Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:O95477 (membrane-bound)
 29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Factor V was purified from the plasma of an activated protein C (APC)-resistant patient who is homozygous for the mutation Arg506-->Gln (factor VR506Q). Factor VR506Q was converted by thrombin into factor Va which was further purified yielding a factor Va preparation that had the same cofactor activity in prothrombin activation as normal factor Va. Inactivation of low concentrations of normal factor Va (< 5 nM) by 0.15 nM APC in the presence of phospholipid vesicles proceeded via a biphasic reaction that consisted of a rapid phase (k = 4.3 x 10(7) M-1s-1), yielding a reaction intermediate with reduced cofactor activity that was fully inactivated during the subsequent slow phase (k = 2.3 x 10(6) M-1s-1). Inactivation of factor VaR506Q proceeded via a monophasic reaction (k = 1.7 x 10(6) M-1s-1). Immunoblot analysis showed that APC-catalyzed inactivation of factor Va occurred via peptide bond cleavages in the heavy chain. The rapid phase of inactivation of normal factor Va was associated with cleavage at Arg506 and full inactivation of factor Va required subsequent cleavage at Arg306. The slow monophasic inactivation of factor VaR506Q correlated with cleavage at Arg306. Cleavage at Arg506 in normal factor Va resulted in accumulation of a reaction intermediate that exhibited 40% cofactor activity in prothrombin activation mixtures that contained a high factor Xa concentration (5 nM). Compared with native factor Va, the reaction intermediate retained virtually no cofactor activity at low factor Xa concentrations (0.3 nM). This demonstrates that factor Va that is cleaved at Arg506 is impaired in its ability to interact with factor Xa. Michaelis-Menten kinetic analysis showed that cleavage at Arg506 in membrane-bound factor Va was characterized by a low Km for factor Va (20 nM) and kcat = 0.96 s-1. For cleavage at Arg306 in factor VaR506Q the kinetic parameters were Km = 196 nM and kcat = 0.37 s-1. This means that differences between APC-catalyzed inactivation of factors Va and VaR506Q become much less pronounced at high factor Va concentrations. When factor VaR506Q was inactivated by APC in the absence of phospholipids, cleavage at Arg679 of the heavy chain also contributed to factor Va inactivation. Comparison of rate constants for APC-catalyzed cleavage at Arg306, Arg506, and Arg679 in the absence and presence of phospholipids indicated that phospholipids accelerated these cleavages to a different extent.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor VaR506Q by activated protein C. 767 48

The B cell antigen receptor is a complex containing the antigen-binding immunoglobulin molecules and the Ig-alpha/Ig-beta heterodimer which presumably connects the B cell antigen receptor to intracellular signaling components. To analyze the functional properties of the cytoplasmic parts of the B cell antigen receptor, we used the K46 B lymphoma line (IgG2a, kappa) to express chimeric molecules composed of the extracellular and transmembrane part of the CD8 alpha molecule and the cytoplasmic sequence of either the Ig-alpha (CD8 alpha/Ig-alpha), the Ig-beta (CD8 alpha/Ig-beta) protein or the membrane-bound gamma 2a heavy chain (CD8 alpha/gamma 2a). From these three types of chimeric molecules only (CD8 alpha/Ig-alpha and CD8 alpha/Ig-beta, but not CD8 alpha/gamma 2a, could transduce signals, thus providing the first evidence that the cytoplasmic tail of Ig-alpha and Ig-beta have a signaling capacity. After cross-linking with anti-CD8 alpha antibodies, both molecules induced a similar increase in intracellular free calcium ion and in MAP kinase phosphorylation. Protein tyrosine kinases, however, were strongly activated via the CD8 alpha/Ig-alpha and only marginally via the CD8 alpha/Ig-beta molecule. This suggests that the Ig-alpha and Ig-beta proteins have distinct roles during signal transduction through the B cell antigen receptor.
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PMID:Differential signaling through the Ig-alpha and Ig-beta components of the B cell antigen receptor. 768 2

