Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.23.5 (cathepsin D)
 4,130 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The determinant specificities of five monoclonal anti-fibronectin antibodies, designated BC7, CE9, BD4, AB3 and CPG1, were defined and mapped within intact human plasma fibronectin by immunoblot analyses with defined fragments of fibronectin. The latter were derived by tryptic, chymotryptic or cathepsin D digestion of the intact molecule and fractionated by DE-cellulose chromatography and gelatin and/or heparin affinity chromatography. Monoclonal BC7 recognizes intrachain disulphide-formed determinants within the 27,000 MW N-terminal domain; monoclonal CE9 recognizes determinants within an 18,000 MW fragment immediately adjacent to the carboxyl end of the gelatin-binding domain; monoclonal BD4 recognizes determinants within the cell-adhesive domain and within 150,000 of the N-terminus; monoclonal AB3 recognizes intrachain disulphide-formed determinants within 35,000 of the COOH-terminus of the intact molecule and detectable only on the alpha-chain polypeptide subunit; and monoclonal CPG1 recognizes determinants present on both chains of the intact molecule and immediately adjacent to the interchain disulphide bonds at the COOH-terminus. None of the epitopes recognized by these monoclonal antibodies is present at alternative regions of the intact molecule. Fab fragments of each of these monoclonal antibodies were incubated with gelatin-coated sheep erythrocytes which had been reacted with a fixed amount of intact plasma fibronectin. When these target particles were incubated with monolayers of human monocytes and the resultant rosettes were quantitated, the Fab fragments of BD4 markedly inhibited the proportion of monocytes binding these fibronectin-bearing targets, whereas none of the other Fab fragments had an inhibitory effect. Thus, monocyte fibronectin receptors which mediate adherence of fibronectin bridges to a target via gelatin recognize regions within the cell-adhesive domains of intact fibronectin but not regions at the amino or carboxy terminals.
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PMID:Identification with monoclonal antibodies of different regions of human plasma fibronectin, including that which interacts with human monocyte fibronectin receptors. 257 23

The polypeptide patterns of MCF-7 human breast cancer cells (MCF-7gpt) and a stably v-H-ras-transfected subclone (MCF-7ras) have been analyzed following estradiol treatment. Since both estradiol and v-H-ras transfection increase tumorigenicity of MCF-7 cells, this study was designed to ascertain if specific changes in polypeptides were common in both treatments. Separation of cellular and secreted polypeptides was accomplished by 2-dimensional polyacrylamide gel electrophoresis, and the consequent patterns were analyzed with computer assistance. Estradiol treatment of the MCF-7gpt cells reduced the number of differences found in the polypeptide patterns between MCF-7gpt and MCF-7ras. Twelve cellular polypeptides were consistently modulated by either estradiol or v-H-ras, with four polypeptides clearly affected in the same way by both treatments. Polypeptides Gchc-0845 (Mr 54,000, pI 6.9) and Gchc-0902 (Mr 52,000, pI 6.3) were suppressed by estradiol and v-H-ras, while Gchc-1240 (Mr 34,000, pI 4.4) and Gchc-1396 (Mr 23,000, pI 5.3) were induced by estradiol and v-H-ras. Sixteen secreted polypeptides were altered by at least 2-fold subsequent to estradiol treatment or v-H-ras transfection. Transfection with v-H-ras had a greater effect than estradiol, stimulating the secretion of eight polypeptides and suppressing the secretion of seven polypeptides compared to estradiol which increased secretion of five polypeptides and decreased secretion of an additional three polypeptides, respectively. Synergistic effects by estradiol and v-H-ras were noted for three polypeptides. The secretion of Gcls-175 (Mr 50,000, pI 5.7) and Gcls-320 (Mr less than 14,000, pI 3.6, p-S2) was increased, while the secretion of Gcls-112 (Mr 76,000, pI 6.9) was decreased. Opposing effects of estradiol and v-H-ras were seen for seven polypeptides including the Mr 48,000 derivative of the Mr 52,000 protein (cathepsin D). These studies support the possibility that an extremely few, but specific polypeptides are regulated in association with quite diverse tumorigenic stimuli in MCF-7 human breast cancer cells.
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PMID:Secreted and cellular polypeptide patterns of MCF-7 human breast cancer cells following either estrogen stimulation or v-H-ras transfection. 264 87

