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Biomedical subjects

M Katan

Publications and source records attributed to M Katan.

At least 37 records · Page 2Linked to original sources
PubMed1997

A ternary metal binding site in the C2 domain of phosphoinositide-specific phospholipase C-delta1.

We have determined the crystal structures of complexes of phosphoinositide-specific phospholipase C-delta1 from rat with calcium, barium, and lanthanum at 2.5-2.6 A resolution. Binding of these metal ions is observed in the active site of the catalytic TIM barrel and in the calcium binding region (CBR) of the C2 domain. The C2 domain of PLC-delta1 is a circularly permuted topological variant (P-variant) of the synaptotagmin I C2A domain (S-variant). On the basis of sequence analysis, we propose that both the S-variant and P-variant topologies are present among other C2 domains. Multiple adjacent binding sites in the C2 domain were observed for calcium and the other metal/enzyme complexes. The maximum number of binding sites observed was for the calcium analogue lanthanum. This complex shows an array-like binding of three lanthanum ions (sites I-III) in a crevice on one end of the C2 beta-sandwich. Residues involved in metal binding are contained in three loops, CBR1, CBR2, and CBR3. Sites I and II are maintained in the calcium and barium complexes, whereas sites II and III coincide with a binary calcium binding site in the C2A domain of synaptotagmin I. Several conformers for CBR1 are observed. The conformation of CBR1 does not appear to be strictly dependent on metal binding; however, metal binding may stabilize certain conformers. No significant structural changes are observed for CBR2 or CBR3. The surface of this ternary binding site provides a cluster of freely accessible liganding positions for putative phospholipid ligands of the C2 domain. It may be that the ternary metal binding site is also a feature of calcium-dependent phospholipid binding in solution. A ternary metal binding site might be a conserved feature among C2 domains that contain the critical calcium ligands in their CBR's. The high cooperativity of calcium-mediated lipid binding by C2 domains described previously is explained by this novel type of calcium binding site.

Animals↗
PubMed1997

Structural mapping of the catalytic mechanism for a mammalian phosphoinositide-specific phospholipase C.

The crystal structures of various ternary complexes of phosphoinositide-specific phospholipase C-delta 1 from rat with calcium and inositol phosphates have been determined at 2.30-2.95 A resolution. The inositol phosphates used in this study mimic the binding of substrates and the reaction intermediate and include D-myo-inositol-1,4,5-trisphosphate, D-myo-inositol-2,4, 5-trisphosphate. D-myo-inositol-4,5-bisphosphate, and D,1-myo-inositol-2-methylene-1,2-cyclićmonophosphonate. The complexes exhibit an almost invariant mode of binding in the active site, each fitting edge-on into the active site and interacting with both the enzyme and the catalytic calcium at the bottom of the active site. Most of the active site residues do not undergo conformational changes upon binding either calcium or inositol phosphates. The structures are consistent with bidentate liganding of the catalytic calcium to the inositol phosphate intermediate and transition state. The complexes suggest explanations for substrate preference, pH optima, and ratio of cyclic to acyclic reaction products. A reaction mechanism is derived that supports general acid/base catalysis in a sequential mechanism involving a cyclic phosphate intermediate and rules out a parallel mechanism where acyclic and cyclic products are simultaneously generated.

Animals↗
PubMed1997

Phosphoinositide-specific phospholipase C: structural basis for catalysis and regulatory interactions.

Phosphoinositide-specific phospholipase C (PI-PLC) isozymes have an important role in cellular responses to a variety of extracellular signals. Recently, the three-dimensional structures of their isolated domains and of the multidomain core, common to all PI-PLCs, have been solved. This provided an insight into the domain organization of PI-PLCs and, together with the structure-function analysis, contributed towards an understanding of the molecular mechanisms of catalysis and regulation.

Journal Article↗
PubMed1996

Structural views of phosphoinositide-specific phospholipase C: signalling the way ahead.

Recent structural studies of mammalian phosphoinositide-specific phospholipase C (PI-PLC) have begun to shed light on the mechanism whereby this family of effector enzymes is able to hydrolyze phospholipid substrates to yield second messengers. PI-PLC isozymes employ a variety of modules (PH domain, EF-hand domain, SH2 domain, SH3 domain and C2 domain) that are common in proteins involved in signal transduction to reversibly interact with membranes and protein components of the signalling pathways.

Binding Sites↗
PubMed1996

Crystal structure of a mammalian phosphoinositide-specific phospholipase C delta.

