scholarly journals Isolation of palmitoyl-CoA hydrolases from human blood platelets

1981 ◽  
Vol 199 (3) ◽  
pp. 639-647 ◽  
Author(s):  
R K Berge ◽  
L E Hagen ◽  
M Farstad
Keyword(s):  
Human Blood ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Cytosol Fraction ◽  
Molecular Weights ◽  
Apparent Homogeneity ◽  
Coa Hydrolase ◽  
Human Blood Platelets

The palmitoyl-CoA hydrolase activity, which in human blood platelets is mainly localized in the cytosol fraction [Berge, Vollset & Farstad (1980) Scand. J. Clin. Lab. Invest. 40, 271--279], was found to be extremely labile. Inclusion of glycerol or palmitoyl-CoA stabilized the activity during preparation. Gel-filtration studies revealed multiple forms of the enzyme with molecular weights corresponding to about 70 000, 40 000 and 24 000. The relative recovery of the mol.wt.-70 000 form was increased by the presence of 20% (v/v) glycerol or 10 microM-palmitoyl-CoA. The three enzyme forms are probably unrelated, since they were not interconvertible. The three different species of palmitoyl-CoA hydrolase were purified by DEAE-cellulose and hydroxyapatite chromatography, isoelectric focusing and high-pressure liquid chromatography (h.p.l.c.) to apparent homogeneity. The three enzymes had isoelectric points (pI) of 7.0, 6.1 and 4.9. The corresponding molecular weights were 27 000--33 000, 66 000--72 000 and 45 000--49 000, calculated from h.p.l.c. and Ultrogel AcA-44 chromatography. The apparently purified enzymes were unstable, as most of the activity was lost during purification. The enzyme with an apparent molecular weight of 45 000--49 000 was split into fractions with molecular weights of less than 10 000 by re-chromatography on h.p.l.c. concomitantly with a loss of activity. The stimulation of the activity by the presence of serum albumin seems to depend on the availability of palmitoyl-CoA, as has been reported for other palmitoyl-CoA hydrolases. [Berge & Farstad (1979) Eur. J. Biochem. 96, 393--401].

Chromatographic Isolation of the LDH-Isoenzymes of Human Blood Platelets and an Investigation of Their Enzyme Kinetics

1968 ◽  
Vol 20 (03/04) ◽  
pp. 301-313 ◽  
Author(s):  
W Schneider ◽  
K Schumacher ◽  
B Thiede ◽  
R Gross
Keyword(s):  
Human Blood ◽  
Blood Platelets ◽  
Deae Cellulose ◽  
Order Reaction ◽  
Kinetic Properties ◽  
Ldh Isoenzymes ◽  
Kinetic Investigations ◽  
Substrate Concentrations ◽  
Chromatographic Isolation ◽  
Human Blood Platelets

SummaryThe LDH-isoenzymes of human blood platelets show a distinct predominance of the isoenzymes 2 and 3 upon chromatography on DEAE-cellulose. Small amounts of LDH-1 are also present, while only traces of LDH-4 and -5 can be detected.Enzyme kinetic investigations of the principal isoenzymes LDH-1, -2 and -3 clearly show that the differences in inhibition constants with pyruvate as substrate which are demonstrable at 25° largely disappear at 37°. On the other hand, the differences among the isoenzymes in their affinity for pyruvate and lactate as substrate as well as in with respect to the optimal substrate concentrations of pyruvate are more marked at 37° than at 25°. Also, the type of inhibition found with lactate as substrate is increasingly the expression of a higher order reaction in going from LDH-1 to LDH-3. A dependence of the LDH distribution pattern upon the metabolism of the cell is discussed. A comparison of our results with thrombocytes with those of other workers with erythrocytes and leucocytes makes it unlikely that the LDH pattern is directly dependent upon the existence of an oxidative metabolism. Rather, the redox potential of the cell could be of importance for the nature of the pattern of isoenzymes and for their differing kinetic properties.

The Effect of Phospholipase C on Platelet Factor 3 in Human Blood Platelets

1977 ◽  
Vol 37 (01) ◽  
pp. 029-035 ◽  
Author(s):  
A-B Otnaess ◽  
H Prydz
Keyword(s):  
Phospholipase C ◽  
Human Blood ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Human Platelets ◽  
Small Reduction ◽  
Platelet Factor ◽  
Human Blood Platelets ◽  
External Attack

SummaryIntact human platelets isolated by gel filtration have been treated with purified phospholipase C. The effect of the enzyme on available and total platelet factor 3 has been tested.The available procoagulant platelet factor 3 was very low. A further small reduction was observed after incubation with phospholipase C when the enzyme was washed away before testing.External attack on platelets by phospholipase C led to a marked inactivation of total platelet factor 3.

