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)

We have reported that mid-region fragments of human parathyroid hormone (hPTH), exemplified by hPTH-(28-48), stimulated [3H]thymidine incorporation into DNA and increased the specific activity of the brain-type isoenzyme of creatine kinase (CK) in both skeletal-derived cell cultures (ROS 17/2.8 cells) and immature rat epiphyseal cartilage and diaphyseal bone, without stimulating cyclic AMP synthesis which is a prerequisite for bone resorption. In the present study, substitution of amino acids in hPTH-(28-48), which resulted in increased resistance to proteolysis, produced variants that stimulated skeletal systems at two orders of magnitude lower concentration than the wild-type fragment. We modified hPTH-(28-48) at Leu-37 by replacement with Met, Thr or Val. Under conditions in which 20% of the native hPTH-(28-48) resisted proteolysis by cathepsin D for 6 h, approx. 40% of the L37V mutant and 70% of the L37T mutant remained intact. Substitution of Met for Phe-34 in addition to Thr for Leu-37, or the substitution of Met for Phe-34 alone, produced 100%-resistant fragments. These variants at residue 34 caused maximal stimulation of CK in ROS 17/2.8 cells at 0.24 nM compared with 24 nM for hPTH-(28-48). The double mutant stimulated CK activity significantly in immature rats, at a minimum dose of 12.5 ng/rat, and caused maximal stimulation at 125 ng/rat, a 10-fold lower dose than for hPTH-(28-48). The effect of the double mutant lasted up to 24 h which differs from the stimulation by hPTH-(28-48) in which CK specific activity returns to the control level at 24 h. This same dose also significantly stimulated CK activity in gonadectomized rats. These results show the advantage of using protease-resistant mid-region variants of hPTH-(28-48) to stimulate bone cells, in terms of lower doses and longer duration of effectiveness, both in vitro and in vivo.
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PMID:Stimulation of creatine kinase activity in rat skeletal tissue in vivo and in vitro by protease-resistant variants of parathyroid hormone fragments. 761 87

A series of enol HIV-1 protease inhibitors which show competitive inhibition and the structure-activity relationship study which led to the design of these compounds are reported. By systematically modifying simple amino acids, Boc-Phe enol and Boc-Tyr enol derivatives yield nanomolar Kiapp values (Kiapp = 0.485 microM and Kiapp = 0.425 microM, respectively). These enols are of low molecular weight (< 500 g/mol) and of non-peptidic nature. The enols are synthesized in a one step chemical synthesis and modifications to increase their potency could easily be performed. Boc-Phe enol and Boc-Tyr enol showed low inhibitory effect on pepsin, Kiapps of 23 and 149 microM, respectively, and Boc-Phe enol showed a Kiapp of 20 microM for cathepsin D. Neither of these two compounds inhibited renin (< 10% inhibition at 200 microM).
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PMID:Synthesis of novel inhibitors of the HIV-1 protease: difunctional enols of simple N-protected amino acids. 792 46

By directly coupling a tetrapeptide to DOTA through an amide bond, we synthesized a novel DOTA derivative, DOTA-glycylglycylglycyl-L-p-nitrophenylalanine amide. We converted this new precursor bifunctional chelating agent to DOTA-glycylglycylglycyl-L-p-isothiocyanatophenylalanine++ + and conjugated it to monoclonal antibody Lym-1. Serum stability studies show that the radiolabeled conjugates are kinetically inert under physiological conditions. The rates of loss of radiometals in human serum are 0.1 +/- 0.1% per day for InIII, 0.02 +/- 0.15% per day for YIII, and 0.3 +/- 0.2% per day for CuII labeled immunoconjugates. In the presence of the liver enzyme cathepsin B, an in vitro digestion of 114mIn-labeled conjugate yields a small fragment containing 114mIn. Characterization of the cleavage products shows that this liver enzyme hydrolyzes the peptide linkage before the phenylalanine residue, freeing the In-DOTA-triglycine complex from the conjugate. However, the liver enzyme cathepsin D does not cleave the linkage over the span of 7 days.
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PMID:Synthesis, metal chelate stability studies, and enzyme digestion of a peptide-linked DOTA derivative and its corresponding radiolabeled immunoconjugates. 821 84

Proteolytic processing of the beta-amyloid precursor proteins (APP) is required for release of the beta/A4 protein and its deposition into the amyloid plaques characteristic of aging and Alzheimer's disease. We have examined the involvement of acidic intracellular compartments in APP processing in cultured human cells. The use of acidotropic agents and inhibitors to a specific class of lysosomal protease, coupled with metabolic labeling and immunoprecipitation, revealed that APP is degraded within an acidic compartment to produce at least 12 COOH-terminal fragments. Nine likely contain the entire beta/A4 domain and, therefore, are potentially amyloidogenic. Treatment with E64 or Z-Phe-Ala-CHN2 irreversibly blocked activities of the lysosomal cysteine proteases cathepsins B and L but did not inhibit the lysosomal aspartic protease cathepsin D and did not alter the production of potentially amyloidogenic fragments. Instead, the inhibitors prevented further degradation of the fragments. Thus, large numbers of potentially amyloidogenic fragments of APP are routinely generated in an acidic compartment by noncysteine proteases and then are eliminated within lysosomes by cysteine proteases. Immunoblot and immunohistochemical analyses confirmed that chronic cysteine protease inhibition leads to accumulation of potentially amyloidogenic APP fragments in lysosomes. The results provide further support for the hypothesis that an acidic compartment may be involved in amyloid formation and begin to define the proteolytic events that may be important for amyloidogenesis.
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PMID:Processing of the beta-amyloid precursor. Multiple proteases generate and degrade potentially amyloidogenic fragments. 834 42

