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)

Net changes in the concentrations of 18 amino acids in perfusate and skeletal muscle were followed during perfusion of hemicorpus preparations from fed rat. Perfusate levels of 16 amino acids showed little change from their initial concentrations during the 1st h, but increased dramatically during the 2nd and 3rd h. Aspartate and glutamate levels decreased continuously throughout the perfusion. Release of alanine and glutamine accounted for approximately 50% of the total change in perfusate amino acids. The increase in perfusate amino acids was derived from net breakdown of muscle proteins and not from leakage from the intracellular pool as evidenced by elevated concentrations of intracellular amino acids in perfused muscle. Addition of insulin to the perfusate did not change the pattern of amino acid release during the 1st h of perfusion. However, during the 2nd and 3rd h the hormone completely prevented the net release of most amino acids and maintained intracellular concentrations of most amino acids at levels found in upperfused tissue. Effects of time of perfusion and insulin on amino acid release were accounted for by changes in the rate of protein turnover. Protein synthesis in gastrocemius and psoas muscles in control perfusions decreased after 1 h to approximately 50% of the initial rate. This decrease was accompanied by a 2-fold increase in the level of ribosomal subunits, indicating development of a block in peptide chain initiation. Addition of insulin maintained the initial rate of synthesis and the in vivo level of ribosomal subunits, demonstrating that the hormone prevented the block in peptide chain initiation from forming. Addition of insulin after 2 h reversed the perfusion-induced block in initiation. Synthesis of the specific muscle protein myosin was increased 45% over the control rate in the presence of insulin. Insulin also produced a 50% decrease in the rate of protein degradation during the 2nd and 3rd h of perfusion. A similar effect was noted when protein synthesis was inhibited by addition of cycloheximide. Higher concentrations of insulin were required to maximally inhibit protein degradation than to increase protein synthesis. Involvement of lysosomal proteases in the effect of insulin on protein degradation was evaluated by measuring cathepsin D activity in psoas muscle homogenates. "Free" enzyme activity increased as a result of perfusion while addition of insulin maintained this activity at the unperfused level. Neither perfusion nor insulin had any effect on total cathepsin D activity. Alterations in protein degradation and lysosomal enzyme activity were not due to changes in levels of adenine nucleotides, GTP, or creatine phosphate.
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PMID:Regulation by insulin of amino acid release and protein turnover in the perfused rat hemicorpus. 83 25

An acid proteinase has been detected in culture supernate of the 9.2.27 murine hybridoma. This enzyme extensively degrades albumin and transferrin during short incubations at pH 3 and below. Limited proteolysis of the 9.2.27 IgG2a appears to occur in the culture supernate. Proteolysis in enhanced at low pH in the presence of urea or 1 M acetic acid. The proteinase activity accumulates in continuous perfusion, total cell recycle cultures, beginning during exponential growth of the hybridoma. It is destroyed by boiling and blocked by pepstatin, but not by inhibitors of cysteine or serine proteinases or by EDTA. The low pH optimum may distinguish this enzyme from the known rat and mouse aspartic acid proteinases including cathepsin D and cathepsin E.
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PMID:A novel acid proteinase released by hybridoma cells. 136 94

We isolated and sequenced a cDNA clone corresponding to the entire coding sequence of rat liver lysosomal cathepsin D. The deduced amino acid sequence revealed that cathepsin D consists of 407 amino acid residues (Mr 44,608) and the 20 NH2-terminal residues seem to constitute a cleavable signal peptide after which 44 amino acid residues follow as a propeptide. Two putative N-linked glycosylation sites and aspartic acid in the active site are as well conserved as those of human lysosomal cathepsin D. In the NH2-terminal sequence analysis of two isolated heavy chains of the mature enzyme, the termini were assigned as tryptophan (118th residue) and glycine (165th or 166th residue), respectively, hence demonstrates that the two heavy chains derive from a split of the single chain of cathepsin D at position between 117th and 118th or between 164th and 165th or 165th and 166th amino acids. We conclude that cathepsin D in rat liver lysosomes is a mixture of three forms composed of a single and two two-chain forms. However, the amounts of the two two-chain forms are low compared with that of the single chain form. Densidometric determination after SDS-PAGE revealed that the two two-chain forms account for less than 5% of the single chain form. There is a 82% similarity in amino acid level between rat and human liver lysosomal cathepsin D.
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PMID:Isolation and sequencing of a cDNA clone encoding rat liver lysosomal cathepsin D and the structure of three forms of mature enzymes. 188 50