A single mouse genomic locus encodes proteins catalyzing three steps of purine synthesis, glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS), and glycinamide ribonucleotide formyltransferase (GART). This gene has 22 exons and spans 28 kilobases. The existence of a second genetic locus and closely related pseudogenes was ruled out by Southern analysis. Mouse tissues express two related classes of messages encoded by this single locus: a trifunctional GARS-AIRS-GART mRNA and a monofunctional GARS mRNA. These transcripts used the same set of multiple transcriptional start sites, and both used the same first 10 exons. CCAAT and TATA elements were not found for this locus. Exon 11, which represented the last coding sequence of the GARS domain, was differentially utilized for the two messages. The trifunctional mRNA was generated by splicing exon 11 to exon 12, the first coding sequence for the AIRS domain with subsequent use of a polyadenylation signal at the end of exon 22. Genomic sequence corresponding to the 3'-UTR of the monofunctional GARS mRNA was contiguous with exon 11, so that the smaller message arose from the recognition of one of the multiple polyadenylation signals present within the intron between exons 11 and 12. Hence, polyadenylation of the primary transcript at a position corresponding to an intron of the genomic locus was responsible for the generation of the monofunctional GARS class of mRNAs. This utilization of an intronic polyadenylation site without alternative exon usage is comparable to the mechanism whereby both secreted and membrane-bound forms of the immunoglobulin mu heavy chain are made from a single genetic locus.
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PMID:Analysis of a mouse gene encoding three steps of purine synthesis reveals use of an intronic polyadenylation signal without alternative exon usage. 782 19

The distribution of membrane-bound organelles was studied in cultured hippocampal neurons after antisense oligonucleotide suppression of the kinesin-heavy chain (KHC). We observed reduced 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) fluorescent staining in neurites and growth cones. In astrocytes, KHC suppression results in the disappearance of the DiOC6(3)-positive reticular network from the cell periphery, and a parallel accumulation of label within the cell center. On the other hand, mitochondria microtubules and microfilaments display a distribution that closely resembles that observed in control cells. KHC suppression of neurons and astrocytes completely inhibited the Brefeldin A-induced spreading and tubulation of the Golgi-associated structure enriched in mannose-6-phosphate receptors. In addition, KHC suppression prevents the low pH-induced anterograde redistribution of late endocytic structures. Taken collectively, these observations suggest that in living neurons, kinesin mediates the anterograde transport of tubulovesicular structures originated in the central vacuolar system (e.g., the endoplasmic reticulum) and that the regulation of kinesin-membrane interactions may be of key importance for determining the intracellular distribution of selected organelles.
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PMID:Kinesin-mediated organelle translocation revealed by specific cellular manipulations. 796 67

The cleavage of human factor V and human factor Va by human activated protein C (APC) was analyzed in the absence and presence of phospholipid vesicles containing 75% phosphatidylcholine (PC) and 25% phosphatidylserine (PS). Membrane-bound human factor V (250 nM) is cleaved by APC (2.5 nM) to give M(r) = 200,000, 70,000, 45,000, and 30,000 fragments and an M(r) = 22/20,000 doublet. These fragments are released after four sequential cleavages of the membrane-bound procofactor at Arg306, Arg506, Arg679, and Lys994. No cofactor activity is observed following thrombin treatment of the membrane-bound APC-cleaved procofactor. In the absence of a membrane surface, no cleavage of factor V by APC is observed, and following thrombin activation factor Va retains full cofactor activity. Membrane-bound human factor Va (600 nM) loses more than 90% of its initial cofactor activity after 10 min of incubation with APC (10.9 nM), and virtually no cofactor activity is observed after 1 h of incubation. Under similar conditions but in the absence of PCPS vesicles, factor Va is cleaved but retains approximately 80% of its initial cofactor activity after 2 h of incubation with APC. In the presence of PCPS vesicles, the APC related loss of activity is correlated with cleavage of the heavy chain and appearance of fragments of M(r) = 45,000, 30,000, and of 28/26,000, and 22/20,000 doublets. These products correspond to three cleavages of the heavy chain (at Arg306, Arg506, and Arg679). Cleavage at Arg506 of factor Va precedes and appears to be required for cleavage at Arg306 and Arg679. In the absence of membrane, proteolysis at Arg506 produces an M(r) = 75,000 fragment which corresponds to the NH2-terminal portion of the human factor Va heavy chain (residues 1-506), and a carboxyl-terminal doublet of M(r) = 28/26,000 (residues 507-709) which is cleaved by APC at Arg679 to generate an M(r) = 22/20,000 doublet and an M(r) = 6,000 peptide. No cleavage of the light chain of the human cofactor is observed in the presence or absence of PCPS vesicles following 2 h of incubation with APC. Our data demonstrate that inactivation of human factor V and human factor Va only occurs in the presence of a membrane surface after cleavage at Arg306. However, while this cleavage site is exposed on membrane-bound human factor V, cleavage at Arg506 on the heavy chain of factor Va appears necessary for complete exposure of the cleavage site at Arg306.
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PMID:The mechanism of inactivation of human factor V and human factor Va by activated protein C. 798 61

