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Biomedical subjects

J Haiech

Publications and source records attributed to J Haiech.

At least 73 records · Page 4Linked to original sources
PubMed1985

Structural variations in actins. Biochemical and immunological tools for probing the structure of rabbit skeletal-muscle and bovine aortic actins.

Structural differences between skeletal-muscle and aortic actins were studied by using biochemical and immunological approaches. By using proteinase digestion we found that three regions of actin show structural differences: (a) in the C-terminal part, (b) the region around residue 227 and (c) the region around residue 167. By using antibodies specific to particular actin conformations we can discriminate between monomeric and filamentous forms of the two actins. Our results show that the minor sequence variations of the N- and C-terminal regions induce structural change in these regions, but also some long-range structural variations in other regions.

Actins↗
PubMed1985

Calcium binding of arterial tropomyosin: involvement in the thin filament regulation of smooth muscle.

Bovine aortic tropomyosin has been isolated by DEAE-Sepharose chromatography following isoelectric precipitation and ammonium sulfate fractionation. A single polypeptide [Mr 36 000 on a sodium dodecyl sulfate (SDS)-polyacrylamide gel] was obtained under different electrophoretic conditions. The amino acid composition of bovine tropomyosin was very similar to that of rabbit skeletal muscle; the amino-terminal residue is blocked. The molecular weight of the native tropomyosin (76 000), which is twice that calculated from the SDS-polyacrylamide gel, suggests that the molecule is a dimer. The diffusion coefficient of 3.4 X 10(-7) cm2 s-1 and the frictional coefficient of 1.7 indicate that the molecule is asymmetric. Comparative high-pressure liquid chromatography peptide mapping of rabbit skeletal and bovine aortic tropomyosins shows primary structure variation. Bovine aortic tropomyosin binds calcium under physiological conditions of pH and ionic strength (22 mol of Ca2+/mol of tropomyosin with a Kd of 1.4 mM). Such a property is not shared by skeletal tropomyosin. In low Mg2+ concentration, both skeletal and aortic actin activations of the skeletal myosin ATPase activity are calcium independent. Addition of aortic tropomyosin to a hybrid actomyosin (aortic actin, skeletal myosin) yields an enhancement of the actin activation of the myosin ATPase activity, but the addition of skeletal tropomyosin yields a decrease of this activity. However, both the enhancement and decrease are calcium dependent. Addition of skeletal or aortic tropomyosin to an actomyosin system, where both actin and myosin come from skeletal muscle, yields only an enhancement of the actin activation of the myosin ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗
PubMed1985

Preparation and characterization of bovine aortic actin.

A functional vascular smooth-muscle actin from bovine aorta was purified to homogeneity by an original method and was able to polymerize. Aortic actin is composed of two major isoforms and at least two minor ones. This actin was not phosphorylated by either cyclic AMP-dependent protein kinase or C kinase. The physical properties of aortic actin were found to be very similar to those of skeletal-muscle actin, except for amino acid composition (three tryptophan residues instead of four). The aortic actin and skeletal-muscle actin differ in the extent of activation of the Mg-dependent ATPase of skeletal-muscle myosin.

Actins↗
PubMed1985

Search for promoter sites of prokaryotic DNA using learning techniques.

Using learning techniques previously described in this journal, we have built an expert system able to point to the start DNA point of a sequence and therefore to recognize a promoter. However, to build this system, we have focused on the TATA box and its environment. We have used this expert system to look for new promoters and also to construct new promoters. The results obtained are discussed.

Base Sequence↗
PubMed1985

Computer search of calcium binding sites in a gene data bank: use of learning techniques to build an expert system.

Using a learning set of 28 sequences able to bind calcium (each sequence is 12 residues long), we have built two filters by learning on this set. The first filter uses a pattern-matching technique and the second one takes into account the environment of amino-acids. These two filters have been used to find new calcium-binding proteins in a data bank. The results are discussed.

Amino Acid Sequence↗
PubMed1985

Identification of the catalytic subunit of brain adenylate cyclase: a calmodulin binding protein of 135 kDa.

The partial purification of the eukaryote adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] catalytic subunit has been achieved by a procedure based on the calmodulin (CaM) sensitivity of the enzyme. Small amounts of rat brain synaptosomal membranes depleted of CaM were solubilized with Lubrol and subjected to a three-step chromatographic procedure involving gel filtration, a CaM-Sepharose affinity step, and fast protein liquid chromatography. About 20% of the adenylate cyclase activity contained in the membranes was recovered in the final enriched fraction with a specific activity of 200 nmol X mg-1 X min-1. The alpha subunits of the adenylate cyclase stimulatory proteins NS were absent from this final fraction. The addition of CaM, of forskolin, or of preactivated NS-containing fractions to this preparation greatly increased the enzyme activity. A CaM-binding polypeptide of 135,000 Da copurified with the adenylate cyclase activity in each of the three steps. Polyacrylamide gel electrophoresis of the final fraction showed that this polypeptide represented 35% of the total protein. We propose that this polypeptide is likely to be the adenylate cyclase catalytic subunit. This enzyme would represent close to 0.5% of the synaptosomal membrane proteins. Its low turnover number would be due to the absence of the alpha subunits of the NS regulatory proteins and would correspond to the enzymic basal level.

Adenylyl Cyclases↗
PubMed1983

Zinc ion binding to human brain calcium binding proteins, calmodulin and S100b protein.

