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111.
112.
S. Gillis B. C. Furie B. Furie H. Patel M. C. Huberty M. Switzer W. B. Foster H. A. Scoble M. D. Bond 《Protein science : a publication of the Protein Society》1997,6(1):185-196
The gamma-carboxyglutamic acid (Gla) domains of the vitamin K-dependent blood coagulation proteins contain 10 highly conserved Gla residues within the first 33 residues, but factor IX is unique in possessing 2 additional Gla residues at positions 36 and 40. To determine their importance, factor IX species lacking these Gla residues were isolated from heterologously expressed human factor IX. Using ion-exchange chromatography, peptide mapping, mass spectrometry, and N-terminal sequencing, we have purified and identified two partially carboxylated recombinant factor IX species; factor IX/gamma 40E is uncarboxylated at residue 40 and factor IX/gamma 36,40E is uncarboxylated at both residues 36 and 40. These species were compared with the fully gamma-carboxylated recombinant factor IX, unfractionated recombinant factor IX, and plasma-derived factor IX. As monitored by anti-factor IX:Ca (II)-specific antibodies and by the quenching of intrinsic fluorescence, all these factor IX species underwent the Ca(II)-induced conformational transition required for phospholipid membrane binding and bound equivalently to phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine. Endothelial cell binding was also similar in all species, with half-maximal inhibition of the binding of 125I-labeled plasma-derived factor IX at concentrations of 2-6 nM. Functionally, factor IX/gamma 36,40E and factor IX/gamma 40E were similar to fully gamma-carboxylated recombinant factor IX and plasma-derived factor IX in their coagulant activity and in their ability to participate in the activation of factor X in the tenase complex both with synthetic phospholipid vesicles and activated platelets. However, Gla 36 and Gla 40 represent part of the epitope targeted by anti-factor IX:Mg(II)-specific antibodies because these antibodies bound factor IX preferentially to factor IX/gamma 36,40E and factor IX/gamma 40E. These results demonstrate that the gamma-carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for any function of factor IX examined. 相似文献
113.
Previous studies in our laboratory have shown that Na absorption across the porcine endometrium is stimulated by PGF2α and cAMP-dependent activation of a barium-sensitive K channel located in the basolateral membrane of surface epithelial cells.
In this study, we identify and characterize this basolateral, barium-sensitive K conductance. Porcine uterine tissues were
mounted in Ussing chambers and bathed with KMeSO4 Ringer solution. Amphotericin B (70 μm) was added to the luminal solution to permeabilize the apical membrane and determine the current-voltage relationship of
the basolateral K conductance after activation by 100 μm CPT-cAMP. An inwardly rectifying current was identified which possessed a reversal potential of −53 mV when standard Ringer
solution was used to bathe the serosal surface. The K:Na selectivity ratio was calculated to be 12:1. Administration of 5
mm barium to the serosal solution completely inhibited the current activated by cAMP under these conditions. In addition to
these experiments, amphotericin-perforated whole cell patch clamp recordings were obtained from primary cultures of porcine
surface endometrial cells. The isolated cells displayed an inwardly rectifying current under basal conditions. This current
was significantly stimulated by CPT-cAMP and blocked by barium. These results together with our previous studies demonstrate
that cAMP increases Na absorption in porcine endometrial epithelial cells by activating an inwardly rectifying K channel present
in the basolateral membrane. Similar patch clamp experiments were conducted using cells from a human endometrial epithelial
cell line, RL95-2. An inwardly rectifying current was also identified in these cells which possessed a reversal potential
of −56 mV when the cells were bathed in standard Ringer solution. This current was blocked by barium as well as cesium. However,
the current from the human cells did not appear to be activated by cAMP, indicating that distinct subtypes of inwardly rectifying
K channels are present in endometrial epithelial cells from different species.
Received: 6 February 1997/Revised: 10 July 1997 相似文献
114.
