scholarly journals Bimane-labelled pepstatin, a fluorescent probe for the subcellular location of cathepsin D

1981 ◽  
Vol 199 (3) ◽  
pp. 611-617 ◽  
Author(s):  
I T W Matthews ◽  
R S Decker ◽  
C G Knight
Keyword(s):  
Fluorescent Probe ◽  
Cathepsin D ◽  
Tight Binding ◽  
Subcellular Location ◽  
Active Conformation ◽  
Apparent Dissociation Constant ◽  
Titration Curves ◽  
Kd Values ◽  
Pepstatin A ◽  
Dissociation Constant Kd

1. Pepstatinyl-cystamine was synthesized. The disulphide bond was cleaved and the pepstatin-bound thiol was made to react with monobromobimane. The fluorescent N-pepstatinyl-S-bimanyl-2-aminoethanethiol was purified. 2. Human cathepsin D showed tight binding of the bimane-labelled pepstatin at pH 3.5. The titration curves were used to determine the apparent dissociation constant, KD; values of approx. 1 x 10(-10) M were obtained. 3. Gel-chromatographic experiments showed that, like that of pepstatin, the binding of N-pepstatinyl-S-bimanyl-1-aminoethanethiol to cathepsin D was strongly pH-dependent. Binding was seen at pH 5.0, but could not be demonstrated at pH 7.4. 4. Cultured human synovial cells were fixed and incubated with the fluorescent inhibitor at pH 5.0 or pH 7.4. When examined by fluorescence microscopy the cells stained at pH 5.0 showed a punctate perinuclear distribution of bimane fluorescence. By contrast, the cells stained at pH 7.4 showed no fluorescence. 5. The distribution of cathepsin D, determined by indirect immunofluorescence at pH 7.4, closely resembled that of the fluorescent inhibitor seen at pH 5.0. 6. We conclude that N-pepstatinyl-S-bimanyl-2-aminoethanethiol is a fluorescent probe selective for the active conformation of cathepsin D.

scholarly journals The binding of Ca2+ ions to pig heart NAD+-isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex

1989 ◽  
Vol 263 (2) ◽  
pp. 453-462 ◽  
Author(s):  
G A Rutter ◽  
R M Denton
Keyword(s):  
Dissociation Constant ◽  
Isocitrate Dehydrogenase ◽  
Binding Sites ◽  
Tight Binding ◽  
Variable Number ◽  
Apparent Dissociation Constant ◽  
Oxoglutarate Dehydrogenase ◽  
Dissociation Constant Kd ◽  
Flow Dialysis ◽  
Dehydrogenase Complex

1. The binding of Ca2+ ions to purified pig heart NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase, freed of contaminating Ca2+ by parvalbumin/polyacrylamide chromatography, has been studied by flow dialysis and by the use of fura-2. 2. For the 2-oxoglutarate dehydrogenase complex, 3.5 mol of Ca2+-binding sites/mol of complex were apparent, with an apparent dissociation constant (Kd value) for Ca2+ of 2.0 microM. These values were little affected by Mg2+ ions, ADP or 2-oxoglutarate. 3. By contrast, binding of Ca2+ to NAD+-isocitrate dehydrogenase (Kd = 14 microM) required ADP, isocitrate and Mg2+ ions. The number of Ca2+-binding sites associated with NAD+-isocitrate dehydrogenase was then 0.9 mol/mol of tetrameric enzyme. 4. The 2-oxoglutarate dehydrogenase complex bound ADP (as ADP3-) to a group of tight-binding sites (Kd = 3.1 microM) with a stoichiometry, 3.3 mol/mol of complex, similar to that for the binding of Ca2+; a variable number of much weaker sites (Kd = 100 microM) for ADP3- was also apparent.

scholarly journals Dinitrophenyl-pepstatins as active-site-directed localization reagents for cathepsin D.