Comparative studies have been performed on the binding properties of zinc ions to human brain calmodulin and S100b protein. Calmodulin is characterized by two sets of Zn2+ binding sites, with KD ranging from 8.10(-5)M to 3.10(-4)M. The S100b protein also exhibited two sets of zinc binding sites, with a much higher affinity. KD = 10(-7) - 10(-6)M. We suggest that S100b protein should no longer be considered only as a "calcium binding protein" but also as a "zinc binding protein", and that Zn2+ ions are involved in the functions of the S100 proteins.

Brain↗
PubMed1983

Phosphorylation and the control of calcium fluxes.

Cell activation, e.g. stimulus-contraction or stimulus-secretion coupling, is brought about by a 100-fold increase in cytosolic free Ca2+ concentration from 0.1 to 10 microM, upon release of Ca2+ from intrareticular or extracellular stores along the concentration gradient. A return to steady state is achieved by either Na+-Ca2+ exchange or ATP-dependent Ca2+ transport against the concentration gradient. Both processes, Ca2+ influx and Ca2+ efflux, are regulated by sophisticated covalent mechanisms. The positive inotropic effect of adrenalin is mediated by the cyclic-AMP-dependent phosphorylation of cardiac sarcolemmal proteins, among which calciductin is the major phosphate acceptor. Upon cyclic-AMP-dependent phosphorylation, the slow Ca2+ channel is activated 3.5 time above its basal low-conductance state, and retains its characteristics, competition by divalent metals, inhibition by La3+ and Ca2+ entry blockers. The adrenalin-induced abbreviation of systole is also explained in terms of the dual phosphorylation of the cardiac sarcoplasmic reticulum calcium pump activator, phospholamban, by cyclic-AMP-dependent protein kinase on the one hand and Ca2+-calmodulin-dependent phospholamban kinase on the other. Calciductin and phospholamban are closely similar acidic proteolipids. A phospholamban-like protein is also found in platelet Ca2+-accumulating vesicles, where its cyclic-AMP-dependent phosphorylation doubles the rate of Ca2+ efflux. These observations raise the possibility that calcium fluxes are regulated by phosphorylation of membrane-bound proteolipids. More generally, phosphorylation modulates K+, Na+ and Ca2+ fluxes through membranes, i.e. the general excitability properties of the cell.

Animals↗
PubMed1983

Ion binding to calmodulin. A comparison with other intracellular calcium-binding proteins.

Over the past few years calcium has emerged as an important bioregulator. Upon external stimulation, the cell generates a transient Ca2+ increase, which is transformed into a cellular event through a molecular cascade. The first step in this cascade is the binding of calcium to proteins present in the cytosol. These proteins capable of binding Ca2+ under physiological conditions all belong to the same evolutionary family that evolved from a common ancestor. However, they strongly differ in the properties of their calcium binding sites. Calmodulin, the ubiquitous calcium binding protein present in all eukaryotic cells, is very close to the ancestor protein, presents four calcium binding sites which bind calcium, magnesium and monovalent ions competitively and is involved in the triggering of cellular processes. Parvalbumin, another member of the family, is more specialized and found mostly in fast-twitch skeletal muscle. It binds calcium and magnesium with high affinity and seems to be involved in muscle relaxation. On the other hand, troponin C which confers Ca2+ sensitivity to acto-myosin interaction exhibits both triggering and relaxing sites. The study of intracellular Ca2+ binding proteins has shown that calcium binding proteins have evolved from a simple common structure to fulfill different functions.

Animals↗
PubMed1981

Effects of cations on affinity of calmodulin for calcium: ordered binding of calcium ions allows the specific activation of calmodulin-stimulated enzymes.

The acid stability of calmodulin has been used to devise a rapid and efficient method of decalcification based on trichloroacetic acid precipitation. Study of the competitive binding of K+, Mg2+, and Ca2+ to the Ca2+-binding sites of calmodulin has allowed determination of the intrinsic binding constants of each of the three cations for the four Ca2+-binding sites. The data are compatible with an ordered binding of Ca2+. If the Ca2+ sites are labeled A, B, C, and D starting at the NH2 terminus, the order of binding is postulated to be B, A, C, and D. The ordered binding properties support the suggestion that calmodulin translates quantitative Ca2+ signals into qualitatively different cellular responses.

Binding Sites↗
PubMed1980

Xenopus laevis oocyte calmodulin in the process of meiotic maturation.

Calcium ions are postulated to be involved in the process of meiotic maturation of amphibian oocytes. Since several of the calcium effects in eukaryotic cells are mediated by calmodulin, the present study was undertaken to assess the presence and level of calmodulin in Xenopus laevis oocytes before and after progesterone treatment. Calmodulin was shown to be present at a concentration of approximately microM in control oocytes cytosol. This level remained stable for 2 h when the maturation promoting factor appeared, and increased to approximately 44 to 59 microM at the time of the germinal vesicle breakdown. Maturation is therefore associated with calmodulin synthesis. Xenopus calmodulin was isolated from oocyte cytosol after heat treatment, anion exchange chromatography, and gel filtration, with a yield of approximately 23%. When compared to mammalian calmodulins, the amphibian protein exhibited the same ultraviolet absorption spectrum, a similar amino acid composition with 1 residue of trimethyllsine, and the same shape conformers in the absence or presence of divalent metals, as shown by different mobilities upon dodecyl sulfate-polyacrylamide gel electrophoresis. The protein migrated faster in the presence than in the absence of Ca2+ ions, Mn2+ and Mg2+ being less effective. It was able to activate calmodulin-deficient myosin light chain kinase. Its high serine content and the tryptic peptide maps obtained after high performance liquid chromatography point, however, to minor differences in the primary structures of mammalian and amphibian calmodulins.

Amino Acids↗