C.H. Pedemonte T.A. Pressley M.F. Lokhandwala A.R. Cinelli 《The Journal of membrane biology》1997,155(3):219-227
Considerable evidence indicates that the renal Na+,K+-ATPase is regulated through phosphorylation/dephosphorylation reactions by kinases and phosphatases stimulated by hormones
and second messengers. Recently, it has been reported that amino acids close to the NH2-terminal end of the Na+,K+-ATPase α-subunit are phosphorylated by protein kinase C (PKC) without apparent effect of this phosphorylation on Na+,K+-ATPase activity. To determine whether the α-subunit NH2-terminus is involved in the regulation of Na+,K+-ATPase activity by PKC, we have expressed the wild-type rodent Na+,K+-ATPase α-subunit and a mutant of this protein that lacks the first thirty-one amino acids at the NH2-terminal end in opossum kidney (OK) cells. Transfected cells expressed the ouabain-resistant phenotype characteristic of
rodent kidney cells. The presence of the α-subunit NH2-terminal segment was not necessary to express the maximal Na+,K+-ATPase activity in cell membranes, and the sensitivity to ouabain and level of ouabain-sensitive Rb+-transport in intact cells were the same in cells transfected with the wild-type rodent α1 and the NH2-deletion mutant cDNAs. Activation of PKC by phorbol 12-myristate 13-acetate increased the Na+,K+-ATPase mediated Rb+-uptake and reduced the intracellular Na+ concentration of cells transfected with wild-type α1 cDNA. In contrast, these effects were not observed in cells expressing
the NH2-deletion mutant of the α-subunit. Treatment with phorbol ester appears to affect specifically the Na+,K+-ATPase activity and no evidence was observed that other proteins involved in Na+-transport were affected. These results indicate that amino acid(s) located at the α-subunit NH2-terminus participate in the regulation of the Na+,K+-ATPase activity by PKC.
Received: 10 July 1996/Revised: 19 September 1996 相似文献
115.
Hiromichi Kawai Hitoshi Yasuda Masahiko Terada Mariko Omatsu-Kanbe Ryuichi Kikkawa 《Journal of neurochemistry》1997,69(1):330-339
Abstract: Three isoforms of catalytic α subunits and two isoforms of β subunits of Na+ ,K+ -ATPase were detected in rat sciatic nerves by western blotting. Unlike the enzyme in brain, sciatic nerve Na+ ,K+ -ATPase was highly resistant to ouabain. The ouabain-resistant α1 isoform was demonstrated to be the predominant form in rat intact sciatic nerve by quantitative densitometric analysis and is mainly responsible for sciatic nerve Na+ ,K+ -ATPase activity. After sciatic nerve injury, the α3 and β1 isoforms completely disappeared from the distal segment owing to Wallerian degeneration. In contrast, α2 and β2 isoform expression and Na+ ,K+ -ATPase activity sensitive to pyrithiamine (a specific inhibitor of the α2 isoform) were markedly increased in Schwann cells in the distal segment of the injured sciatic nerve. These latter levels returned to baseline with nerve regeneration. Our results suggest that α3 and β1 isoforms are exclusive for the axon and α2 and β2 isoforms are exclusive for the Schwann cell, although axonal contact regulates α2 and β2 isoform expressions. Because the β2 isoform of Na+ ,K+ -ATPase is known as an adhesion molecule on glia (AMOG), increased expression of AMOG/β2 on Schwann cells in the segment distal to sciatic nerve injury suggests that AMOG/β2 may act as an adhesion molecule in peripheral nerve regeneration. 相似文献
116.
布氏田鼠肥满度分析和小型兽类肥满度指标K与KWL(重长指标)的比较 总被引:22,自引:0,他引:22
通过对两个肥满度指标的理论和生物学意义分析,以及对布氏田鼠肥满度的研究和实际应用的讨论,认为描述动物的肥满度时,重长指标KWL优于指标K。两指标的最大差别是成体的KWL值大于幼体,而成体的K值小于幼体。布氏田鼠肥满度没有性别差异;有异著的年龄差异,成体鼠的肥满度高于幼鼠;有显著的季节变化,鼠种群春季肥满度最高,夏季降低,秋季回升;有显著的年际变化,高数量年的肥满度高于低数量年。 相似文献
117.
