scholarly journals Equilibrium and kinetic studies of oxygen binding to fragments of Lymnaea stagnalis (freshwater snail) haemocyanin obtained by proteolytic digestion

1983 ◽  
Vol 209 (2) ◽  
pp. 519-526 ◽  
Author(s):  
A Dawson ◽  
E J Wood
Keyword(s):  
Lymnaea Stagnalis ◽  
Functional Unit ◽  
Polyacrylamide Gel Electrophoresis ◽  
Kinetic Studies ◽  
Freshwater Snail ◽  
Dissociation Rate ◽  
Exchange Chromatography ◽  
Hill Coefficient ◽  
Oxygen Binding ◽  
O2 Binding

Functional fragments of the haemocyanin from the gastropod mollusc Lymnaea stagnalis (freshwater snail) were obtained by partial digestion with trypsin and plasmin. The fragments were purified by ion-exchange chromatography and characterized by detergent/polyacrylamide-gel electrophoresis and crossed immunoelectrophoresis. Three types of single-functional unit fragment were isolated from the trypsin digest, and two immunologically distinct three-functional unit fragments and a single-functional unit fragment were isolated from the plasmin digest. The O2-binding behaviour of the fragments was investigated by equilibrium and kinetic methods. Over the pH range 7.0-8.2, in the presence of 10-20 mM-CaCl2, all of the single-functional unit fragments displayed non-co-operative O2 binding and showed no evidence of a Bohr or a salt effect. A Hill coefficient of less than 1.0 was obtained with one of the two three-functional unit fragments studied, whereas both of these fragments displayed a Bohr effect. Functional heterogeneity of the fragments was indicated by the variation in the O2 affinity, the P50 (partial pressure of O2 at half saturation) ranging between 0.26 and 0.77 kPa (approx. 2-6 mmHg). Stopped-flow data reflected the O2 equilibrium behaviour. Thus there was a fall in the value of the O2 dissociation rate constant from approx. 15 to 1s-1 in parallel with the increase in O2 affinity.

scholarly journals Properties of antithrombin-thrombin complex formed in the presence and in the absence of heparin

1983 ◽  
Vol 213 (2) ◽  
pp. 345-353 ◽  
Author(s):  
A Danielsson ◽  
I Björk
Keyword(s):  
Rate Constants ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Dissociation Rate ◽  
Exchange Chromatography ◽  
Stable Complex ◽  
Intermediate Step ◽  
First Order ◽  
Activity Measurements ◽  
Dissociation Rate Constants

Purification of antithrombin-thrombin complex by ion-exchange chromatography on DEAE-agarose resulted in predominantly monomeric complex, whereas purification on matrix-linked heparin produced large amounts of aggregated complex. Monomeric antithrombin-thrombin complexes formed in the presence and in the absence of heparin had similar conformations and heparin affinities. Moreover, the first-order dissociation rate constants, measured by thrombin release, of these complexes were similar, 2.3 × 10(-6)-3.4 × 10(-6)S-1, regardless of whether newly formed or purified complex was analysed. Similar dissociation rate constants were also obtained for purified complex formed with or without heparin, from analyses by dodecyl sulphate/polyacrylamide-gel electrophoresis of the release of modified antithrombin, cleaved at the reactive-site bond. No dissociation of intact antithrombin from the complex was detected by activity measurements or by gel electrophoresis. Aggregation of the complex was found to be accompanied by a decrease in apparent dissociation rate. The similar properties of antithrombin-thrombin complexes formed with or without heparin support the concept of a catalytic role for the polysaccharide in the antithrombin-thrombin reaction. Furthermore, the results indicate that the reaction between enzyme and inhibitor involves the rapid formation of an irreversible, kinetically stable, complex that dissociates into active thrombin and modified, inactive, antithrombin by a first-order process with a half-life of about 3 days. The inhibition thus resembles a normal proteolytic reaction, one intermediate step of which is very slow.

scholarly journals The interleukin-1 receptor in Raji human B-lymphoma cells. Molecular characterization and evidence for receptor-mediated activation of gene expression

1989 ◽  
Vol 260 (3) ◽  
pp. 657-663 ◽  
Author(s):  
R Horuk ◽  
J A McCubrey
Keyword(s):  
Molecular Mass ◽  
Interleukin 1 ◽  
Polyacrylamide Gel Electrophoresis ◽  
Kinetic Studies ◽  
Cell Types ◽  
Biochemical Properties ◽  
Dissociation Rate ◽  
Raji Cells ◽  
B Lymphoma Cells ◽  
Sds Page

