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1.
Preincubation of normal human skin fibroblasts with tunicamycin, which inhibits N-glycosylation of glycoproteins, resulted in a dose-dependent and reversible inhibition of binding and internalization of homologous low-density lipoproteins by the cells. The degradation of the internalized lipoproteins was not affected by the drug. Comparative studies with fibroblasts deficient in low-density-lipoprotein receptors indicated that tunicamycin exerts its inhibitory effect only via the receptor-mediated high-affinity binding and uptake of lipoproteins. These results suggest that expression of low-density-lipoprotein receptors on the cell surface of human skin fibroblasts depends on intact N-glycosylation. 相似文献
2.
In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules 总被引:8,自引:6,他引:2
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![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The mouse anterior pituitary tumor cell line, AtT-20, targets secretory proteins into two distinct intracellular pathways. When the DNA that encodes trypsinogen is introduced into AtT-20 cells, the protein is sorted into the regulated secretory pathway as efficiently as the endogenous peptide hormone ACTH. In this study we have used double-label immunoelectron microscopy to demonstrate that trypsinogen colocalizes in the same secretory granules as ACTH. In vitro mutagenesis was used to test whether the information for targeting trypsinogen to the secretory granules resides at the amino (NH2) terminus of the protein. Mutations were made in the DNA that encodes trypsinogen, and the mutant proteins were expressed in AtT-20 cells to determine whether intracellular targeting could be altered. Replacing the trypsinogen signal peptide with that of the kappa-immunoglobulin light chain, a constitutively secreted protein, does not alter targeting to the regulated secretory pathway. In addition, deletion of the NH2-terminal "pro" sequence of trypsinogen has virtually no effect on protein targeting. However, this deletion does affect the signal peptidase cleavage site, and as a result the enzymatic activity of the truncated trypsin protein is abolished. We conclude that neither the signal peptide nor the 12 NH2-terminal amino acids of trypsinogen are essential for sorting to the regulated secretory pathway of AtT-20 cells. 相似文献
3.
Redesigning trypsin via genetic engineering 总被引:1,自引:0,他引:1
C S Craik S Roczniak S Sprang R Fletterick W Rutter 《Journal of cellular biochemistry》1987,33(3):199-211
4.
Structural characterization of the rat carboxypeptidase A1 and B genes. Comparative analysis of the rat carboxypeptidase gene family 总被引:7,自引:0,他引:7
E Clauser S J Gardell C S Craik R J MacDonald W J Rutter 《The Journal of biological chemistry》1988,263(33):17837-17845
Nucleotide sequencing of a rat carboxypeptidase B (CPB) cDNA and direct sequencing of the CPB mRNA via primer extension on pancreatic polyadenylated RNA has yielded the complete amino acid sequence of rat CPB. The rat enzyme is synthesized as a precursor species containing a large amino-terminal fragment (108 amino acids) that contributes a putative signal sequence and an activation peptide. The mature form of rat CPB is homologous to bovine CPB (77% identity); the amino acids in bovine CPB which have been previously implicated in catalysis or ligand binding are invariant in the rat orthologue. The rat CPB cDNA was used as a probe for the isolation of the rat CPB gene. Detailed characterization of three overlapping rat genomic clones demonstrated that the coding region for the rat CPB precursor is sequestered in 11 exons which are dispersed throughout 34 kilobase pairs of genomic DNA. The nucleotide sequence of a large part of the gene has been determined including that of the exons, the exon/intron boundaries, and the 5' flanking region. We also report the partial nucleotide sequence of the rat CPA1 gene. Comparative analysis of the structural organization of the rat CPB, rat CPA1, and rat CPA2 genes (Gardell, S. J., Craik, C. S., Clauser, E., Goldsmith, E. J., Stewart, C.-B., Graf, M., and Rutter, W. J. (1988) J. Biol. Chem. 263, 17828-17836) reveals that, with one exception, the number, position, and sequence composition of the exons in these three carboxypeptidase genes are conserved in spite of considerable divergence with respect to the lengths of their corresponding intervening sequences. Conserved sequences in the 5' flanking regions of the rat CPA1, CPA2, CPB, and other pancreas-specific genes have been identified. 相似文献
5.
Genetic analysis of cystic fibrosis: linkage of DNA and classical markers in multiplex families 总被引:3,自引:3,他引:0
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![点击此处可从《American journal of human genetics》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A M Bowcock J Crandall L Daneshvar G M Lee B Young V Zunzunegui C Craik L L Cavalli-Sforza M C King 《American journal of human genetics》1986,39(6):699-706
Linkage of cystic fibrosis (CF) to DNA and classical markers was studied in 36 families of two or three generations with at least two living affected children. Among the 79 affected children, no recombinants were detected between the disease and the markers MET and pJ3.11, previously shown to be linked to CF. No linkage between the human trypsin gene family (which appears to include at least 10 members) and CF was found, although not all genes of the trypsin family have been screened yet. In one of the CF families, recombination between MET and pJ3.11 was detected in an unaffected sib. Data from our families suggest that the gene order of markers among chromosome 7q is: (7cen;p8.33)collagen(COL1A2);DOCR1-917;paraoxonase+ ++(PON);(MET-cf-J3.11);T-cell receptor beta chain (TCRB);qter. There was no evidence for (or against) either postzygotic selection or meiotic drive to explain the high frequency of CF in Caucasian populations. 相似文献
6.