Subcultured rat fibroblasts secreted a cathepsin L precursor when maintained for 24 h in serum-free medium containing 20 mM ammonium ions. The precursor was identified by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using polyclonal antibodies to cathepsin L. The molecular mass of the precursor was found to be approximately 39 kDa, which confirms the result originally reported by Y. Nishimura et al. (1988, Arch. Biochem. Biophys. 263, 107-116). Treatment of the precursor containing medium with cathepsin D at pH values ranging from 3.5 to 5.5 caused a limited cleavage of the precursor molecule. The resultant polypeptides are an unstable intermediate form with Mr 35,000 and a stable single chain form of cathepsin L showing a Mr about 32,500. The cathepsin D-mediated conversion was strongly accelerated by Hg2+ ions. A further proteolytic cleavage of the 32.5-kDa polypeptide has not been observed. The enzymatic activity toward Z-Phe-Arg-NHMec at pH 5.5 increased during the conversion, indicating that active cathepsin L was formed from an inactive precursor molecule.
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PMID:The processing of a cathepsin L precursor in vitro. 275 13

A novel effective procedure for the purification of cathepsin D inhibitor from potatoes (PDI) was developed. The amino acid sequence of PDI was determined by analysis of the cyanogen bromide digest and of the limited tryptic and chymotryptic digest of the protein. The inhibitor is a single polypeptide chain protein consisting of 188 residues with a simple sugar moiety attached to Asn-19. The tentative disulfide pairings are also suggested. The sequence data clearly indicate that PDI is homologous with the soybean trypsin inhibitor (STI) (Kunitz) family. The active center of PDI for trypsin inhibition was identified as Pro-Val-Arg-Phe in analogy to STI.
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PMID:Primary structure of cathepsin D inhibitor from potatoes and its structure relationship to soybean trypsin inhibitor family. 275 67

Cathepsin E (EC 3.4.23.--) has been isolated from rat spleen. The procedure included autolysis at pH 4.2 which was probably the reason why we isolated a polypeptide of Mr 42 kDa instead of 90 kDa. The latter is reported in the literature to be the Mr of native cathepsin E. The enzyme dissociates under reducing conditions in two identical monomers. In our preparation a mechanism different from reduction must be active producing the 42 kDa polypeptide. This enzyme was hard to distinguish from cathepsin D (EC 3.4.23.5.) which shows similar properties such as size, substrate specificity, stability in 6 M urea, and dependence of the activity on pH. The clear distinction between the two enzymes was proven on the basis of immunochemical reactions. Antibodies to both cathepsins, D and E, did not show any crossreaction with the nonrelated antigen.
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PMID:Isolation and some properties of a cathepsin E type proteinase from rat spleen. 277 49

The specificity of action of bovine brain cortex cathepsin D (EC 3.4.23.5) and high-Mr aspartic endopeptidase (EC 3.4.23.-) was studied with the vasoactive peptides renin substrate tetradecapeptide (RSTP), substance P (SP), and angiotensins I and II, and with model peptides--Lys-Pro-Ala-Glu-Phe-Phe (NO2)-Ala-Leu (I), Gly-Gly-His-Phe (NO2)-Phe-Ala-Leu-NH2 (II), and Abz-Ala-Ala-Phe-Phe-pNA (III). Cerebral aspartic peptidases show identical substrate specificity, cleaving the Leu10-Leu bond in RSTP and Phe-Phe in SP and peptide I-III, and not splitting angiotensins I and II. Because of the higher catalytic efficiency of cathepsin D (Kcat value), the specificity constants (Kcat/Km) for cathepsin D-catalyzed hydrolysis of substrates 1-111 are much higher than those for the high-Mr enzyme. High-Mr aspartic peptidase shares a number of properties with cathepsin D (sensitivity to pepstatin, substrate specificity, pH activity profile) and shows partial immunological identity; however, high-Mr aspartic peptidase has a specific activity 7-10 times lower than that of cathepsin D. The kinetic parameters of proteolysis of model peptides presented indicate that the high-Mr enzyme may be a complex of a single-chain cathepsin D with another polypeptide, although the possibility that it is an independent aspartic peptidase cannot be excluded.
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PMID:Substrate specificity of cerebral cathepsin D and high-Mr aspartic endopeptidase. 328 13

Evidence is presented that fibronectin (FN) polypeptide chain contains a latent proteinase. Human plasma FN was cleaved with cathepsin D into three main fragments: 140-kDa and 70-kDa single-chain and 140-kDa double-chain polypeptides. Their separation was achieved according to their affinity for heparin-Sepharose. A single-chain 140-kDa fragment (H-1) was eluted in the first peak. This peptide corresponds to the already described fragment that originates from the central part of FN; it contains a low-affinity heparin-binding site, one free SH group, and a cell-binding site. After reduction and further purification by preparative polyacrylamide gel electrophoresis, this fragment revealed a spontaneous decomposition, which could be attributed to proteolytic degradation. The subfragments, ranging from 25 to 95 kDa, yielded the same proteolytically active doublet of 28-30 kDa when tested by NaDodSO4/polyacrylamide gel electrophoresis in a gel containing copolymerized gelatin or fibrinogen. The proteolytic activity was inhibited by specific SH proteinase inhibitors. The proteinase forms a labeled complex after its incubation with 125I-labeled cystatin. Neither FN, cathepsin D, nor any products from previous purification steps were proteolytically active under the conditions of the assay. It was suggested that the same fragment may also yield an inhibitor, since structural analogies were found between the cell-binding region of FN and SH proteinase inhibitors.
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PMID:Potential proteolytic activity of human plasma fibronectin. 352 28