Enrique A. Gonzlez Froiln Vzquez Jos Ignacio Garabal Jorge Blanco 《Microbiology and immunology》1995,39(12):937-942
Fimbriae isolation by means of thermal shock was applied to fifteen K88-positive (three K88ab, nine K88ac and three K88ad) Escherichia coli reference strains belonging to serotypes O8:K87, O32, O45, O138:K81, O141:K85, O147:K89, O149:K91, and O157, as well as to ten K88-positive enterotoxigenic strains isolated from porcine diarrhea in Spain, all of them belonging to the O149 serogroup. Fimbriae were removed from the bacterial cells by thermal shock at 60 C and then precipitated using ammonium sulfate. The final amount of K88 antigen and the purification degree were not related to the serogroup of the bacteria or to the antigen variant but were related to the buffer used for isolation. Phosphate buffer containing urea was shown to be more effective than Tris-HCl for isolation of K88 antigen. The molecular weights by SDS-PAGE for K88ab, K88ac, and K88ad were 28.5, 29.2, and 31.0 kDa, respectively. All enterotoxigenic E. coli strains isolated in Spain showed the K88ac variant. 相似文献
118.
脱落酸对线粒体Na^+—K^+ATPase活性的影响 总被引:3,自引:0,他引:3
在提取大豆(Glycine m ax)黄化子叶的线粒体时,于清洗和悬浮介质中加入ABA,发现40 μm ol/L(±) ABA 对线粒体膜结合Na+ -K+ ATPase 活性及线粒体吸氧速率有明显的促进作用。ABA 可使16℃与27℃培养的大豆子叶线粒体膜结合Na+ -K+ ATPase Arrhenius图的折点温度分别下降6.3℃和5.9℃,并将该酶的底物动力学曲线由正协同曲线转变为非协同曲线。表明ABA 可降低线粒体的膜相变温度 相似文献
119.
TheSaccharomyces cerevisiae killer toxin K1 is a secreted α/β-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage
process. The first step involves toxin binding to β-1,6-d-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any
of theKRE genes whose products are involved in synthesis and/or assembly of cell wall β-d-glucans. After binding to the yeast cell wall, the killer toxin is transferred to the cytoplasmic membrane, subsequently
leading to cell death by forming lethal ion channels. In an attempt to identify a secondary K1 toxin receptor at the plasma
membrane level, we mutagenized sensitive yeast strains and isolated killer-resistant (kre) mutants that were resistant as spheroplasts. Classical yeast genetics and successive back-crossings to sensitive wild-type
strain indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), renderingkrel2 mutants incapable of binding significant amounts of toxin to the membrane. Sincekrel2 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic
membranes from akrel2 mutant and from an isogenicKre12+ strain and analyzed the membrane protein patterns by 2D-electrophoresis using a combination of isoelectric focusing and SDS-PAGE.
Using this technique, three different proteins (or subunits of a single multimeric protein) were identified that were present
in much lower amounts in thekre12 mutant. A model for K1 killer toxin action is presented in which the gene product ofKRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation. 相似文献
120.
Limited proteolysis of native proteins: the interaction between avidin and proteinase K. 总被引:1,自引:1,他引:0 下载免费PDF全文
D. Ellison J. Hinton S. J. Hubbard R. J. Beynon 《Protein science : a publication of the Protein Society》1995,4(7):1337-1345
Avidin is a tetramer of 16-kDa subunits that have a high affinity for biotin. Proteolysis of native apoavidin by proteinase K results in a limited attack at the loop between beta-strands 3 and 4, involving amino acids 38-43. Specifically, sites of proteolysis are at Thr 40-Ser 41 and Asn 42-Glu 43. The limited proteolysis results in an avidin product that remains otherwise intact and which has enhanced binding for 4'-hydroxyazobenzene-2-benzoic acid (HABA), a chromogenic reporter that can occupy the biotin-binding site. Saturation of the biotin-binding site with the natural ligand protects avidin from proteolysis, but saturation with HABA enhances the rate of proteolysis of the same site. Analysis of the three-dimensional structures of apoavidin and holoavidin reveals that the 3-4 loop is accessible to solvent and scores highly in an algorithm developed to identify sites of proteolytic attack. The structure of holoavidin is almost identical to the apoprotein. In particular, the 3-4 loop has the same structure in the apo and holo forms, yet there are marked differences in proteolytic susceptibility of this region. Evidence suggests that the 3-4 loop is rather mobile and flexible in the apoprotein, and that it becomes constrained upon ligand binding. In one crystal structure of the apoprotein, this loop appears constrained by contacts with symmetry-related molecules. Structural analyses suggest that the "lid" to the biotin-binding site, formed by the 3-4 loop, is displaced and made more accessible by HABA binding, thereby enhancing its proteolytic susceptibility. 相似文献