Interactions of human blood platelets with three circulating plasma fibrinogens of different molecular weights

1987 ◽  
Vol 47 (6) ◽  
pp. 683-692 ◽  
Author(s):  
L.I. Thorsen ◽  
B. Holm ◽  
F. Brosstad ◽  
N.O. Solum Research
Keyword(s):  
Human Blood ◽  
Blood Platelets ◽  
Molecular Weights ◽  
Human Blood Platelets

scholarly journals Purification and properties of four species of lysyl oxidase from bovine aorta

1979 ◽  
Vol 177 (1) ◽  
pp. 203-214 ◽  
Author(s):  
Herbert M. Kagan ◽  
Kathleen A. Sullivan ◽  
Theodore A. Olsson ◽  
Anne L. Cronlund
Keyword(s):  
Metal Ion ◽  
Lysyl Oxidase ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Active Species ◽  
Molecular Weights ◽  
Subunit Molecular Weight ◽  
Apparent Homogeneity ◽  
Multiple Species

Lysyl oxidase of bovine aorta was resolved into four enzymically active species by elution from DEAE-cellulose with a salt gradient in 6m-urea, consistent with purification results obtained with enzyme of other tissues [Stassen (1976) Biochim. Biophys. Acta438, 49–60]. In the present study, each of the four peaks of activity was purified to apparent homogeneity by subsequent chromatography on gel-filtration media in 6m-urea. Each enzyme is eluted as a species with mol.wt. approx. 30000 under these conditions, although lysyl oxidase polymerizes to a series of multimers with molecular weights ranging up to 1000000 in the absence of urea. The apparent subunit molecular weight of each enzyme species determined by electrophoresis in sodium dodecyl sulphate and 8m-urea is approx. 32000–33000. The amino acid compositions of the purified forms of lysyl oxidase are similar to each other, although sufficient differences exist to conclude that each is a unique molecular species. Incorporation of α-toluenesulphonyl fluoride into the purification scheme does not alter the resolution of enzyme into four species, suggesting that proteolysis during isolation is not the basis of the heterogeneity. The similar sensitivities of each form of enzyme to chelating agents and to semicarbazide and isoniazid indicate that each requires the participation of a metal ion, presumably Cu2+, and of a carbonyl compound for enzyme function. The present study describes a method for the purification of multiple species of lysyl oxidase and reveals that significant chemical differences exist between the different enzyme forms.

On the Mechanism by which Cell Contact Induces the Release Reaction of Blood Platelets; the Effect of Cationic Polymers

1972 ◽  
Vol 27 (01) ◽  
pp. 121-133 ◽  
Author(s):  
P Massini ◽  
E. F Lüscher
Keyword(s):  
Human Blood ◽  
Calcium Ions ◽  
Blood Platelets ◽  
Cell Contact ◽  
Second Phase ◽  
Cationic Polymers ◽  
Release Reaction ◽  
Per Se ◽  
Human Blood Platelets

SummaryHuman blood platelets are aggregated by the basic polymers polylysine and DEAE- dextran. Under certain conditions a second phase of aggregation, concomitant with the release reaction, is elicited. The presence of ADP, calcium ions and a plasmatic cofactor within the primary aggregates are necessary for the induction of the release reaction. These experiments demonstrate that cell contact per se does not lead to a release reaction ; in order to become effective it must take place in the presence of ADP.

Interaction of Vasopressin with Human Blood Platelets: Dependency on Mg2+

1986 ◽  
Vol 56 (03) ◽  
pp. 260-262 ◽  
Author(s):  
Isabella Roos ◽  
Fabrizia Ferracin ◽  
Alfred Pletscher
Keyword(s):  
Phosphatidic Acid ◽  
Human Blood ◽  
Arginine Vasopressin ◽  
Specific Binding ◽  
Blood Platelets ◽  
Bivalent Cations ◽  
Platelet Membranes ◽  
Maximal Rise ◽  
Human Blood Platelets ◽  
Stimulation Of

SummaryArginine-vasopressin (AVP) in the presence of Mg2+ but not in the absence of bivalent cations led to accumulation of [32P]-phosphatidic acid ([32P]-PA) in human blood platelets. Mg2+ also enhanced the specific binding of [3H]-AVP to intact platelets. The concentrations of the cation which enabled AVP to cause half maximal rise of [32P]-PA and those inducing half maximal [3H]-AVP-binding were of the same order. It is concluded that the stimulation of phosphatidyl inositide breakdown by AVP in presence of Mg2+ is at least partially due to a Mg2+-induced enhancement of specific AVP-binding to the platelet membranes.

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