomic DNA, we have found probands with complete OTC deficiency from two unrelated families in whom the same TaqI restriction endonuclease site has been altered because of independent, but not necessarily identical, mutations in the OTC gene, suggesting that this site may be a relative hotspot for mutation at a location that is critical for normal gene function. This TaqI alteration has allowed the identification of the individual in each family in whom the mutation originated as well as the exclusion of a recurrence of OTC deficiency in a male fetus at risk for the disease. OTC deficiency joins the growing list of genetic disorders for which Southern blot analysis allows accurate heterozygote detection and prenatal diagnosis in conditions for which they were not previously available. 相似文献
125.
The nucleotide sequence for human argininosuccinate synthetase cDNA was determined by analysis of six clones isolated from a single experiment. The sequence covered 1623 nucleotides including 76 bases of poly(A) and contained a 1236 nucleotide open reading frame encoding a protein of 46,434 daltons. In one cDNA isolate, a cloning artifact or perhaps RNA polymerase error involving addition of an A in a region of six A's within the coding sequence was documented. Single base variations in the 3' untranslated region were examined in detail since detection of DNA polymorphisms in the cDNAs could imply over-expression of both alleles at the active locus in canavanine-resistant cells, i.e. a trans-acting mechanism for enzyme overproduction. However, the sequence from five cDNAs suggested some single base artifacts, and DNA polymorphism remains uncertain. The occurrence of three tandem arginine codons in the 5' untranslated region of the cDNA suggested the possibility of an interaction of arginyl-tRNA with mRNA to regulate RNA processing or half-life as a mechanism for arginine-mediated repression. 相似文献
126.
Cloning and sequencing of the genes for Shiga toxin from Shigella dysenteriae type 1. 总被引:48,自引:11,他引:37
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N A Strockbine M P Jackson L M Sung R K Holmes A D O''''Brien 《Journal of bacteriology》1988,170(3):1116-1122
127.
The region of the envelope gene of human immunodeficiency virus type 1 responsible for determination of cell tropism. 总被引:23,自引:20,他引:3
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A J Cann M J Churcher M Boyd W O''''Brien J Q Zhao J Zack I S Chen 《Journal of virology》1992,66(1):305-309
Different isolates of human immunodeficiency virus type 1 (HIV-1) vary in the cell tropisms they display, i.e., the range of cell types in which they are able to establish a productive infection. Here, we report on the phenotypes of recombinants between two molecularly cloned strains of HIV-1. Our results prove that the envelope glycoprotein gp120 is solely responsible for the difference in cell tropism between the two parental isolates and that no other genes or sequences are involved in determining the cell tropism of these strains. The region of the envelope involved in the determination of cell tropism includes sequences which encode the V3 loop of gp120. Control of cell tropism by this region of the virus env gene is a general phenomenon which applies to many different HIV-1 isolates. 相似文献
128.
129.
Yellow Fever Vaccine. V. Antibody Response in Monkeys Inoculated with Graded Doses of the 17D Vaccine 总被引:1,自引:1,他引:0
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Richard A. Mason Nicola M. Tauraso Robert K. Ginn Thomas C. O''''Brien Roy W. Trimmer 《Applied microbiology》1972,23(5):908-913
A dosage equal to or greater than approximately 3.4 Dex (decimal exponent, log(10)) weanling mouse intracerebral 50% lethal dose (LD(50)) was sufficient to elicit a yellow fever antibody response, as determined by the plaque neutralization (PN) test, in better than 90% of vaccinated rhesus monkeys. Lower dosages were progressively less effective in terms of PN titers and the PN and hemagglutination-inhibition serological conversion rates observed. A dose of between 3.4 and 4.2 Dex weanling mouse intracerebral LD(50), or one-tenth to one times the dosage recommended for man, provided an optimal antibody response in monkeys. In rhesus monkeys, in contrast to the findings for man, pre-existing yellow fever antibody did not interfere with the antibody response to yellow fever vaccine. The PN test was felt to be a more sensitive and specific indicator of yellow fever antibody in rhesus monkeys after vaccination than the hemagglutination inhibition or complement fixation tests. 相似文献
130.
Helix handedness of Leptospira interrogans as determined by scanning electron microscopy. 总被引:1,自引:0,他引:1
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Representative serovars and strains of the seven genetic groups of Leptospira interrogans, and two previously studied serovars, were all found to form exclusively right-handed helices as determined by scanning electron microscopy. No change in handedness occurred in cells grown in a minimal medium (Tween-80 albumin) compared to cells grown in a rich medium (rabbit serum). The right-handedness of the organisms was related to the evolution, cell wall structure, and the mechanism of motility of L. interrogans. 相似文献