We have investigated the IgE heavy chain isoforms produced in vivo by analyzing the epsilon mRNA species present in unstimulated PBL and expressing them individually in a myeloma cell line. Seven epsilon mRNA species were identified by using reverse transcription-PCR, cloning, and sequencing analysis. These species included the classical secreted (epsilon CH4-S) and membrane-bound (epsilon CH4-M1'-M2) IgE and five alternatively spliced epsilon transcripts. At the protein level, the five alternatively spliced epsilon transcripts (epsilon CH4*, epsilon CH4-M2', epsilon CH4'-1, epsilon CH4'-1-M2, and epsilon CH3-13-CH4) corresponded to four epsilon heavy chain isoforms, in which various parts of the CH4 domain were replaced by new stretches of amino acids at the carboxyl termini. The same epsilon mRNA species also were present in the IgE producing myeloma cell line U266. However, except for the classical membrane and secreted IgE, the corresponding proteins could not be identified. To further characterize the epsilon CH4-S, epsilon CH4*, epsilon CH4-M2', epsilon CH4'-1, and epsilon CH4-M1'-M2 species, we expressed them as chimeric mouse/human anti-4-hydroxy-5-iodo-3-nitrophenacetyl Abs in a mouse myeloma cell line. Only the classical secreted and membrane isoforms were found to be secreted or expressed on the cell surface, respectively, and the other forms were retained within the cells and degraded. These data suggest that some of the epsilon mRNA isoforms produced by PBL are aberrantly spliced mRNAs, the protein products of which are eliminated by post-translational events.
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PMID:Characterization and expression of alternatively spliced IgE heavy chain transcripts produced by peripheral blood lymphocytes. 799 41

Clinical manifestations of arterial and venous thrombosis in a family with protein C deficiency was associated with two mutations in the light chain of protein C: Glu20-->Ala and Val34-->Met. Further studies showed that the mutation Glu20-->Ala which eliminated a gamma-carboxylation site was exclusively responsible for the anticoagulant defect of activated protein C (APC). Membrane-bound human factor Va is inactivated by APC after two sequential cleavages of the heavy chain at Arg506 and Arg306. Human factor Va inactivation by human recombinant APC (rAPC) and a mutant molecule with an alanine instead of a glutamic acid at position 20 (rAPC(gamma 20A)) was investigated in the presence and absence of phospholipid vesicles. During a 2-hour incubation period of the cofactor with either rAPC or rAPC(gamma 20A). In the absence of a membrane surface, factor Va is cleaved quantitatively at Arg506 and retains approximately 60% of its initial cofactor activity. After a 2-hour incubation period with rAPC membrane-bound factor Va has no cofactor activity, whereas in the presence of a membrane surface and rAPC(gamma 20A) factor Va retains 60% of its initial cofactor activity. The completed loss in factor Va cofactor activity upon incubation of the membrane-bound cofactor with phospholipid vesicles and rAPC is associated with cleavages at Arg506 and Arg306, whereas membrane-bound factor Va cleavage at Arg306 by rAPC(gamma 20A) is impaired, resulting in a cofactor that is cleaved at Arg506. Slow cleavage at Arg306 occurs when membrane-bound factor Va is incubated with rAPC(gamma 20A) and only small amounts of fragments of M(r) = 45,000 and 30,000 are noticed. Our data show that the genetic defect which leads to the absence of a gamma-carboxylation site at Glu20 impairs membrane binding of human APC, which in turn is required for cleavage of factor Va at Arg306 and inactivation of the cofactor. The consequence of impaired membrane-dependent cleavage at Arg306 is manifested in vivo by venous and arterial thrombosis.
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PMID:Loss of membrane-dependent factor Va cleavage: a mechanistic interpretation of the pathology of protein CVermont. 804 58

A protease purified from the venom of the elapid snake Naja naja oxiana converts human blood coagulation factor Va into a molecule (factor VaNO) with greatly reduced cofactor activity. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the venom protease cleaved a small peptide from the heavy chain of factor Va and reduced the apparent M(r) from 105,000 to 101,000. This peptide was isolated by high performance liquid chromatography on a reversed-phase column. Amino acid sequence analysis of the peptide indicated that the venom enzyme cleaved the peptide bond between His682 and Asp683, thus removing 27 amino acids from the carboxyl-terminal part of the heavy chain. The cofactor activities of factors Va and VaNO were compared by measuring their abilities to support factor Xa-catalyzed prothrombin activation in the presence of phospholipids and calcium ions. Both factor Va molecules stimulated the binding of factor Xa to negatively charged phospholipids. However, the amounts of factor Va required for half-maximal incorporation of factor Xa into the membrane-bound factor Xa-Va complex were much lower for native factor Va (0.25 nM) than for factor VaNO (2.01 nM). At saturating concentrations of factor Va or factor VaNO the kcat values for prothrombin activation were 114 s-1 for factor Va and 128 s-1 for factor VaNO. The Km values for prothrombin determined under these conditions were 0.24 and 0.83 microM for prothrombinase complexes with native factor Va and factor VaNO, respectively. Direct binding studies revealed that factors Va and VaNO bind with equal affinity to phospholipids. These data indicate that factor VaNO is impaired in its ability to interact with factor Xa and prothrombin. Together with the structural data this implies that the carboxyl-terminal Asp683-Arg709 domain of the heavy chain is required for optimal interaction of factor Va with factor Xa and prothrombin.
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PMID:Functional properties of human factor Va lacking the Asp683-Arg709 domain of the heavy chain. 805 Nov 66