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

The amino acid sequence of porcine spleen cathepsin D heavy chain has been determined and, hence, the complete structure of this enzyme is now known. The sequence of heavy chain was constructed by aligning the structures of peptides generated by cyanogen bromide, trypsin, and endo-proteinase Lys C cleavages. The structure of the light chain has been published previously. The cathepsin D molecule contains 339 amino acid residues in two polypeptide chains: a 97-residue light chain and a 242-residue heavy chain, with a combined Mr of 36,779 (without carbohydrate). There are two carbohydrate units linked to asparagine residues 70 and 192. The disulfide bond arrangement in cathepsin D is probably similar to that of pepsin, because the positions of six half-cystine residues are conserved. The active site aspartyl residues, corresponding to aspartic acid-32 and -215 of pepsin, are located at residues 33 and 224 in the cathepsin D molecule. The amino acid sequence around these aspartyl residues is strongly conserved. Cathepsin D shows a strong homology with other acid proteases. When the sequence of cathepsin D, renin, and pepsin are aligned, 32.7% of the residues are identical. The homology is observed throughout the length of the molecules, indicating that three-dimensional structures of all three molecules are similar.
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PMID:Amino acid sequence of porcine spleen cathepsin D. 658 85

Interleukin-6 (IL-6), a four-helix bundle protein, is a multifunctional cytokine which plays an important role in the regulation of the immune system, hematopoiesis, and inflammatory response, as well as in the pathogenesis of multiple myeloma. We have previously shown that a single-disulfide variant of human IL-6, lacking 22 N-terminal amino acids and the disulfide bond connecting Cys-45 and Cys-51 in the 185-residue chain of the wild-type protein, fully retains the conformational, stability, and functional properties of the full-length human IL-6 [Breton et al. (1995) Eur. J. Biochem. 227, 573-581]. In this study, we have investigated the conformational and stability properties of mutant IL-6 at acidic pH (A-state). Using far- and near-ultraviolet (UV) circular dichroism (CD), fluorescence emission, and second-derivative absorption spectroscopy, we have established that mutant IL-6 at pH 2.0 fully retains the helical secondary structure of the native protein at pH 7.5, while the tertiary interactions are much weaker. At variance from the native species, mutant IL-6 in the A-state binds 1-anilinonaphthalene-8-sulfonic acid (ANS), a property considered most typical of a protein in the molten globule state. The pH-induced conformational change from the native to the A-state, monitored either by near-UV CD or by ANS-binding measurements, shows a transition midpoint at pH approximately 4.5, thus indicating that the partial unfolding of the protein is mediated by the titration of glutamic and/or aspartic acid residues. At pH 2.0, the thermal denaturation of mutant IL-6 occurs as a broad process of low cooperativity with a transition at 50-60 degrees C, whereas at pH 7.5 the thermal unfolding is cooperative and characterized by a transition midpoint at 65 degrees C. Of interest, the unfolding of the A-state is not complete even up to approximately 85 degrees C. The urea-mediated unfolding profile of mutant IL-6, measured by far-UV CD, is essentially identical at both pH 7.5 and 2.0, with a midpoint of the cooperative unfolding transition at 5.5 +/- 0.1 M denaturant. Both thermal and urea denaturations of the A-state are complex and cannot fit to a two-state model for unfolding. The unusual stability of mutant IL-6 in acid is also reflected by the resistance to proteolysis at pH 3.6-4.0 by Staphylococcus aureus V8 protease or cathepsin D, an acid protease released by machrophages upon inflammatory stimulation. It is suggested that the molten globule state of IL-6 at acidic pH can play a role in the biological activity of this cytokine, which can exert its activity also at mildly acidic pH, as in inflammation sites.
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PMID:Acid-induced molten globule state of a fully active mutant of human interleukin-6. 878 6