In a previous paper [Horuk, Huang, Covington & Newton (1987) J. Biol. Chem. 262, 16275-16278] we reported that there were fundamental differences in the biochemical properties of the interleukin-1 (IL-1) receptor between Raji and EL4 cell lines. In the present study we have investigated the basis for these differences. Kinetic studies measuring the on and off rates of IL-1 receptor binding revealed that the low-affinity IL-1-binding sites observed in Raji cells, compared with EL4 cells, result from a combination of a lower association rate and a higher dissociation rate in the Raji cells. The turnover of the Raji IL-1 receptor, measured by inhibiting protein synthesis with cycloheximide, was much faster than that of the EL4 IL-1 receptor, with a half-time of 2 h as against 5 h. Treatment of 125I-IL-1-labelled IL-1 receptors in Raji and EL4 cells with neuraminidase decreased their molecular mass by approx. 2-5 kDa as assessed by SDS/polyacrylamide-gel electrophoresis (PAGE). The covalently labelled IL-1 receptors in both cell types were sensitive to treatment with endoglycosidase F, which decreased their molecular mass on SDS/PAGE by 12-13 kDa. Incubation of Raji cells with maximally stimulating doses of IL-1 resulted in an increase in the nascent RNA levels of several genes, including the IL-2 receptor and the proto-oncogenes c-Ha-ras and c-myc.

scholarly journals Purification and characterization of N-acetylglutamate 5-phosphotransferase from pea (Pisum sativum) cotyledons

1981 ◽  
Vol 195 (1) ◽  
pp. 71-81 ◽  
Author(s):  
G McKay ◽  
P D Shargool
Keyword(s):  
Pisum Sativum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Kinetic Studies ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Coomassie Blue ◽  
Km Value ◽  
Acetylglutamate Kinase

N-Acetylglutamate 5-phosphotransferase (acetylglutamate kinase, EC 2.7.2.8) has been isolated from pea (Pisum sativum) cotyledons and purified 312-fold by using heat treatment, (NH4)2SO4 fractionation, affinity chromatography on ATP--Sepharose and ion-exchange chromatography on DEAE-cellulose. This preparation was shown on polyacrylamide-gel electrophoresis to yield one band staining with Coomassie Blue. The enzyme was shown by a variety of techniques to be composed of two different kinds of subunits, of mol.wts. 43000 and 53000 respectively. These subunits are arranged to give either a dimeric or tetrameric enzyme composed of equal numbers of each type of subunit. The dimeric and tetrameric enzyme forms are thought to be interconvertible, the equilibrium between these forms being influenced by the type of ligand bound to the subunits. Kinetic studies performed on the purified enzyme, indicated a random Bi Bi type of mechanism. The enzyme displayed apparent negative co-operativity with respect to one of its substrates, N-acetylglutamate; as a result, two Km values were found for this substrate, one at 1.9 X 10(-3) M and the other at 6.2 X 10(-3) M. A single Km value for ATP was found to be 1.7 X 10(-3) M. Allosteric regulation by arginine was also shown. A model, based on the Koshland, Némethy & Filmer [(1966) Biochemistry 5, 365-385] Sequential model, which adequately describes the kinetic and structural properties of N-acetylglutamate 5-phosphotransferase, is presented.

Equilibrium and kinetic studies of oxygen binding by gastropod (Lymnaea stagnalis) haemocyanin

1981 ◽  
Vol 9 (5) ◽  
pp. 446-447 ◽  
Author(s):  
ANNE DAWSON ◽  
DAVID A. BAXENDALE ◽  
EDWARD J. WOOD
Keyword(s):  
Lymnaea Stagnalis ◽  
Kinetic Studies ◽  
Oxygen Binding

scholarly journals Purification and properties of an alginate lyase from a marine bacterium

1976 ◽  
Vol 159 (3) ◽  
pp. 707-713 ◽  
Author(s):  
I W Davidson ◽  
I W Sutherland ◽  
C J Lawson
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Alginate Lyase ◽  
Kinetic Studies ◽  
Defined Medium ◽  
Exchange Chromatography ◽  
Bacterial Cells ◽  
Purification And Properties ◽  
Primary Composition

An unidentified pseudomonad isolated by enrichment procedures from decomposing seaweed was grown in defined medium containing sodium alginate as the sole carbon source. The alginate lyase recovered from disrupted bacterial cells was purified by a procedure of (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. From sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis experiments a mol.wt. of about 50 000 was determined. The enzyme was active against both algal and bacterial alginate preparations. Kinetic studies together with analysis of the unsaturated oligouronide products of alginate lyase action indicated the enzyme was specific for guluronic acid-containing regions of the macromolecular substrate. The specificity of the enzyme can be used to give information about the primary composition of alginate samples.

scholarly journals Equilibrium and kinetic studies of oxygen binding to the haemocyanin from the freshwater snail Lymnaea stagnalis

1982 ◽  
Vol 207 (1) ◽  
pp. 145-153 ◽  
Author(s):  
A Dawson ◽  
E J Wood
Keyword(s):  
Rate Constant ◽  
Lymnaea Stagnalis ◽  
Relaxation Times ◽  
Oxygen Affinity ◽  
Dissociation Rate ◽  
Dissociation Rate Constant ◽  
Oxygen Binding ◽  
Maximum Slope ◽  
Cacl2 Concentration ◽  
Ph Range

