ABSTRACT
Cathepsins B and L are thought to function extracellularly in pathological conditions. pH-Activity profiles of cathepsin B, measured in phosphate and acetate-Mes-Tris buffers of constant ionic strength, indicated that cathepsin B is sensitive to specific buffer ions, as previously reported for cathepsin L. In assessing the activity of these enzymes in vitro the influence of the buffer must therefore be taken into account. In Hank's balanced salt solution, a buffer modeling the extracellular fluid, the half-life of activated human liver cathepsin B at 37 degrees C is 245 +/- 11.3 s, at pH 7.2, and 857 +/- 50.1 s, at pH 6.8 (the peritumor pH), indicating that cathepsin B is markedly stable under these conditions. The stability was increased by the additional presence of proteins. Without immediate activation, however, the stabilities of both cathepsins B and L were markedly decreased, a large proportion of their activity being lost before it could be measured. Enzymes injected into the extracellular space in the unactivated state would therefore survive for only a very short time in their native conformation. It is proposed that the active site thiolate-imidazolium ion pair contributes substantially to the stability of cathepsins B and L to extracellular ionic conditions.
Subject(s)
Cathepsin B/metabolism , Cathepsins/metabolism , Cysteine Endopeptidases/metabolism , Endopeptidases , Extracellular Space/enzymology , Buffers , Cathepsin B/chemistry , Cathepsin L , Cathepsins/chemistry , Cysteine Endopeptidases/chemistry , Enzyme Activation/physiology , Half-Life , Humans , Hydrogen-Ion Concentration , Liver/enzymology , Protein ConformationABSTRACT
The activity of cathepsin L is affected by ionic strength, resulting in the measured pH optimum being higher in acetate-4-morpholineethane sulfonic acid (MES)-Tris buffers of constant ionic strength than in phosphate buffers of constant molarity (and hence varying ionic strength). In acetate-MES-Tris and phosphate buffers of constant ionic strength across the pH range, the catalytic constant, kcat, generally peaked at ca. pH 6.5 and essentially independently of ionic strength. Km values, of ca. 5 microM, manifested a slight rising trend with increasing ionic strength, with a sharp increase to 20-25 microM, specifically at pH 6.5 and I = 0.4. At physiological ionic strengths, the specific buffer ions present affected the activity of mature cathepsin L, kcat/Km declining above pH 6.5 in phosphate buffer, but only above pH 7 in acetate-MES-Tris buffer. In Hanks' balanced salt solution, a model of the extracellular fluid, measured values at pH 7.2 were kcat, 18.9 s-1; Km, 13.5 microM; and kcat/Km, 1.4 x 10(6) M-1 s-1. The stability of cathepsin L in the physiological pH range was also differentially affected by the specific buffer ions, generally in parallel with the enzyme activity. In Hanks' balanced salt solution, mature cathepsin L was substantially active and stable, having a half-life of 179 s at pH 7.2 and 657 s at pH 6.8 (the peritumor pH).
Subject(s)
Cathepsins/metabolism , Endopeptidases , Extracellular Space , Acetates , Alkanesulfonic Acids , Buffers , Cathepsin L , Cysteine Endopeptidases , Enzyme Stability , Humans , Hydrogen-Ion Concentration , Isotonic Solutions , Kidney/enzymology , Morpholines , Osmolar Concentration , Phosphates , TromethamineABSTRACT
A method has been developed for the preparation of protoplasts from both the leaves and tubers of Callilepis laureola, a plant used extensively as a medicament by black people in South Africa. The cellular vacuoles from these protoplasts were isolated and tested for the presence of the nephrotoxic substance, atractyloside, by thin layer chromatography and immunoassay. Both methods indicate that the vacuole of C. laureola is the primary site of storage for atractyloside in the cells of the tuber.
