Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.23.5 (cathepsin D)
4,130 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A 6-h procedure for the isolation of bovine cathepsin D is described. The procedure involves essentially only two steps; three-phase partitioning in t-butanol/water/ammonium sulfate followed by affinity chromatography on pepstatin-agarose. The major advantage of this new method over previous methods is the greatly reduced time required to obtain comparably pure cathepsin D.
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PMID:Isolation of cathepsin D using three-phase partitioning in t-butanol/water/ammonium sulfate. 281 38

The development of a model of chronic myocardial ischemic injury (MII) in rabbits by administering increasing doses of isoproterenol (ISO) is described. Repeated s.c. injections of increasing doses of ISO (0.5 mg/kg, on day 1 to 15.5 mg/kg, on day 15) resulted in an increase in serum glucose, free fatty acids and creatine phosphokinase. Examination of hearts from ISO-treated rabbits revealed marked hypertrophy of the left ventricle and an increase in total water content. Biochemical analysis showed an increase in left ventricular hydroxyproline and a decrease in ATP and glycogen content following ISO-treatment. Ion measurements revealed extensive accumulation of Na and Ca, with the Ca being preferentially accumulated in the mitochondria. Measurement of subcellular organelle marker enzymes showed decreases in the sarcolemmal Na+-K+-stimulated (ouabain-sensitive), mitochondrial (azide-sensitive) and sarcoplasmic reticulum ATPase activities in the ISO-treated animals. Analysis of lysosomal enzyme activities in myocardial homogenates showed significant decreases in the latency of N-acetyl-beta-glucosaminidase and cathepsin D. The above biochemical alterations in ISO-induced MII generally parallel changes previously seen in the rabbit following acute coronary artery ligation. The present model allows the study of MII uncomplicated by some uncertainties arising from the surgical or anesthetic procedures employed in acute "open-chest" preparations and would permit long-term follow-up studies of pharmacological interventions. The susceptibility of the rabbit to experimental atherosclerosis should allow the development of an experimental model of MII which more closely approximates the clinical situation.
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PMID:Myocardial ischemic injury induced by isoproterenol in the rabbit: biochemical and chemical alterations. 385 Jul 74

The synthesis of 10 analogues of pepstatin modified so that statine is replaced by 4-amino-3-hydroxy-3,6-dimethylheptanoic acid (Me3Sta) or 4-amino-3-hydroxy-3-methyl-5-phenylpentanoic acid (Me3AHPPA) residues is reported. Both the 3S,4S and 3R,4S diastereomers of each analogue were tested as inhibitors of the aspartic proteases, porcine pepsin, cathepsin D, and penicillopepsin. In all cases the 3R,4S diastereomer (rather than the 3S,4S diastereomer) of the Me3Sta and Me3AHPPA derivatives was found to be the more potent inhibitor of the aspartic protease (Ki = 1.5-10 nM for the best inhibitors), in contrast to the results obtained with statine (Sta) or AHPPA derivatives, where the 3S,4S diastereomer is the more potent inhibitor for each diastereomeric pair of analogues. The Me3Sta- and Me3AHPPA-containing analogues are only about 10-fold less potent than the corresponding statine and AHPPA analogues and 100-1000-fold more potent than the corresponding inhibitors lacking the C-3 hydroxyl group. Difference NMR spectroscopy indicates that the (3R,4S)-Me3Sta derivative induces conformational changes in porcine pepsin comparable to those induced by the binding of pepstatin and that the (3S,4S)-Me3Sta derivatives do not induce the difference NMR spectrum. These results require that the C-3 methylated analogues of statine-containing peptides must inhibit enzymes by a different mechanism than the corresponding statine peptides. It is proposed that pepstatin and (3S)-statine-containing peptides inhibit aspartic proteases by a collected-substrate inhibition mechanism. The enzyme-inhibitor complex is stabilized, relative to pepstatin analogues lacking the C-3 hydroxyl groups, by the favorable entropy derived when enzyme-bound water is returned to bulk solvent.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of aspartic proteases by pepstatin and 3-methylstatine derivatives of pepstatin. Evidence for collected-substrate enzyme inhibition. 392 73

Candida albicans was able to produce a keratinolytic proteinase (KPase) when cultivated in a medium containing human stratum corneum as a nitrogen source. The KPase was purified to 108.5-fold by ion-exchange chromatography and gel filtration. The molecular weight of the enzyme was estimated to be 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration through Sephacryl S-200, while the isoelectric point was determined to be at pH 4.5. The enzyme had an optimum pH of 4.0 and was "inactive" below pH 2.5 and above pH 6.0. The activity of KPase after preincubation at various temperatures was stable up to 50 degrees C. The keratinolytic activity was not affected by the addition of nonionic detergents and divalent cations. The enzyme was a glycoprotein and contained a high content of aspartic acid residues (172/1000). Pepstatin and chymostatin inhibited the activity in a dose-dependent manner; however, neither the other group specific inhibitors tested nor the pepsin specific inhibitors, DAN or EPNP, showed any effect on the enzyme. From these inhibitory profiles, this enzyme was determined to be a carboxyl proteinase such as cathepsin D. Among the various substrates for proteolytic enzymes, KPase digested human stratum corneum as much as albumin and hemoglobin. In the three fractions (water soluble, keratin filamentous, and membranous) prepared from human stratum corneum, the keratin filamentous fraction was more susceptible to degradation by KPase than the other two fractions were. KPase also digested much less human fingernail (13%) than human stratum corneum, but did not show any signs of there being any digestion of human scalp hair. These studies suggest that KPase from C. albicans may play an important role in superficial infection by affecting the human stratum corneum of the skin and nail.
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PMID:Isolation and characterization of proteinase from Candida albicans: substrate specificity. 620 88