The binding of oxygen by the haemocyanin of the gastropod Lymnaea stagnalis was studied by equilibrium and kinetic methods. The studies were performed under conditions in which the haemocyanin molecule was in the native state. Over the pH range 6.8-7.6, in the presence of 10mM-CaCl2 the haemocyanin bound O2 cooperatively. Over this pH range the haemocyanin molecule displayed a normal Bohr effect whereby the O2 affinity of the molecule decreased with a fall in the pH of the solution. The maximum slope of the Hill plot (hmax.) was 3.5, obtained at pH 7.5. An increase in the CaCl2 concentration from 5 to 20 mM at pH 6.8 resulted in a slight increase in the oxygen affinity, with hmax. remaining virtually unchanged. At constant pH and CaCl2 concentration, an increase in NaCl concentration from 0 to 50 mM resulted in a small decrease in O2 affinity, but a significant increase in the value of hmax. from 3.5 to 8.6. Temperature-jump relaxation experiments over a range of O2 concentrations produced single relaxation times. The dependence of the relaxation time on the reactant concentrations indicated a simple bimolecular binding process. The calculated association and dissociation rate constants for this process at pH 7.5 are 29.5×10(6) M-1 X S-1 and 49 S-1 respectively. The association rate constant kon was found to be essentially independent of pH and CaCl2 concentration. The dissociation rate constant, koff, however, increased with a decrease in the pH, but was also independent of CaCl2 concentration. These results indicate that the stability of the haemocyanin-O2 complex is determined by the dissociation rate constant.

scholarly journals Fragmentation of a mollusc haemocyanin with plasmin and immunological identification of the fragments

1981 ◽  
Vol 197 (1) ◽  
pp. 23-29 ◽  
Author(s):  
W J Gullick ◽  
E J Head ◽  
E J Wood
Keyword(s):  
Time Course ◽  
Lymnaea Stagnalis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Pond Snail ◽  
Oxygen Binding ◽  
Experimental Conditions ◽  
Gastropod Mollusc ◽  
Molecular Weights ◽  
Binding Domains ◽  
Crossed Immunoelectrophoresis

Haemocyanin from the gastropod mollusc Lymnaea stagnalis (pond snail) was partially digested with plasmin under a variety of experimental conditions and the products of digestion analysed by detergent/polyacrylamide-gel electrophoresis and crossed immunoelectrophoresis. Fragments were obtained corresponding to one, two, three, four and five oxygen-binding domains, one domain having a mol.wt. of approx. 50000 and containing 2 ions of Cu. The fragments obtained after extensive digestion were non-identical immunologically, and summation of their molecular weights allowed a minimal mol.wt. of 413000 to be calculated for the original, undigested, eight-domain polypeptide chain. The use of mild-digestion conditions allowed the time course and sequence of the digestion to be monitored. An initial cleavage gave a three-domain and a five-domain fragment. The three-domain fragment was resistant to further digestion. The five-domain fragment could be digested further to give, successively a four-domain, a three-domain, and finally a two-domain fragment, single-domain units being cleaved. These data form the basis for a proposed sequence for the different domains in the original chain.

Responses to Hypersaline Exposure in the Euryhaline Crayfish PAcifastacus Leniusculus: II. Modulation of Haemocyanin Oxygen Binding In Vitro and In Vivo

1982 ◽  
Vol 99 (1) ◽  
pp. 447-467
Author(s):  
MICHÈLE G. WHEATLY ◽  
B. R. MCMAHON
Keyword(s):  
Salt Effect ◽  
Ionic Composition ◽  
Inorganic Ions ◽  
Pacifastacus Leniusculus ◽  
Oxygen Binding ◽  
Complete Saturation ◽  
O2 Delivery ◽  
O2 Binding

The effect of 48 h of hypersaline exposure (25, 50 and 75% SW) on haemocyanin oxygenation properties in the euryhaline crayfish Pacifastacus leniusculus was investigated in vitro and in vivo. In vitro significant increases in affinity and cooperativity were measured, although the magnitude of the Bohr shift was unaffected. In vitro dialysis of haemolymph against physiological salines of variable ionic composition proved that these changes were only partly attributable to altered levels of haemolymph ions, implicating the existence of modulators other than H+ and inorganic ions, the possible identities of which are discussed. Significant depressions of both pre- and postbranchial oxygen tensions (Pv, Ov, O2 and Pa, Oa, O2) were observed, but O2 delivery was maintained by utilization of the venous reserve and by an increase in haemocyanin O2 affinity. This occurred despite a concomitant acidosis whose effect on O2 affinity was directly opposed by the ‘salt’ effect. Under hypersaline conditions, haemocyanin played an increasingly important role in O2 delivery in vivo. Despite a reduction in the concentration of combined O2 at complete saturation of the pigment (CmaxHCyOHCyO2). indicating lowered haemocyanin concentration, compensatory changes in O2-binding and cardiac output precluded an impairment to O2 transfer. Equilibration at the tissues (Et,Ot,O2) in FW was less effective than at the gills (Eb,Ob,O2 but progressively improved with hypersaline exposure reversing this trend. Although effects of increased salinity on O2 equilibrium characteristics were qualitatively similar in vivo and in vitro, some interesting quantitative differences are discussed.

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