Mammalian phosphoinositide-specific phospholipase C enzymes (PI-PLC) act as signal transducers that generate two second messengers, inositol-1,4,5-trisphosphate and diacylglycerol. The 2.4-A structure of phospholipase C delta 1 reveals a multidomain protein incorporating modules shared by many signalling proteins. The structure suggests a mechanism for membrane attachment and Ca2+-dependent hydrolysis of second-messenger precursors. The regulation and reversible membrane association of PI-PLC may serve as a model for understanding other multidomain enzymes involved in phospholipid signalling.

Amino Acid Sequence↗
PubMed1996

The control of inositol lipid hydrolysis.

Hydrolysis of PIP2 by specific PLC enzymes is involved in the regulation of different cellular processes by many extracellular signals. The need stringently to control this reaction is reflected by the fact that there are many PLC isozymes and multiple mechanisms linking these isozymes to various receptors. For two of the three PLC families found in mammalian cells (PLC beta and gamma), the components of the main regulatory pathways have been identified. PLC beta isozymes are regulated through G protein coupled receptors. Their activity is stimulated by interaction with alpha subunit from the Gq family and interaction with G protein beta gamma subunits. PLC gamma isozymes are regulated through receptor and non-receptor tyrosine kinases. The combination of SH2 dependent complex formation with phosphorylated tyrosine kinases and the subsequent phosphorylation of PLC gamma leads to stimulation of its activity. Although components that stimulate PLC beta and gamma isozymes have been identified, the molecular mechanism of stimulation remains largely unknown. Each signalling component operating within this general framework represents a family of related proteins. It is not clear what all the functional differences between members of the same family may be and to what extent they could determine specificity of individual signalling pathways. Similarly, it is not known to what extent alterations in PLC function/expression contribute to human pathologies. In the context of oncology, there is evidence for upregulation of PLC gamma in parallel with increased expression of the EGF receptor (Artega et al. 1991). However, it is not clear yet whether this is causally involved or a bystander effect.

Animals↗
PubMed1995

Phospholipase C delta 1 requires a pleckstrin homology domain for interaction with the plasma membrane.

The structural requirements of phospholipase C delta 1 for interaction with the plasma membrane were analysed by immunofluorescence after microinjection into living cells. Microinjection of deletion mutants revealed that the region required for membrane attachment and binding of inositol 1,4,5-trisphosphate in vitro corresponded to the pleckstrin homology domain, a structural module described in more than 90 proteins.

3T3 Cells↗
PubMed1995

Mutations within a highly conserved sequence present in the X region of phosphoinositide-specific phospholipase C-delta 1.

Phosphoinositide-specific phospholipase C (PI-PLC) enzymes have considerable structural similarity within limited regions (X and Y) implicated in catalysis. The role of residues contained within a highly conserved sequence present in the X region was investigated by site-directed mutagenesis of PLC-delta 1 isoenzyme. Seven residues (Ser-308, Ser-309, Ser-310, His-311, Thr-313, Tyr-314, and Gln-319) were individually replaced by alanine or glutamine (His-311). Replacement of two residues, His-311 and Tyr-314, resulted in a dramatic reduction of enzyme activity. The kcat of hydrolysis of phosphatidylinositol 4,5-bisphosphate by H311A and Y314A mutants was reduced 1000- and 10-fold respectively, with little effect on Km. Further analysis of H311A and Y314A mutants, using limited proteolysis and circular dichroism, had shown that no major structural alterations had occurred. Since site-directed mutagenesis demonstrated the importance of histidine residues, their role in enzyme function was also analysed by chemical modification with diethyl pyrocarbonate. This modification of histidine residues resulted in the reduction of enzyme activity and also indicated that more than one residue could be important.

Amino Acid Sequence↗
PubMed1994

Purification and properties of zinc-metallophospholipase C from Pseudomonas fluorescens.

Phospholipase C produced by Pseudomonas fluorescens, isolated as a laboratory contaminant, has been purified to apparent homogeneity by ammonium sulphate fractionation, anion-exchange and size-exclusion chromatographies. The apparent molecular mass of the purified polypeptide was 39.5 kDa. Purified preparations of phospholipase C were used to characterize its enzymic properties and to obtain amino acid sequence of the N-terminus of the molecule. The P. fluorescens phospholipase C hydrolysed PtdEtn, PtdCho and PtdSer (PtdEtn > PtdCho >> PtdSer) and was relatively thermostable. The enzyme was inactivated in the presence of chelating agent o-phenanthroline and the activity restored after addition of zinc. Properties of this enzyme and in particular the requirement for zinc ions for the activity, revealed similarity with the well characterised Bacillus cereus phospholipase C. Similarities with other bacterial and mammalian enzymes reported to be related to the B. cereus type are discussed.

Amino Acid Sequence↗
PubMed1993

Structural requirements of phosphatidylinositol-specific phospholipase C delta 1 for enzyme activity.