1983 ◽  
Vol 211 (1) ◽  
pp. 139-147 ◽  
Author(s):  
I T W Matthews ◽  
R S Decker ◽  
W Hornebeck ◽  
C G Knight
Keyword(s):  
Fluorescence Microscopy ◽  
Active Site ◽  
Cathepsin D ◽  
Enzyme Inhibitor ◽  
Ph Value ◽  
Specific Binding ◽  
Tight Binding ◽  
Water Soluble ◽  
Synovial Cells ◽  
Active Conformation

1. N-Pepstatinyl-N'-dinitrophenyl-1,6-diaminohexane, a potential active-site-directed localization reagent for cathepsin D, was found to bind non-specifically to immuno-precipitates containing cathepsin D. 2. Three new water-soluble localization reagents were synthesized, by using NN'-bis-(3-aminopropyl)piperazine, 3-oxa-1,5-diamino-pentane or 3,6-dioxa-1,8-diamino-octane, as spacer arms between the pepstatin and dinitrophenyl moieties. 3. The hydrophilic dinitrophenyl-pepstatins were all tight-binding inhibitors of cathepsin D at pH 3.5, but showed little or no binding to immuno-precipitates containing the inactive enzyme at pH 7.4. 4. Gel-chromatographic experiments showed that, at pH 5.0, all the dinitrophenyl-pepstatins were bifunctional reagents able to bind cathepsin D and anti-dinitrophenyl antibody at the same time. Enzyme-inhibitor-antibody complexes were not formed at pH 7.4, thus confirming that the reagents were active-site-directed. 5. Cultured human synovial cells were fixed and incubated with the dinitrophenyl-pepstatins at pH 5.0 or pH 7.4. After washing briefly, the cells were incubated at the appropriate pH value with anti-dinitrophenyl antibody labelled with fluorescein. When examined by fluorescence microscopy the cells stained at pH 5.0 showed fluorescent perinuclear granules, which were not seen in the cells treated at pH 7.4. The distribution of cathepsin D, determined by indirect immuno-fluorescence at pH 7.4, closely resembled that revealed by the dinitrophenyl-pepstatins at pH 5.0. 7. NN'-(3-Pepstatinylaminopropyl-3′-dinitrophenylaminopropyl)piperazine gave the most intense lysosomal staining and showed no non-specific binding. We conclude that this reagent is suitable for the subcellular localization of the active conformation of cathepsin D.

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.

Essential role for cathepsin D in bleomycin-induced apoptosis of alveolar epithelial cells

2004 ◽  
Vol 287 (1) ◽  
pp. L46-L51 ◽  
Author(s):  
Xiaopeng Li ◽  
Heather Rayford ◽  
Ruijie Shu ◽  
Jiaju Zhuang ◽  
Bruce D. Uhal
Keyword(s):  
Enzymatic Activity ◽  
Cathepsin D ◽  
Alveolar Epithelial Cells ◽  
Type Ii ◽  
Ang Ii ◽  
Nuclear Fragmentation ◽  
Alveolar Epithelial ◽  
Induced Apoptosis ◽  
Pepstatin A

Our earlier studies showed that bleomycin-induced apoptosis of type II alveolar epithelial cells (AECs) requires the autocrine synthesis and proteolytic processing of angiotensinogen into ANG II and that inhibitors of ANG-converting enzyme (ACEis) block bleomycin-induced apoptosis (Li X, Zhang H, Soledad-Conrad V, Zhuang J, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 284: L501–L507, 2003). Given the documented role of cathepsin D (CatD) in apoptosis of other cell types, we hypothesized that CatD might be the AEC enzyme responsible for the conversion of angiotensinogen into ANG I, the substrate for ACE. Primary cultures of rat type II AECs challenged with bleomycin in vitro showed upregulation and secretion of CatD enzymatic activity and immunoreactive protein but no increases in CatD mRNA. The aspartyl protease inhibitor pepstatin A, which completely blocked CatD enzymatic activity, inhibited bleomycin-induced nuclear fragmentation by 76% and reduced bleomycin-induced caspase-3 activation by 47%. Antisense oligonucleotides against CatD mRNA reduced CatD-immunoreactive protein and inhibited bleomycin-induced nuclear fragmentation by 48%. A purified fragment of angiotensinogen (F1–14) containing the CatD and ACE cleavage sites, when applied to unchallenged AEC in vitro, yielded mature ANG II peptide and induced apoptosis. The apoptosis induced by F1–14 was inhibited 96% by pepstatin A and 77% by neutralizing antibodies specific for CatD (both P < 0.001). These data indicate a critical role for CatD in bleomycin-induced apoptosis of cultured AEC and suggest that the role(s) of CatD in AEC apoptosis include the conversion of newly synthesized angiotensinogen to ANG II.

