Creatine kinase. Relations of trypsin susceptibility to substrate binding

By using sodium dodecyl sulphage/polyacrylamide-gel electrophoresis it was shown that rabbit muscle creatine kinase, both in a homogenate and purified, appears to be composed of a mixture of two peptides (mol.wts. 42100 and 40300) differing in length by about 15 amino acids. It is found that low concentrations of proteinase K from the fungus Tritirachium album can cleave about 38 amino acids from each chain of creatine kinase, leaving two large fragments (mol.wts 37700 and 35500). Scission of the whole enzyme was found to be concomitant with complete loss of enzyme activity. MgADP in the presence of absence of creatine slowed the rate of proteolysis by about 50%, but the transition-state analogue complex creatine-NO3--MgADP appeared to protect completely. The time course for the proteolytic inactivation in the presence of this complex, but not in its absence, was biphasic.

mentioning

confidence: 99%

Heterogeneity of rabbit muscle creatine kinase and limited proteolysis by proteinase K

Williamson¹,

Greene²,

Cherif³

et al. 1977

“…When digestion was allowed to proceed beyond 30 min, the 20000-Mr band gradually disappeared as it was digested to form small peptides. Whatever the complex kinetic explanation behind the biphasic digestion rate [which was also observed by Jacobs & Cunningham (1968)], the linear portion is clearly a function of, and related to, the NAD+ concentration.…”

Section: Protective Effect Of Nad+ On Tryptic Digestionmentioning

confidence: 60%

“…Substrate protection from proteolysis has been reported for other ADP-ribosylating toxins (Kandel et al, 1974;Tweten et al, 1985) and for other unrelated proteins (e.g. Jacobs & Cunningham, 1968;Trayser & Colowick, 1961), and has been exploited successfully in the calculation of binding constants.…”

Section: Digestion With Trypsinmentioning

confidence: 99%

“…A method based on using changes in the rate of tryptic digestion at different ligand concentrations was used successfully by Jacobs & Cunningham (1968) to calculate binding constants for the interaction of creatine kinase with its substrates. Jacobs & Cunningham (1968) used a pH-stat to measure the rate of digestion of creatine kinase.…”

Section: Protective Effect Of Nad+ On Tryptic Digestionmentioning

confidence: 99%

See 1 more Smart Citation

Binding of NAD+ by cholera toxin

Galloway

Heyningen

1987

1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.

“…The binding of substrates or substrate analogues to an enzyme is often accompanied by a readily detectable change in its susceptibility to proteinase digestion and thermal inactivation (Zyk et al, 1969;Adelman et al, 1968;Jacobs & Cunningham, 1968;Rupley, 1967;Lui & Cunningham, 1966;Trayser & Colowick, 1961). It has been proposed by Zyk et al (1969) that the binding of such ligands may cause a change in the conformation of the enzyme structure that is reflected in stabilization towards proteolysis and thermal inactivation, and the term 'conformative response' has been suggested by Citri & Zyk (1967) to describe conformational changes that accompany specific ligand binding.…”

mentioning

confidence: 99%

Rabbit erythrocyte purine nucleoside phosphorylase. Differential-inactivation studies

Savage¹,

Sṕencer²

1979

1. Qualitative studies on the stability of rabbit erythrocyte purine nucleoside phosphorylase showed a marked decrease in the susceptibility of the enzyme to thermal inactivation and digestion by proteinases of different specificities in response to certain of its substrates. 2. The extent to which inosine stabilizes the enzyme against thermal and proteolytic inactivation is related in a quantitative manner to the concentration of this substrate; it is proposed that differences in the rates of inactivation of the enzyme may reflect substrate-induced conformational changes in the enzyme structure that could alter the binding properties of the enzyme in a kinetically significant way. 3. A synergistic effect in the stabilization of the enzyme is observed in response to both substrates, inosine and phosphate, when the enzyme is inactivated with Pronase. 4. In the presence of substrate an increased rate of inactivation after reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) is reported. 5. Differential-inactivation studies were also carried out with calf spleen purine nucleoside phosphorylase, and the results are discussed in relation to the kinetic properties displayed by this enzyme.