Cathepsin D occurs in two forms, a single polypeptide chain (Mr 44 000) and a non-covalent complex of two peptides of Mr 14 000 and 30 000 that is derived by proteolytic processing of the 44 000 polypeptide. The two forms from bovine spleen are closely similar in secondary structure content, in aromatic amino acid environment and in the two step denaturation behaviour. Enzyme activity is lost irreversibly on denaturation but conformation can be partially regained. The two separated chains will only refold partially and this is related to their positions in the overall structure of cathepsin D. It is suggested that the processing step is related to protein turnover.
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PMID:Conformation and processing of cathepsin D. 383 Feb 70

Multiple biosynthetic forms of glucocerebrosidase were immunoprecipitated after synthesis in vitro using cell-free translation or in vivo using pulse-chase conditions in porcine kidney cells or human fibroblasts. The initial product in vitro was a 52-kDa polypeptide. When canine pancreatic microsomes were present during translation, the nascent polypeptide crossed the microsomal membrane and increased its mass to 60 kDa. Treatment of the 60-kDa polypeptide with endoglycosidase H to remove high mannose carbohydrate yielded a 51-kDa polypeptide. Thus, the membrane-translocated molecule was apparently a high mannose glycoprotein from which a signal peptide had been cleaved, as observed for the lysosomal protease cathepsin D (Erickson, A. H., and Blobel, G. (1979) J. Biol. Chem. 254, 11771-11774). Treatment of pancreatic microsomes or microsomes from porcine kidney cells with protease did not decrease the size of the polypeptide, which shows that this form is not a transmembrane protein bearing a cytoplasmic domain susceptible to digestion. The in vitro product synthesized in the presence of microsomal membranes was indistinguishable from the in vivo product synthesized during pulse-labeling of cultured porcine kidney cells. Following a 2-h chase period, the 60-kDa product was converted to a 59-kDa polypeptide. The major form of glucocerebrosidase detected after a 24-h chase period was a 56-kDa polypeptide, which in turn was converted to a 55-kDa polypeptide by 72 h. The same forms were precipitated from human fibroblasts but the rate of processing was accelerated in this cell type. Limited treatment of the 60-kDa form of glucocerebrosidase with endoglycosidase H suggested that high mannose carbohydrate is added to at least four sites on the polypeptide chain. By 24 h after synthesis, conversion to endoglycosidase H-resistant complex carbohydrate had occurred. Thus, both polypeptide and carbohydrate processing steps are involved in the biosynthesis of glucocerebrosidase.
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PMID:Biosynthesis of the lysosomal enzyme glucocerebrosidase. 393 53

The proteolytic maturation of cathepsin D polypeptides was studied in lysosomes isolated from metabolically labeled fibroblasts. In lysosomes isolated from fibroblasts labeled with [35S]methionine, 70-95% of labeled cathepsin D polypeptides were represented by a Mr = 47,000 polypeptide after a 20-min pulse and 75-min chase. When these lysosomes were incubated in vitro, up to 70% of the Mr = 47,000 polypeptide was processed to mature cathepsin D polypeptides. The processing was dependent on the integrity of the lysosomes, had an optimum between pH 6 and 7, and could be stimulated by dithiothreitol and ATP. The noncleavable ATP analogue, adenosine 5'-(beta, gamma-imido)triphosphate, and GTP, CTP, and UTP could not substitute for ATP. The ATP-dependent stimulation was associated with an acidification of lysosomes. It was inhibited by agents that dissipate the lysosomal pH gradient (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, N,N'-dicyclohexylcarbodiimide, nigericin, NH4Cl). A stimulatory effect of ATP was observed also at pH 5.5. The stimulation at pH 5.5 was not associated with acidification of lysosomes and was resistant to protonophores. Inhibitors of lysosomal cysteine proteinases and N-ethylmaleimide inhibited the processing. In the presence of ATP the processing activity was partially protected from inhibition by N-ethylmaleimide. In conclusion, the maturation of cathepsin D in lysosomes depends on cysteine proteinases and is stimulated by the ATP-driven acidification of lysosomes. In addition, ATP stimulates maturation at pH 5.5 by a mechanism not involving the proton pump.
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PMID:Processing of human cathepsin D in lysosomes in vitro. 397 22


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