Thermal Denaturation Kinetics of Yeast Proteins in Whole Cells of Saccharomyces cerevisiae and Kluyveromyces fragilis

SummaryHeat inactivation of thrombin at 54° C followed first order kinetics with a rate constant of 1.0 min−1 approximately. Addition of heparin resulted in protection against thermal denaturation and, at the same time, rendered denaturation kinetics more complex. Analysis of the biphasic curve of heat inactivation in the presence of heparin revealed that the rate constants of the second phase changed systematically with heparin concentrations. Namely, at 4.5 × 10−6M, 9 × 10−6M, 1.8 × 10−5M and 3.6 × 10−5M heparin concentrations, the rate constants were 0.27 min−1, 0.17 min−1, 0.11 min−1 and 0.06 min−1, respectively.Sulfate as well as phosphate ions displayed also enzyme protection against heat inactivation, however, the same effect was obtained already at a heparin concentration, lower by three orders of magnitude.The kinetics of enzyme denaturation was not affected by calcium ions, whereas in the presence of heparin the inactivation rate of thrombin changed, i. e. calcium ions abolished the biphasic character of time course of thermal denaturation.Thus, the data suggest that calcium ions contribute to the effect of heparin on thrombin.

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Functional Fractionation of Platelets: Aggregation Kinetics and Glycoprotein Labeling of Differing Platelet Populations

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657259 ◽

1982 ◽

Vol 48 (02) ◽

pp. 211-216 ◽

Cited By ~ 12

Author(s):

V M Haver ◽

A R L Gear

Keyword(s):

Galactose Oxidase ◽

Aggregation Kinetics ◽

Maximal Rate ◽

Membrane Glycoproteins ◽

Whole Cells ◽

Reversible Aggregation ◽

Significant Difference ◽

Small Doses ◽

Major Loss ◽

Kinetics Of

SummaryPlatelet heterogeneity has been studied with a technique called functional fractionation which employs gentle centrifugation to yield subpopulations (“reactive” and “less-reactive” platelets) after exposure to small doses of aggregating agent. Aggregation kinetics of the different platelet populations were investigated by quenched-flow aggregometry. The large, “reactive” platelets were more sensitive to ADP (Ka = 1.74 μM) than the smaller “less-reactive” platelets (Ka = 4.08 μM). However, their maximal rate of aggregation (Vmax, % of platelets aggregating per sec) of 23.3 was significantly lower than the “less-reactive” platelets (Vmax = 34.7). The “reactive” platelets had a 2.2 fold higher level of cyclic AMP.Platelet glycoproteins were labeled using the neuraminidase-galactose oxidase – [H3]-NaBH4 technique. When platelets were labeled after reversible aggregation, the “reactive” platelets showed a two-fold decrease in labeling efficiency (versus control platelets). However, examination of whole cells or membrane preparations from reversibly aggregated platelets revealed no significant difference in Coomassie or PAS (Schiff) staining.These results suggest that the large, “reactive” platelets are more sensitive to ADP but are not hyperaggregable in a kinetic sense. Reversible aggregation may cause a re-orientation of membrane glycoproteins that is apparently not characterized by a major loss of glycoprotein material.

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