The processing of antigenic peptides for presentation by MHC molecules to T cells, may depend upon the function of a second, consensus sequence in or near the T cell-presented epitope. One such processing-regulating sequence appears to be composed of amino acids Leu, Ile, Val, Phe, and Met recurring in a fashion to form a longitudinal, hydrophobic strip when the excised peptide is coiled as an alpha-helix. Such a hydrophobic strip-of-helix may: (a) scavenge peptides from lumens onto lipid membranes of digestion vesicles, (b) stabilize peptides there as protease-resistant helices, (c) specify recognition by the antigenic peptide-binding sites of chaperonin proteins, transmembranal transporters, or MHC molecules. By circular dichroism and electron paramagnetic resonance, we demonstrated that peptides with recurrent hydrophobic residues potentially forming longitudinal strips adsorbed to, and partially coiled as helices on, di-O-hexadecyl, D-L-alpha-phosphatidylcholine (DHPC) vesicles. Cathepsin B or cathepsin D cleavages of three such peptides were identified. With either enzyme, it made no significant difference whether a peptide substrate was in solution or bound to vesicles in terms of efficiency and specificity of peptide bond cleavages. We conclude that protease resistance, per se, of membrane-adsorbed, helically coiled peptides is not a major factor in the selection for T cell presentation of epitopes in peptides which have a motif with a longitudinal hydrophobic strip.
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PMID:Adsorption and helical coiling of amphipathic peptides on lipid vesicles leads to negligible protection from cathepsin B or cathepsin D. 838 60

In order to characterize the intracellular processing event of lysosomal cathepsin B, the proenzyme was purified from the rat liver microsomal contents using a Con A-Sepharose column, a Sepharose-Gly-Phe-GlySc column, and an anti-cathepsin B IgG column. The purified proenzyme gave a single protein band of 39 kDa on SDS/polyacrylamide gel electrophoresis. The proenzyme showed no appreciable enzymatic activity. When the purified proenzyme was incubated with the cathepsin B-free tritosomal contents, prepared by treatment of the tritosomal contents with anti-cathepsin B IgG Sepharose, at pH 3.0, 30 degrees C, a remarkable increase of enzymatic activity was observed. Immunoblot analysis showed that the proenzyme was completely converted to the active intermediate form of 31 kDa after 1 h incubation. These processing and activation events were blocked in the presence of pepstatin. When the proenzyme was incubated with the cathepsins B- and D-free tritosomal contents, prepared by treatment of the cathepsin B-free tritosomal contents with anti-cathepsin D IgG Sepharose, the processing and activation did not occur. These results indicate that cathepsin D is involved in the processing and activation of procathepsin B in rat liver lysosome. In the NH2-terminal sequence analysis of the 31 kDa form, the terminal was assigned as proline (66th residue). Since the NH2-terminus of the mature single-chain form of cathepsin B (29 kDa) ends at leucine (80th residue), the NH2-terminus of the 31 kDa form is 14 amino acid residues longer than that of the single-chain form.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and processing of rat liver procathepsin B. 848 12

Via a combination of chemical and enzymatic synthesis, new hexapeptide substrates convenient for use in activity assessment of several aspartyl proteinases--porcine pepsin, human pepsin, gastricsin, and cathepsin D--were prepared. These peptide derivatives, o-aminobenzoyl-Ala-Ala-Phe-Phe-Ala-Ala-p-nitroanilide and N-(o-aminobenzoyl-Ala-Ala-Phe-Phe-Ala-Ala)-N'-2,4-dinitrophenyl ethylenediamine, contain a fluorescent o-aminobenzoyl moiety as well as p-nitroaniline or N-2,4-dinitrophenyl ethylenediamine--the groups that cause fluorescence quenching. Aspartyl proteinases hydrolyze the Phe-Phe peptide bond in the substrates, which diminishes quenching due to separation of the fluorescent and quenching moieties and leads to an increase in the fluorescence intensity of o-aminobenzoyl residue. Abz-Ala-Ala-Phe-Phe-Ala-Ala-Ded, being fairly well hydrolyzed by HIV proteinase, might be used for assay of this enzyme.
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PMID:Fluorogenic peptide substrates for assay of aspartyl proteinases. 871 88