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.
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PMID:Dinitrophenyl-pepstatins as active-site-directed localization reagents for cathepsin D. 634 85

Renin and cathepsin D were purified by seven-step procedures involving five steps common to both enzymes. These common five steps were extraction of freeze-dried kidney powder in 30% methoxyethanol-water, diethylaminoethyl-cellulose (DEAE-cellulose) batch absorption and elution, pepstatin-aminohexyl-Sepharose chromatography, Sephadex G-100 chromatography, and DEAE-cellulose chromatography. The renin component was purified further by passage through an anti-rat spleen cathepsin D immunoglobulin G-Sepharose (IgG-Sepharose) column followed by carboxymethyl-Sephadex (CM-Sepharose) chromatography which separated two renin components. Cathepsin D activity obtained by the fifth step was purified by passage through an anti-rat kidney renin IgG-Sepharose column followed by DEAE-Sephacel chromatography which separated three cathepsin D components. The homogeneity of renin and cathepsin D preparations was demonstrated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The two components of renins showed molecular weights of 42 000 and 36 000 by gel filtration and 38 000 and 36 000 by SDS gel electrophoresis, respectively. They showed isoelectric points of 5.35 and 5.65 by electrofocusing in 5% polyacrylamide gels. Their optimum pHs of enzyme activity were 6.5 as determined by using nephrectomized rat plasma as a substrate. Their specific angiotensin I (Ang I) generation activities were 158 and 146 micrograms of Ang I (microgram of protein)-1 h-1, respectively, which correspond to 1100 and 1020 Goldblatt units (mg of protein)-1 h-1. The three cathepsins showed molecular weights of 41 000, 43 000, and 41 000 by gel filtration and 46 000, 45 000, and 46 000 by SDS gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Rat kidney renin and cathepsin D: purification and comparison of properties. 636 Feb 7

Solid-phase synthesis was used for the preparation of pyroglutamyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (I) and glycyl-glycyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (II), two water-soluble and sensitive chromophoric substrates of chicken pepsin, hog pepsin A, and bovine spleen cathepsin D. The kinetic constants of hydrolysis of the p-nitrophenylalanyl-phenylalanyl bond of the substrates were measured by difference spectrophotometry at 308 nm (delta epsilon = 860 M-1 cm-1) and by ninhydrin colorimetry (substrate I, epsilon 570 = 2.31 X 10(4) M-1 cm-1). The pH optimum of cleavage is 5 for the pepsins and 3.7 for cathepsin D. Since all three proteinases still have a significant activity at pH 5.5-6 a new, simple assay was designed for submicrogram quantities of pepsins in the presence of pepsinogens without interference of the latter. The method is particularly suitable for the analyses of the zymogen activation mixtures.
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PMID:Chromophoric peptide substrates for activity determination of animal aspartic proteinases in the presence of their zymogens: a novel assay. 641 4

Immersion of rat scrotum into water (41 degrees C) for 30 min leads to gross impairment of spermatogenesis. During incubation in Ringer-Krebs solution at 33 degrees C for 1h, fragments of the decapsulated testes taken from the heated rats secrete to the medium a high amount of substances that increase the optical density of the incubation liquid at 270 nm. This phenomenon can be recorded shortly after scrotum heating is completed. The optical density increases further 12 and 24 h after heating. During incubation, fragments of the testes heated in vivo also secrete a high amount of the lysosomal enzymes, acid phosphatase and cathepsin D. It is assumed that post-thermal degeneration of the spermatogenic epithelium may be determined by a change in the permeability of cellular and lysosomal membranes.
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PMID:[Early changes in the permeability of cellular and lysosomal membrane in rat testes after local heating of the scrotum]. 650 82

Experiments on rats were made to study the effect of water-soluble antioxidants on the permeability of lysosomal membranes of liver cells and liver structure under burn. Antioxidants were injected intraperitoneally shortly after burn, whereas examination was performed after one day. It has been discovered that one day after burn there takes place an appreciable destabilization of lysosomal membranes with the release of a lysosomal matrix enzyme, cathepsin D to the cytoplasm. Liver structure had undergone substantial changes by that time. After administration of water-soluble antioxidants lysosomal membranes got stabilized while liver structure manifested but insignificant disorders.
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PMID:[Effect of water-soluble antioxidants on the permeability of lysosomal membranes and on the structure of the liver in rats with thermal burns]. 662 25

The binding of folic acid as a model compound and methotrexate as a representative of antifolates to bovine fibrinogen with the aid of 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide was investigated in order to study the possibility of using fibrinogen as a drug carrier. Soluble modified fibrinogen derivatives containing 0.03-0.1 mg of folic acid or methotrexate per mg of protein were obtained under optimal conditions. These derivatives retained the ability to form fibrin clot by the action of thrombin and to copolymerize with native fibrinogen to the three dimensional fibrin network. At higher concentrations of water soluble carbodiimide, higher temperature and low pH highly cross-linked derivatives of fibrinogen and folic acid (or methotrexate) were formed which were insoluble in water and salt solutions (pseudofibrin). The modified fibrin was extensively proteolytically cleaved by plasmin, pepsin, trypsin and cathepsin D, whereas the proteolysis of insoluble pseudofibrin was very slow.
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PMID:Chemical binding of folic acid and methotrexate to bovine fibrinogen. 668 28


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