scholarly journals Interaction of human cathepsin D with the inhibitor pepstatin

1976 ◽  
Vol 155 (1) ◽  
pp. 117-125 ◽  
Author(s):  
C G Knight ◽  
A J Barrett
Keyword(s):  
Cathepsin D ◽  
Specific Activity ◽  
Dissociation Constants ◽  
Equilibrium Dialysis ◽  
Tight Binding ◽  
Extinction Coefficients ◽  
Titration Curves ◽  
Human Cathepsin

1. Because of the proposed role of cathepsin D in a variety of biological and pathological processes, the characteristics of inhibition by the potentially useful agent, pepstatin, were determined. 2. The β and γ forms of human cathepsin D, separated by isoelectric focusing, have identical specific extinction coefficients and specific activity in the degradation of haemoglobin. 3. Cathepsin D showed tight binding of 1 mol of pepstatin per 43000 g of protein, indicating that titration with the inhibitor represents a useful method for determination of absolute concentrations of the enzyme. 4. The titration curves were used to determine apparent dissociation constants (KD) for the binding of pepstatin and pepstatin methyl ester at pH3.5; values of approx. 5 } 10(-10)M were obtained. 5. Pepstatinyl-[3H]glycine was synthesized and shown to have a KD similar to that of pepstatin. Gel-chromatographic experiments showed that the binding of pepstatin and its derivatives is strongly pH-dependent. 6. The effect of pH on the KD for pepstatinyl-glycine was determined by equilibrium dialysis. As the pH was raised from 5.0 to 6.4, KD rose from 5 } 10(-10)M to 2 } 10(-6)M. 7. The catalytic activity of cathepsin D declines essentially to zero on going from pH5.0 to pH7.0, and we suggest that the binding site for substrate and pepstatin is abolished by a conformational change in the enzyme molecule. 8. The data indicate that, in biological experiments near neutral pH, large molar excesses of pepstatin over cathepsin D will be required for efficient inhibition.

scholarly journals Role of thiols in degradation of proteins by cathepsins

1982 ◽  
Vol 204 (2) ◽  
pp. 471-477 ◽  
Author(s):  
T Kooistra ◽  
P C Millard ◽  
J B Lloyd
Keyword(s):  
Cathepsin D ◽  
Limited Proteolysis ◽  
Lysosomal Storage ◽  
Protein Substrates ◽  
Disulphide Bonds ◽  
Protection Method ◽  
Opening Up ◽  
Lysosome Membrane

The effects of thiols on the breakdown of 125I-labelled insulin, albumin and formaldehyde-treated albumin by highly purified rat liver cathepsins B, D, H and L at pH 4.0 and 5.5 were studied. At both pH values degradation was strongly activated by the thiols cysteamine, cysteine, dithiothreitol, glutathione and 2-mercaptoethanol, and its rate increased with increasing thiol concentration. Preincubation of the protein substrates with 5 mM-glutathione did not affect concentration. Preincubation of the protein substrates with 5 mM-glutathione did not affect the rate of degradation by cathepsin D or L, and determination of free thiol groups after incubation of the proteins in the presence of glutathione but without cathepsin showed that their disulphide bonds were stable under the incubation conditions. Sephadex G-75 chromatography of the acid-soluble products of insulin digestion by cathepsin D or L suggested that thiols can reduce disulphide bonds in proteins after limited proteolysis. The resultant opening-up of the protein structure would lead to further proteolysis, so that the two processes (proteolysis and reduction) may act synergistically. By using the osmotic protection method it was shown that, at a physiological pH, cysteamine, and its oxidized form cystamine, can cross the lysosome membrane and thus may well be the physiological hydrogen donor for the reduction of disulphides in lysosomes. The results are discussed in relation to the lysosomal storage disease cystinosis.

Determination of acids and their strength in isobutyl methyl ketone

1982 ◽  
Vol 47 (4) ◽  
pp. 1203-1215 ◽  
Author(s):  
Vladimír Dostál ◽  
Zdeněk Stránský ◽  
Jan Slouka
Keyword(s):  
Potentiometric Titration ◽  
Malonic Acid ◽  
Methyl Ketone ◽  
Dissociation Constants ◽  
Weak Acids ◽  
Titration Curves ◽  
Derivatives Of

The dissociation constants of nitrophenoxazines in isobutyl methyl ketone (MIBK) were determined and correlated with the HNP values in acetone. Of the derivatives studied, the strongest acid is 1,3,7-trinitrophenoxazine (pK = 19.8), the weakest, 1-nitrophenoxazine (pK > 26). The compounds have no tendency to homo- or heteroconjugation, and were used as indicators in determination of weak acids in MIBK. Some derivatives of malonic acid and of 1,2,4-triazine as well as the intermediates used in their synthesis were determined; their HNP and pK values were established. The shape of the potentiometric titration curves can be of assistance in solving some structure problems.

Alterations and Role of Human Cathepsin D in Cancer Metastasis

1996 ◽  
Vol 49 (1-3) ◽  
pp. 106-116 ◽  
Author(s):  
Henri Rochefort ◽  
Emmanuelle Liaudet ◽  
Marcel Garcia
Keyword(s):  
Cathepsin D ◽  
Cancer Metastasis ◽  
Human Cathepsin

Distribution of hexokinase and phosphoenolpyruvate carboxykinase along the rabbit nephron

1981 ◽  
Vol 240 (6) ◽  
pp. F492-F500 ◽  
Author(s):  
A. Vandewalle ◽  
G. Wirthensohn ◽  
H. G. Heidrich ◽  
W. G. Guder
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Specific Activity ◽  
Dominant Role ◽  
Distal Tubule ◽  
Fresh Tissue ◽  
Pars Recta ◽  
Proximal Tubular ◽  
Gluconeogenic Enzyme

Radiochemical microprocedures were developed for the determination of hexokinase and phosphoenolpyruvate carboxykinase (PEPCK) activity in single microdissected segments of the mature rabbit nephron dissected from fresh tissue after collagenase treatment. All results were related to tubular length and tubular protein content. Hexokinase activity was found to be lowest in the proximal convoluted tubule and to increase along the following nephron segments, with highest activity in the connecting tubule. The gluconeogenic enzyme PEPCK, on the other hand, was exclusively found in the proximal tubule. Early and late portions of the convoluted segment exhibited the same specific activity, but only 50% was found in the pars recta. All other renal structures exhibited only insignificant activity of PEPCK. The results show that renal glucose metabolism and gluconeogenesis are clearly separated. As previously shown for the cytosolic rat enzyme, rabbit mitochondrial PEPCK is also exclusively a proximal tubular enzyme, thus confirming the dominant role of this segment in mammalian renal gluconeogenesis. The high activity of hexokinase in the segments of the distal tubule points to the role of glucose as metabolic fuel, glycogen precursor, and other glucose-6-phosphate-using pathways in these structures.

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