The murine T cell hybridoma line, MBI-1.15, secretes a 17-kDa protein which decreases binding activity of the CD23 molecule for its natural ligand, IgE. This protein, denoted epsilon receptor-modulating protein (epsilon RMP), was previously characterized and shown to be a novel serine protease. The present studies show that, in addition to modulating CD23, epsilon RMP costimulates with IL-4 the de novo synthesis and secretion of IgE and IgG 1 by cultured B cells. Since such costimulating activity is reminiscent of a similar synergism with IL-4 previously observed with cell membranes from activated T cells, we examined isolated membranes from the epsilon RMP-producing MBI-1.15 T cell line for comparable activity; indeed, as shown herein, MBI-1.15 cell membranes do exhibit this synergism. Furthermore, we show that a monoclonal antibody (mAb), 2E5B, specific for the 17-kDa soluble form of epsilon RMP, blocks the costimulating activities of both the soluble epsilon RMP and MBI-1.15 T cell membranes for IL-4-induced de novo synthesis of IgE by cultured B cells. This anti-epsilon RMP mAb also detects a 36-kDa membrane-bound protein species which appears to be related to soluble epsilon RMP by immunochemical criteria. The membrane-bound proteins, present on MBI-1.15 T cells, induce germ-line IgE heavy chain transcripts (I epsilon) in I-29 B cells independently of IL-4, and this inductive event is also specifically blocked by the 2E5B anti-epsilon RMP mAb. These findings suggest that T cell membrane-bound epsilon RMP molecules are crucial proteins involved in contact-dependent B cell class switching in the course of IgE biosynthesis. Finally, both IL-4 and epsilon RMP induce I epsilon on I-29 B cells, but neither molecule by itself can induce class switching to IgE synthesis by splenic B cells. This clearly suggests that both epsilon RMP and IL-4 have another important molecular effect (which may or may not be identical) on B cells, that is essential for class switching, but only when both molecules are present simultaneously is the complete mechanism of class switching manifested.
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PMID:Identification of a T cell membrane protein possibly involved in IL-4-induced B cell immunoglobulin class switching to IgE. 811 70

The interactions of the isolated heavy and light chains of factor Va with factor Xa were evaluated using active-site-modified factor Xa [(carboxytetramethyl)rhodamine-Glu-Gly- Arg-factor Xa (ctr-EGR-Xa)]. The Kd for the factor Va heavy-chain interaction with ctr-EGR-Xa was 60 microM. A series of monoclonal antibodies directed against bovine factor Va were tested for their ability to inhibit thrombin formation in an assay using the fluorescent thrombin inhibitor dansylarginine N,N-(3-ethyl-1,5-pentanediyl)amide (DAPA). Monoclonal antibody alpha BFV-4, which recognizes the light chain of the cofactor, was found to inhibit the formation of thrombin. Similarly, monoclonal antibody alpha BFV-5, which is directed against the heavy chain of the cofactor, was found to inhibit thrombin formation. In contrast, monoclonal antibody alpha BFV-1, also directed against the heavy chain of the cofactor, did not inhibit thrombin generation by the prothrombinase complex. Monoclonal antibodies alpha BFV-4 and alpha BFV-5 inhibited the interaction of active-site-modified radiolabeled factor Xa (125I-Xa-EGR) with factor Va bound to PC/PS-coated microtiter wells, whereas nonimmune mouse IgG did not have any effect on the 125I-Xa-EGR.membrane-bound factor Va interaction. The antibodies effect upon the phospholipid-independent interaction between the cofactor and ctr-EGR-Xa was evaluated by analytical ultracentrifugation. Both alpha BFV-4 and alpha BFV-5 inhibited the phospholipid-independent interaction between factor Va and ctr-EGR-Xa.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Contribution of the heavy and light chains of factor Va to the interaction with factor Xa. 820 89


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