The catalytic activity and inhibitor binding energetics of enzymes are often pH-dependent properties. Aspartic proteases comprise an important class of enzyme targets for structure-based drug design. We have performed a complete thermodynamic study of pepstatin binding to plasmepsin II, an aspartic proteinase found in Plasmodium falciparum, using isothermal titration calorimetry and circular dichroism. Thermodynamic parameters (DeltaG, DeltaH, DeltaCp, and DeltaS) were measured as functions of both pH and temperature. In the pH range from 4.5 to 7.0, pepstatin binding is accompanied by proton transfer between the solvent and the complex. We used thermodynamic proton linkage theory to derive both the pH-independent binding energetics for pepstatin and the number and pKa values of ionizable residues whose pKa values change during ligand binding. These residues were identified as the two catalytic aspartates, with pKas of 6.5 and 3.0, and His 164, with a pKa of 7.5, based on the three-dimensional structure of the pepstatin-plasmepsin II complex. At pH 5.0, where the protease has optimum activity, the proton transfer process contributes almost 40% of the total binding free energy change and the total charge of the active-site aspartic acid residues is -1. These experimental results provide direct measurement for the protonation states of the catalytic aspartates in the presence of bound ligands. Comparison of the thermodynamic and structural data for pepstatin binding with human cathepsin D, a lysosomal aspartic protease that shares 35% sequence identity with plasmepsin II, suggests that the energetic differences between these two proteins are due to a higher interdomain flexibility in plasmepsin II.
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PMID:Dissection of the pH dependence of inhibitor binding energetics for an aspartic protease: direct measurement of the protonation states of the catalytic aspartic acid residues. 940 50

This review covers the unique catalytic and molecular properties of three proteolytic enzymes and a glycosidase from Aspergillus. An aspartic proteinase from A. saitoi, aspergillopepsin I (EC 3.4.23.18), favors hydrophobic amino acids at P1 and P'1 like gastric pepsin. However, aspergillopepsin I accommodates a Lys residue at P1, which leads to activation of trypsinogens like duodenum enteropeptidase. Substitution of Asp76 to Ser or Thr and deletion of Ser78, corresponding to the mammalian aspartic proteinases, cathepsin D and pepsin, caused drastic decreases in the activities towards substrates containing a basic amino acid residue at 1. In addition, the double mutant T77D/G78(S)G79 of porcine pepsin was able to activate bovine trypsinogen to trypsin by the selective cleavage of the K6-I7 bond of trypsinogen. Deuterolysin (EC 3.4.24.39) from A. oryzae, which contains 1g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. It was concluded that His128, His132, and Asp164 provide the Zn2+ ligands of the enzyme according to a 65Zn binding assay. Deuterolysin is a member of a family of metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH + D" motif and an aspartic acid as the third zinc ligand. Acid carboxypeptidase (EC 3.4.16.1) from A. saitoi is a glycoprotein that contains both N- and O-linked sugar chains. Site-directed mutagenesis of the cpdS, cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. A. saitoi carboxypeptidase indicated that Ser153, Asp357, and His436 residues were essential for the enzymic catalysis. The N-glycanase released high-mannose type oligosaccharides that were separated on HPLC. Two, which had unique structures of Man10 GlcNAc2 and Man11GlcNAc2, were characterized. An acidic 1,2-alpha-mannosidase (EC 3.2.1.113) was isolated from the culture of A. saitoi. A highly efficient overexpression system of 1,2-alpha-mannosidase fusion gene (f-msdS) in A. oryzae was made. A yeast mutant capable of producing Man5GlcNAc2 human-compatible sugar chains on glycoproteins was constructed. An expression vector for 1,2-alpha-mannosidase with the "HDEL" endoplasmic reticulum retention/retrieval tag was designed and expressed in Saccharomyces cerevisiae. The first report of production of human-compatible high mannose-type (Man5GlcNAc2) sugar chains in S. cerevisiae was described.
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PMID:Unique catalytic and molecular properties of hydrolases from Aspergillus used in Japanese bioindustries. 1083 Apr 77