We describe the training of divers to recognise and remove bias in estimating lengths of fish underwater. Divers were asked to allocate objects, from a population (N=50) with a known length frequency distribution, to ten 100 mm size classes. Observed and expected distributions were then compared and the divers informed of their errors. Training continued until divers consistently produced length frequency distributions that were not significantly different from the expected distribution (=0.8) by the one sample Kolmogorov-Smirnov (K-S) test. Divers were trained in five trials, but after six months they had lost all their ability and had to be retrained. Three trained divers observing the same population of the large reef fish Plectropomus leopardus (Serranidae) produced length frequency distributions that were not significantly different (P>0.1) on 67% of occasions. Data collected by divers can be used to detect small but real differences in length frequency distributions of populations when analysed using the two sample K-S test. We suggest a means of determining within site variation in length frequency relative to between site variation. 相似文献
7.
G H Swift C S Craik S J Stary C Quinto R G Lahaie W J Rutter R J MacDonald 《The Journal of biological chemistry》1984,259(22):14271-14278
8.
Inhibition of the HIV-1 and HIV-2 proteases by curcumin and curcumin boron complexes 总被引:3,自引:0,他引:3
Zhihua Sui Rafael Salto Jia Li Charles Craik Paul R. Ortiz de Montellano 《Bioorganic & medicinal chemistry》1993,1(6):415-422
Curcumin, a relatively non-toxic natural product isolated from Curcuma longa, is a modest inhibitor of the HIV-1 (1050 = 100 μM) and HIV-2 (IC50 = 250 μM) proteases. Simple modifications of the curcumin structure raise the IC50 value but complexes of the central dihydroxy groups of curcumin with boron lower the IC50 to a value as low as 6 μM. The boron complexes are also time-dependent inactivators of the HIV proteases. The increased affinity of the boron complexes may reflect binding of the orthogonal domains of the inhibitor in intersecting sites within the substrate-binding cavity of the enzyme, while activation of the ,β-unsaturated carbonyl group of curcumin by chelation to boron probably accounts for time-dependent inhibition of the enzyme. 相似文献
9.
Elín Gudmundsdóttir Rémi Spilliaert Qing Yang Charles S. Craik Jón B. Bjarnason Agusta Gudmundsdóttir 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1996,113(4):795-801
The cDNAs encoding two different Atlantic cod elastases have been isolated and sequenced. The predicted amino acid sequences revealed two preproelastases, consisting of a signal peptide, an activation peptide and a mature enzyme of 242 and 239 amino acids. Amino acid sequence identity between the two cod elastases was 60.1% and identity with mammalian elastases ranged from 50–64%. The two cod elastases contain all the major structural features common to serine proteases, such as the catalytic triad His57, Asp102 and Ser195. Both cod elastases have a high content of methionine, consistent with previous findings in psychrophilic fish enzymes. 相似文献
10.
Defining the level of human immunodeficiency virus type 1 (HIV-1) protease activity required for HIV-1 particle maturation and infectivity. 总被引:16,自引:8,他引:8
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![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The human immunodeficiency virus type 1 (HIV-1) protease is the enzyme required for processing of the Gag and Gag-Pol polyproteins to yield mature, infectious virions. Although the complete absence of proteolytic activity prevents maturation, the level of activity sufficient for maturation and subsequent infectivity has not been determined. Amino acid substitutions that reduce catalytic activity without affecting substrate recognition have been engineered into the active site of the HIV-1 protease. The catalytic efficiency (kcat) of the HIV-1 protease is decreased 4-fold when threonine 26 is replaced by serine (T26S) and approximately 50-fold when alanine 28 is replaced by serine (A28S). Genes containing these mutations were cloned into a proviral vector for analysis of their effects on virion maturation and infectivity. The results show that virions containing the T26S protease variant, in which only 25% of the protease is active, are very similar to wild-type virions, although slight reductions in infectivity are observed. Virions containing the A28S protease variant are not infectious, even though a limited amount of polyprotein processing does occur. There appears to be a linear correlation between the level of protease activity and particle infectivity. Our observations suggest that a threshold of protease activity exists between a 4-fold and 50-fold reduction, below which processing is insufficient to yield infectious particles. Our data also suggest that a reduction of protease activity by 50-fold or greater is sufficient to prevent the formation of infectious particles. 相似文献