Phosphatidylinositol-specific phospholipase C delta 1 isozyme of phosphoinositol-specific phospholipase C has been used for studies of structural requirements for the catalytic function. The enzyme was expressed in a bacterial system and purified to homogeneity. Using a combination of deletion mutant analysis and limited proteolysis, it was found that the large proportion of the molecule participated in formation of a catalytic domain (residues 139-756); it included regions of high and low conservation with other phospholipase-C molecules. These studies also showed that the residues spanning regions of conservation, designated as X and Y, were exposed and highly susceptible to proteolysis by trypsin. Two of the fragments resulting from the cleavage (30 kDa and 40 kDa) interacted and, under non-denaturing conditions, formed a protein of 70 kDa.

Amino Acid Sequence↗
PubMed1990

Characterization of a cellular factor which interacts functionally with Oct-1 in the assembly of a multicomponent transcription complex.

Induction of transcription of the immediate-early (IE) genes of herpes simplex virus involves the assembly of a DNA-binding complex containing the viral protein Vmw65 and the cellular transcription factor Oct-1. We show that Oct-1 is not sufficient for complex formation and that another cellular factor(s) which is absolutely required for complex formation can be separated from Oct-1 under native conditions. We have purified this factor by approximately 100-fold using DNA-cellulose, ion-exchange and size-exclusion chromatographies. The assay used throughout the purification procedure follows the ability of the cellular factor to form a complex when added to purified Oct-1, Vmw65 and an IE specific DNA probe. The complex forming factor (CFF) had a sedimentation coefficient of about 4.4 S (i.e. molecular mass of about 70K, under non-denaturing conditions) and the polypeptide profile of highly purified CFF demonstrated two major species with molecular masses of 80K and 70K. Unequivocal association of either of these two species with CFF activity could not presently be demonstrated due to the sensitivity of CFF to denaturation. CFF, when tested on its own or in the presence of Vmw65, did not bind to the IE-specific consensus motif. We have also used deletion mutants of Oct-1 to show that the POU domain of this protein was sufficient for CFF-dependent complex formation with Vmw65. Deletions of the POU specific region of Oct-1 significantly reduced the complex forming ability, although detectable levels of complex were reconstituted using Vmw65, CFF and just the homeodomain of Oct-1.

Animals↗
PubMed1989

A novel inositol-phospholipid-specific phospholipase C. Rapid purification and characterization.

A novel bovine brain inositol-phospholipid-specific phospholipase C has been identified on the basis of chromatographic behaviour and purified to apparent homogeneity by a rapid three-step procedure. The purified enzyme has a molecular mass of 85 kDa on SDS/polyacrylamide gel electrophoresis and a specific activity of 24 mumol.min-1.mg-1. The enzyme is dependent on Ca2+ and shows a marked preference for inositol phospholipid substrates. The unique nature of this polypeptide was confirmed through partial protein sequence analysis.

Amino Acid Sequence↗
PubMed1988

Determination of the primary structure of PLC-154 demonstrates diversity of phosphoinositide-specific phospholipase C activities.

Protein sequence analysis of a bovine brain phosphoinositide-specific phospholipase C (PI-PLC; PLC-154) has permitted the isolation of a cDNA that appears to code for this protein. Transient expression of this cDNA in COS-1 cells demonstrates that the cDNA encodes a functional phospholipase C that migrates at approximately 150,000 daltons. A transcript of approximately 7 kb is observed in RNA derived from bovine brain and a related transcript of the same size is present in certain human cell lines. Southern blot analysis indicates that one or possibly two genes hybridize with a PLC-154 probe. Regions of homology between PLC-154 and the previously described PLC-148 allow the assignment of a putative catalytic domain to the central region of PLC-154.

Amino Acid Sequence↗
PubMed1987

Purification of phosphoinositide-specific phospholipase C from a particulate fraction of bovine brain.

The coupling of various agonist receptors to the hydrolysis of phosphoinositides has generated much interest in the nature of the phospholipase C that is activated. Here we report the purification of a bovine brain phospholipase C derived from the particulate fraction. A 1000-fold purification was achieved by a combination of heparin-Sepharose, DEAE-cellulose and gel-permeation chromatography. The purified enzyme appears to be monomeric and under denaturing conditions shows a single staining major polypeptide of molecular mass 154 kDa in SDS gels. The enzyme is specific for phosphoinositides although it shows a marked preference for the polyphosphoinositides. With phosphatidylinositol 4,5-bisphosphate as substrate the enzyme expresses a specific activity of greater than 100 mumol min-1 mg-1. The phospholipase C is activated by Ca2+ (0.1-10 microM). The behaviour of this particulate enzyme is discussed in the context of a agonist-induced phosphatidylinositol hydrolysis.

Animals↗