The relationship between skeletal muscle aspartyl protease activity (APA) and wasting was investigated in male DBA/2 mice inoculated with L1210 tumor cells. Using the peptidic substrate H-Pro-Thr-Glu-Phe-Phe(NO2)-Arg-Leu-OH, which is specific for aspartyl proteases, proteases, proteolytic activity was detected in a number of tissues including muscle by using a crude extraction procedure for isolation of lysosomal enzymes. Biochemical characterization and increased muscle levels following either fasting or injection of endotoxin (ETX) suggest that the enzyme is likely cathepsin D. The wasting syndrome accompanying the tumor was measured by comparing the weight of the skinned hind limb in treated and control animals. DBA/2 mice inoculated intraperitoneally with L1210 cells developed multiple solid tumors in the peritoneum and ascites; maximal tumor burden was reached by 16 days. There was a significant reduction in hind limb weight (16 +/- 2%; mean +/- SE) and significant increase (31 +/- 8%) in muscle APA associated with the development of ascites and solid tumors. Plasma APA activity was substantially increased (240 +/- 33%), while liver and spleen APA were increased (10-20%) but not significantly. Chronic pepstatin administration, 30 mg.kg-1.day-1, for 7 days concurrent with the initiation of observable ascites and solid tumor formation (7 days post-inoculation), completely inhibited hind limb weight loss and alleviated the tumor-dependent increase of APA in both plasma and muscle without altering tumor development. Delaying the administration of pepstatin by 3 days resulted in less of an inhibition (33 +/- 13%) of hind limb weight loss. Thus, cathepsin D or a similar aspartyl protease appears to be of key importance in the wasting syndrome associated with cachexia.
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PMID:Muscle aspartyl protease (cathepsin D) activity: detection using a chromophoric substrate and relation to wasting in DBA/2 mice implanted with leukemic L1210 tumor cells. 902 34

An acidic proteinase was purified from human kidney cortex. The enzyme showed a molecular mass of 31 kDa by SDS-PAGE, 36 kDa by gel filtration, and isoelectric points of 5.2 and 6.1. The optimum pH for hydrolysis of bovine hemoglobin was about 3.5. Reverse-phase HPLC analysis of the incubation mixture of the enzyme with human plasma showed the presence of an active peptide on rat uterus muscle with the same retention time as the methionyl-lysyl-bradykinin (MLBK) standard. The specific activities were 2.91 micrograms MLBK equivalent mg-1.min-1 at pH 3.5 and 2.15 micrograms MLBK equivalent mg-1.min-1 at pH 6.0. All the enzymatic activities of this human kidney proteinase were inhibited by pepstatin A. Intramolecularly quenched fluorogenic substrates with amino acid sequences of human kininogen were used to determine the cleavage points. On the N-terminal sequences (Abz-Leu-Met-Lys-Arg-Pro-Eddnp and Abz-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Eddnp) the cleavage occurred at the Leu-Met linkage, and on the C-terminal sequences (Abz-Phe-Arg-Ser-Ser-Arg-Eddnp and Abz-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp) the cleavage occurred at the Arg-Ser linkage. Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp++ + was hydrolyzed by the renal acidic proteinase and yielded the peptide Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (Abz-bradykinin). Kinectic parameters were determined using Abz-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Eddnp (K(m) = 0.69 +/- 0.08 microM; Kcat = 0.052 +/- 0.0095 s-1; Kcat/K(m) = 0.075 +/- 0.005 microM-1.s-1) and Abz-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp (K(m) = 1.56 +/- 0.16 microM; Kcat = 0.0048 +/- 0.0001 s-1; Kcat/K(m) = 0.003 +/- 0.0003 microM-1.s-1). Human liver cathepsin D had no activity on C-terminal sequences and human pepsin hydrolyzed them at the Ser-Ser bond. The results suggest that the renal acid proteinase is distinct from human pepsin and human liver cathepsin D and releases MLBK from human kininogen.
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PMID:Characterization of kininogenase activity of an acidic proteinase isolated from human kidney. 927 60

The substrate specificities and kinetic properties of proteinase A, an intracellular aspartic proteinase from the yeast Saccharomyces cerevisiae, were determined using a series of synthetic chromogenic peptides with the general structure P5-P4-P3-P2-Phe-(NO2)Phe-P2'-P3' [P5, P4, P3, P2, P2', P3' are various amino acids; (NO2)Phe is p-nitro-L-phenylalanine]. The nature of the residues occupying the NH2-terminal region of the substrate had a strong influence on the kinetic constants. Among those tested, Ala-Pro-Ala-Lys-Phe-(NO2)-Phe-Arg-Leu had the best kinetic constants (Km = 0.012 mM, kcat = 14.4 s-1, kcat/Km = 1,200 M-1.s-1). Compared with such aspartic proteinases as pepsin, cathepsin D, and renin, the substrate specificity of proteinase A was unique. Based on these results, a novel fluorescent substrate, MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH2, was developed for the sensitive measurement of proteinase A.
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PMID:Substrate specificities and kinetic properties of proteinase A from the yeast Saccharomyces cerevisiae and the development of a novel substrate. 964 56


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