scholarly journals Large Pore Mesoporous Silica and Organosilica Nanoparticles for Pepstatin A Delivery in Breast Cancer Cells

Molecules ◽  
2019 ◽  
Vol 24 (2) ◽  
pp. 332 ◽  
Author(s):  
Saher Rahmani ◽  
Jelena Budimir ◽  
Mylene Sejalon ◽  
Morgane Daurat ◽  
Dina Aggad ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Mesoporous Silica ◽  
Silica Nanoparticles ◽  
Cancer Cells ◽  
Cathepsin D ◽  
Breast Cancer Cells ◽  
Potent Inhibitor ◽  
Mesoporous Silica Nanoparticles ◽  
Sol Gel ◽  
Pepstatin A

(1) Background: Nanomedicine has recently emerged as a new area of research, particularly to fight cancer. In this field, we were interested in the vectorization of pepstatin A, a peptide which does not cross cell membranes, but which is a potent inhibitor of cathepsin D, an aspartic protease particularly overexpressed in breast cancer. (2) Methods: We studied two kinds of nanoparticles. For pepstatin A delivery, mesoporous silica nanoparticles with large pores (LPMSNs) and hollow organosilica nanoparticles (HOSNPs) obtained through the sol–gel procedure were used. The nanoparticles were loaded with pepstatin A, and then the nanoparticles were incubated with cancer cells. (3) Results: LPMSNs were monodisperse with 100 nm diameter. HOSNPs were more polydisperse with diameters below 100 nm. Good loading capacities were obtained for both types of nanoparticles. The nanoparticles were endocytosed in cancer cells, and HOSNPs led to the best results for cancer cell killing. (4) Conclusions: Mesoporous silica-based nanoparticles with large pores or cavities are promising for nanomedicine applications with peptides.

Kinetics of in vivo inhibition of tissue cathepsin d by pepstatin A

1988 ◽  
Vol 20 (9) ◽  
pp. 917-920 ◽  
Author(s):  
Gaetano Leto ◽  
Francesca Maria Tumminello ◽  
Nicola Gebbia ◽  
Luciano Rausa
Keyword(s):  
Cathepsin D ◽  
Kinetics Of ◽  
Pepstatin A

Synthesis and characterization of a new fluorescent probe for measuring potassium

1990 ◽  
Vol 258 (2) ◽  
pp. F438-F443 ◽  
Author(s):  
K. Golchini ◽  
M. Mackovic-Basic ◽  
S. A. Gharib ◽  
D. Masilamani ◽  
M. E. Lucas ◽  
...  
Keyword(s):  
Aqueous Solutions ◽  
Fluorescent Probe ◽  
Membrane Vesicles ◽  
Excitation Peak ◽  
Synthetic Procedure ◽  
K Concentration ◽  
Dissociation Constant Kd ◽  
K Transport ◽  
Micromolar Range

