ABSTRACT
Many cellular proteins are bound to the surfaces of membranes and participate in various cell signaling responses. Interactions between this group of proteins are in part controlled by the membrane surface to which the proteins are bound. This review focuses on the effects of pressure on membrane-associated proteins. Initially, the effect of pressure on membrane surfaces and how pressure may perturb the membrane binding of proteins is discussed. Next, the effect of pressure on the activity and lateral association of proteins is considered. We then discuss how pressure can be used to gain insight into these types of proteins.
Subject(s)
Humans , Hydrostatic Pressure , Membrane Proteins/metabolism , Isoenzymes/chemistry , Isoenzymes/metabolism , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Membrane Proteins/chemistry , Phospholipase C delta , Protein Binding , Static Electricity , Type C Phospholipases/chemistry , Type C Phospholipases/metabolismABSTRACT
Phospholipase C-gamma1, containing two SH2 and one SH3 domains which participate in the interaction between signaling molecules, plays a significant role in the growth factor-induced signal transduction. However, the role of the SH domains in the growth factor-induced PLC-gamma1 regulation is unclear. By peptide-mass fingerprinting analysis, we have identified SHIP1 as the binding protein for the SH3 domain of PLC-gamma1. SHIP1 was co-immunoprecipitated with PLC-gamma1 and potentiated EGF-induced PLC-gamma1 activation. However, inositol 5'-phosphatase activity of SHIP1 was not required for the potentiation of EGF-induced PLC-gamma1 activation. Taken together, these results suggest that SHIP1 may function as an adaptor protein which can potentiate EGF-induced PLC-gamma1 activation without regards to its inositol 5'-phosphatase activity.
Subject(s)
Animals , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , COS Cells/enzymology , Chlorocebus aethiops , Enzyme Activation , Epidermal Growth Factor/pharmacology , Immunoprecipitation , Inositol 1,4,5-Trisphosphate/metabolism , Molecular Sequence Data , Type C Phospholipases/chemistry , Phosphoric Monoester Hydrolases/chemistry , Protein Binding , Signal Transduction , src Homology Domains/physiologyABSTRACT
5'-upstream region of the phospholipase C-beta2 gene, 810 bp, was cloned and characterized. S1 nuclease mapping and primer extension analyses revealed that a single transcriptional start site locates at 284 nucleotides upstream from the beginning of translation. The 5-upstream region lacks both TATA motif and typical initiator sequence, but retains GC-rich segment. Two putative regulatory regions, a negative region (-636/-588) and a positive region (-98/ -13) were identified in the upstream region of PLC-beta2 gene. We suggest that the transcription of PLC-beta2 may be regulated by binding of regulatory proteins to the negative and/or positive regulatory regions located in the upstream of the gene.
Subject(s)
Single-Strand Specific DNA and RNA Endonucleases/metabolism , Base Sequence , Cells, Cultured , Chloramphenicol O-Acetyltransferase/metabolism , Cloning, Molecular , Conserved Sequence , Gene Deletion , Isoenzymes/chemistry , Molecular Sequence Data , Mutagenesis, Site-Directed , Type C Phospholipases/chemistry , Promoter Regions, Genetic , Protein Binding , Transcription, Genetic , TransfectionABSTRACT
Since glycosylphosphatidylinositol is the most common form of attachment of proteins to membranes in T. cruzi, and that this parasite depends on surface-mediated interactions for survival within the vector and mammalian host, it is probable that a drug which interfers with the metabolism of glycosylphosphatidylinositol (GPI) could be successfully employed in chemotherapy. Over the last few years several groups have been characterizing this mode of attachment in T. cruzi and more recently we have been concentrating our efforts on the identification of candidate precursors for protein anchors in metacyclic trypomastigotes. Previously detected GPI heterogeneity regarding solubilization of a major stage-specific antigen (1G7-Ag) by phospholipase C led us to investigate whether biosynthetic precursors with similar properties could also be identified. Two glycolipid species whose migration properties resemble glycolipids A and C of T. brucei were amenable to biosynthetic radiolabelling with palmitic acid, inositol, ethanolamine, glucosamine and mannose. Following purification, these species were submitted to classical GPI diagnostic treatments. In both cases digestion with GPI-specific phospholipase D (GPIPLD) produced phosphatidic acid and treatment with either mild base or phospholipase A2 (PLA2) produced free fatty acid, indicating an acylation at least at position 2 of the glycerol. The glycolipid A-like species proved to be susceptible to solubilization by PIPLC of B. thuriengiensis and by GPIPLC of T. brucei and the glycolipid C-like material proved to be fully resistant to both lipases. Although the glycolipid A-like species indeed presents these and other properties compatible with a precursor for the chemically characterized 1G7-Ag anchor, the PLC-resistant species which is completely insensitive to nitrous acid deamination might be an exception to the general finding of a non-acetylated glucosamine in the GPI moieties so far described
Subject(s)
Antigens, Protozoan , Phosphatidylinositols/chemistry , Glycolipids/chemistry , Trypanosoma cruzi/immunology , Type C Phospholipases/chemistry , Base Sequence , Carbohydrate Sequence , Fatty Acids , Molecular Sequence DataABSTRACT
Listeria monocytogenes is a model intracellular pathogen which escapes from a host cell vacuole, grows intracytoplasmically, and spreads cell to cell without an extracellular phase. A number of genes necessary for pathogenicity have been discovered, two of which encode phospholipases C, a PI-PLC and a broad-range PLC. Single and double mutants were constructed with in-frame deletions in one or both PLCs. Characterization of the strains indicated that the two PLCs may have overlapping functions as the double mutant was 500-fold less virulent while the single mutants had a negligible effect on virulence. The role of the PLCs appears to be multifactorial as PI-PLC has a role in escaping from the initial host vacuole and the broad-range PLC appears to have a role in cell to cell spreading
Subject(s)
Rats , Listeria monocytogenes/pathogenicity , Type C Phospholipases/chemistry , Protein Kinase C/metabolismABSTRACT
Phospholipase C activity was demonstrated from axystelma esculantum root tuber. The optimum enzyme activity was found at pH 5.5 and temperature 30°C. Enzyme activity was stimulated with Triton x - 100 [1mM] 25%, sodium deoxycholate [1mM] 65%, cobalt chloride [5mM] 162.5% and Zinc chloride [10mM] 180%. Enzyme activity showed 100% inhibition with EDTA [1mM] and Calcium chloride [10mM]. Oxystelma esculantum crude Phospholipase C enzyme activity was found to be heat labile