alpha-chymotrypsin and fructose-1-6-diphosphate

Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of four alpha- and four beta-subunits, each of them carrying catalytic and regulatory bindings sites for MgATP. In this paper, various photoaffinity labels, such as 8-azidoadenosine 5'-triphosphate, 8-azido-1,N6-ethenoadenosine 5'-triphosphate, and 8-N3-3'(2')-O-biotinyl-8-azidoadenosine 5'-triphosphate have been used to study their interaction with the enzyme in the dark and during irradiation. All nucleotidetriphosphates function as phosphate donor forming fructose 1,6-bisphosphate from fructose 6-phosphate. However, the kinetic analysis revealed distinctly differences between them. Photolabeling causes a decrease in enzyme activity to a similar extent, and ATP acts as competitive effector to inactivation. Three bifunctional diazidodiadeninedinucleotides (8-diN3AP4A, monoepsilon-8-diN3AP4A, and diepsilon-8-diN3AP4A) were applied for studying the spatial arrangement of the nucleotide binding sites. No cross-linking of the subunits was obtained by irradiation of the enzyme with 8-diN3AP4A. Photolabeling with diepsilon-8-diN3AP4A resulted in the formation of two alpha-beta cross-links with different mobilities in the SDS-polyacrylamide gel electrophoresis, while monoepsilon-8-diN3AP4A yielded only one alpha-beta cross-link. Because an interfacial location of the catalytic sites between two subunits is less likely, we suggest that the formation of cross-linked subunits may be the result of specific interactions of the bifunctional photolabels with regulatory sites at the interface of both subunits.

Topics: Adenosine Triphosphate; Binding Sites; Blotting, Western; Chymotrypsin; Cross-Linking Reagents; Darkness; Electrophoresis, Polyacrylamide Gel; Fructosediphosphates; Fructosephosphates; Kinetics; Light; Magnesium; Phosphofructokinase-1; Photoaffinity Labels; Protein Subunits; Saccharomyces cerevisiae

Cathepsin L was capable of destroying rabbit muscle aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) activity towards the substrate fructose 1,6-bisphosphate. The rate of loss of activity towards this substrate was stimulated (approx. 2-fold) by physiological concentrations of ATP and to a lesser degree by GTP, CTP, UTP, ADP and cyclic AMP, while PPi and Pi decreased the rate of inactivation. Other proteinases (cathepsin B, cathepsin D, trypsin and chymotrypsin) also decreased aldolase activity toward fructose 1,6-bisphosphate more rapidly in the presence of ATP and more slowly in the presence of Pi. Cathepsin L, at higher concentrations, was capable of inactivating aldolase activity towards fructose 1-phosphate and extensively degrading the enzyme; these reactions were not affected by ATP and Pi. The thermostability of aldolase was also unaffected by these ligands. ATP and Pi had no effect on the rates of hydrolysis of other proteins (hemoglobin, bovine serum albumin, casein and azocasein) by cathepsin L. These data indicate that the effects of ATP and Pi are due to interactions of these ligands with aldolase that make the enzyme more vulnerable to limited but not extensive proteolysis; these ligands do not directly affect cathepsin L activity.

Topics: Adenosine Triphosphate; Animals; Cathepsin D; Cathepsin L; Cathepsins; Chymotrypsin; Cysteine Endopeptidases; Endopeptidases; Fructose-Bisphosphate Aldolase; Fructosediphosphates; Humans; Liver; Muscles; Myocardium; Rabbits; Rats