A new fluorescent probe, 6,7-(4-methyl)coumaro-[2.2.2] cryptand, has been developed for measuring K+. This compound was made by fusing [2.2.2] cryptand with 4-methylcoumarin through 2 ethoxy bridges at the 6 and 7 positions. The probe has a fluorescent excitation peak at 340 nm and an emission peak at 420 nm. In aqueous solutions, increasing the K+ concentration from 0 to 10 mM causes the fluorescence intensity to increase by 143%. The dissociation constant (Kd) for K+ in aqueous solutions is 1.9 mM. In 100% methanol, the Kd for K+ decreases to 0.012 mM, making it possible to measure K+ concentrations in the micromolar range. Na+, tetramethylammonium, NH4+, Ca2+, and Mg2+ have a minimal affect on the fluorescence of the probe in the absence of K+. The coefficient of variation for the measurement of K+ by use of this new dye is less than 1%. In this report, the synthetic procedure is described and the spectral properties of the probe are characterized. Experiments are described demonstrating the use of this probe 1) in measuring K+ in aqueous solutions from 0 to 10 mM and in a microfluorometric assay to measure K+ from 0.0005 to 0.003 mM and 2) in monitoring K+ transport in rabbit proximal tubule brush-border membrane vesicles.

scholarly journals The effects of calcium ions and pH on bovine prothrombin fragment 1. Intrinsic fluroescence studies

1979 ◽  
Vol 177 (3) ◽  
pp. 879-886 ◽  
Author(s):  
M E Scott ◽  
K A Koehler ◽  
R G Hiskey
Keyword(s):  
Ph Dependence ◽  
Tight Binding ◽  
Ph Titration ◽  
Prothrombin Fragment ◽  
Ph Values ◽  
Titration Curves ◽  
Or Groups ◽  
Effects Of Ph ◽  
Induced Changes ◽  
Apparent Association

The effects of pH and Ca2+ on the intrinsic fluorescence of bovine prothrombin fragment 1 were investigated to deduce the nature of protein functional groups involved in Ca2+ binding to fragment 1. From pH values of 9 to 3, increasing the H3O+ concentration results in quenching of the fluorescence of fragment 1. Reversible pH-titration curves are obtained which appear to consist of two regions. From pH 4 to pH6.5 a broad titration curve is obtained, whereas from pH6.5 to 9 a more pronounced titration behaviour is evidenced by a group or groups on fragment 1 with an apparent pKa of approx. 7.5. In contrast, the apparent association constant for Ca2+ and fragment 1 shows a sharp pH-dependence in the region between pH7 and 8 with tighter Ca2+ binding at higher pH values. A PKa of approx. 7.5 can be estimated for the group or groups on fragment 1 linked to the tight binding of Ca2+. Both H3O+ and Ca2+ result in blue-shifts in the wave-lengths of fragment-1 emission. These results are interpreted in terms of H+ - and Ca2+ - induced changes in the conformation of fragment 1 as a result of surface-charge neutralization.

scholarly journals Nature and properties of human platelet vasopressin receptors

1986 ◽  
Vol 233 (3) ◽  
pp. 631-636 ◽  
Author(s):  
D Vittet ◽  
A Rondot ◽  
B Cantau ◽  
J M Launay ◽  
C Chevillard
Keyword(s):  
Arginine Vasopressin ◽  
Protein Concentration ◽  
Binding Capacity ◽  
Rat Kidney ◽  
Human Platelets ◽  
Single Population ◽  
Liver Membrane ◽  
Kd Values ◽  
Vasopressin Receptors ◽  
Dissociation Constant Kd

The binding of 3H-labelled [8-arginine]vasopressin to human platelets or crude platelet membranes was examined. Both preparations specifically bound [8-arginine]vasopressin. The binding increased linearly with protein concentration, it was temperature- and time-dependent, saturable and could be reversed to a large extent by EDTA (10 mM). In this latter case, addition of an excess of MgCl2 (20 mM) restored the initial level of binding. Intact platelets and membranes derived from these platelets presented a single population of binding sites with a dissociation constant (Kd) of 1.3 +/- 0.2 and 1.8 +/- 0.3 nM and a maximal binding capacity of 142 +/- 48 and 270 +/- 17 fmol/mg of protein, respectively. The Kd values of various analogues correlated well with those determined on rat liver membrane V1 vasopressin receptors but not with those determined on rat kidney membrane V2 receptors.

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