A Caenorhabditis elegans gene (asp-1) and cDNA that encode a homologue of cathepsin D aspartic protease were cloned and characterized. The asp-1 mRNA is transcribed from a single exon, and it begins with the SL1 trans-splice leader sequence. The protein (ASP-1) is expressed as a 396-amino acid, 42.7-kDa pre-pro-peptide that is post-translationally processed into a approximately 40-kDa lysosomal protein. ASP-1 shares approximately 60% sequence identity with the aspartic protease precursor from the nematode Strongyloides stercoralis. The amino acid sequences adjacent to the two active site aspartic acid residues in ASP-1 are 100% identical to those in other eukaryotic aspartic proteases. In addition, ASP-1 contains conserved, potential disulfide bond-forming cysteine residues and N-glycosylation sites. The asp-1 gene is exclusively transcribed in the intestinal cells, with the highest levels of expression observed at late embryonic and early larval stages of development. asp-1 transcription is not observed in adult nematodes or mature larvae. Furthermore, transcription predominantly occurs in eight anterior cells of the intestine (int6-int8). Analyses of ASP-1 nucleotide and amino acid sequences revealed the presence of five additional C. elegans aspartic proteases.
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PMID:Aspartic proteases from the nematode Caenorhabditis elegans. Structural organization and developmental and cell-specific expression of asp-1. 1085 22

The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase, which hydrolyzes internalized insulin and generates the major primary end product A(1--21)-B(1--24) insulin resulting from a major cleavage at residues Phe(B24)-Phe(B25). This study addresses the nature of the relevant endopeptidase activity in rat liver that is responsible for most receptor-mediated insulin degradation in vivo. The endosomal activity was shown to be aspartic acid protease cathepsin D (CD), based on biochemical similarities to purified CD in 1) the rate and site of substrate cleavage, 2) pH optimum, 3) sensitivity to pepstatin A, and 4) binding to pepstatin A-agarose. The identity of the protease was immunologically confirmed by removal of greater than 90% of the insulin-degrading activity associated with an endosomal lysate using polyclonal antibodies to CD. Moreover, the elution profile of the endosomal acidic insulinase activity on a gel-filtration TSK-GEL G3000 SW(XL) high performance liquid chromatography column corresponded exactly with the elution profile of the immunoreactive 45-kDa mature form of endosomal CD. Using nondenaturating immunoprecipitation and immunoblotting procedures, other endosomal aspartic acid proteases such as cathepsin E and beta-site amyloid precursor protein-cleaving enzyme (BACE) were ruled out as candidate enzymes for the endosomal degradation of internalized insulin. Immunofluorescence studies showed a largely vesicular staining pattern for internalized insulin in rat hepatocytes that colocalized partially with CD. In vivo pepstatin A treatment was without any observable effect on the insulin receptor content of endosomes but augmented the phosphotyrosine content of the endosomal insulin receptor after insulin injection. These results suggest that CD is the endosomal acidic insulinase activity which catalyzes the rate-limiting step of the in vivo cleavage at the Phe(B24)-Phe(B25) bond, generating the inactive A(1--21)-B(1--24) insulin intermediate.
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PMID:Endosomal proteolysis of internalized insulin at the C-terminal region of the B chain by cathepsin D. 1177 65


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