Limited proteolysis of rabbit cardiac procathepsin D in a cell-free system

1986 ◽  
Vol 250 (4) ◽  
pp. C589-C596 ◽  
Author(s):  
A. M. Smarel ◽  
S. W. Worobec ◽  
A. G. Ferguson ◽  
R. S. Decker ◽  
M. Lesch
Keyword(s):  
Cathepsin B ◽  
Cathepsin D ◽  
Intracellular Transport ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Cysteine Proteases ◽  
Proteolytic Processing ◽  
Reaction Products ◽  
Free System

Rabbit cardiac cathepsin D is initially synthesized as an inactive, apparent molecular weight (Mr) 53,000, pI 6.6 precursor (procathepsin D) that is proteolytically processed during intracellular transport to produce the Mr 48,000 isoforms of active cathepsin D found in cardiac lysosomes. To examine potential proteases responsible for intracellular proteolytic processing, biosynthetically labeled procathepsin D was isolated from rabbit ventricular tissue perfused for 30 min with [35S]methionine. Procathepsin D was then incubated in vitro (40 degrees C, 1-240 min) with active cathepsin D, papain, and cathepsin B, either singly or sequentially, and the reaction products analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. Incubation of 35S-labeled procathepsin D with active cathepsin D produced a single reaction product (Mr 51,000; pI 6.2). This limited proteolysis occurred at pH 3-5 and was inhibited by pepstatin. Incubation of 35S-labeled procathepsin D with papain or cathepsin B produced a major reaction product (Mr 48,000; pI 6.4) and a minor form (Mr 50,000; pI 6.0). These reactions occurred at pH 4-7 and were inhibited by leupeptin but not pepstatin. Only the Mr 48,000, pI 6.4 products of papain and cathepsin B-mediated proteolysis comigrated with the most basic isoform of active cathepsin D found in cardiac tissue. In addition, the Mr 51,000 intermediate produced by cathepsin D was susceptible to further limited proteolysis by cysteine proteases with resultant production of a Mr 48,000 product. Thus the intracellular proteolytic processing of rabbit cardiac procathepsin D does not result solely from autocatalysis but requires at least one other protease, possibly cathepsin B.

Transport and proteolytic processing of rabbit cardiac cathepsin D

1985 ◽  
Vol 248 (1) ◽  
pp. C135-C144
Author(s):  
A. M. Samarel ◽  
A. G. Ferguson ◽  
S. W. Worobec ◽  
M. Lesch
Keyword(s):  
Gel Electrophoresis ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Proteolytic Processing ◽  
Time Dependent ◽  
Low Density ◽  
Enzyme Maturation

Rabbit cardiac cathepsin D is synthesized as a 53,000-mol wt precursor that undergoes limited proteolysis at an unknown intracellular site to a 48,000-mol wt active form. To examine the site of proteolytic processing, isolated perfused rabbit hearts were fractionated by differential centrifugation 150 or 300 min after pulse labeling with [35S]methionine. Newly synthesized precursor and processed cathepsin D were quantitatively isolated from each fraction by extraction, immunoadsorption, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After 30-min pulse perfusions, all of the 35S-labeled cathepsin D was present as precursor, with the greatest amounts found in low-density subcellular fractions. Proteolytic processing of cathepsin D precursor occurred after chase perfusions that were coincident with the subcellular redistribution of newly synthesized enzyme from sites of synthesis to heavier subcellular structures. Pulse-chase perfusions with chloroquine (10 microM) inhibited precursor proteolytic processing and the time-dependent subcellular redistribution of newly synthesized cathepsin D. The data are consistent with a model for cardiac lysosomal enzyme maturation in which limited proteolytic processing occurs coincident with or soon after the transport of precursors to an acidic intracellular compartment. The results thus suggest that cathepsin D proteolytic processing occurs within cardiac lysosomes.

Effects of cysteine protease inhibitors on rabbit cathepsin D maturation

1989 ◽  
Vol 257 (6) ◽  
pp. C1069-C1079 ◽  
Author(s):  
A. M. Samarel ◽  
A. G. Ferguson ◽  
R. S. Decker ◽  
M. Lesch
Keyword(s):  
Protease Inhibitors ◽  
Cysteine Protease ◽  
Cathepsin D ◽  
Intracellular Transport ◽  
Cardiac Fibroblasts ◽  
Primary Cultures ◽  
Cysteine Proteases ◽  
Proteolytic Processing ◽  
Dependent Manner ◽  
Cysteine Protease Inhibitors

To examine the effects of cysteine protease inhibitors on cathepsin D intracellular transport, proteolytic processing, and secretion, primary cultures of rabbit cardiac fibroblasts were grown to confluence and exposed (24 h) to media containing leupeptin (0-10 mM), E 64 (0-10 mM), or chloroquine (0-50 microM). Cathepsin D maturation was then evaluated in pulse-chase biosynthetic labeling experiments. None of the three agents affected the charge modification of procathepsin D (Mr 53,000) within the Golgi apparatus. However, all three agents interfered with the subsequent proteolytic processing of procathepsin D isoforms to active cathepsin D (Mr 48,000). Both leupeptin and E 64 caused the intracellular accumulation of large amounts of a Mr 51,000 processing intermediate (not detectable in control fibroblasts). Trace amounts of this intermediate were also detected in chloroquine-treated cells. Combined activity assay and radioimmunoassay of cell lysates indicated that this partially processed form of cathepsin D possessed proteolytic activity. Whereas low medium concentrations of leupeptin (10-100 microM) but not E 64 appeared to stimulate procathepsin D secretion, neither agent appeared to have a major effect on the rate of proenzyme secretion at doses required to inhibit proteolytic maturation (1-10 mM). Furthermore, pretreatment of cells with 10 mM leupeptin appeared only to delay, but not prevent, the intracellular transport of cathepsin D to lysosomes. In contrast, chloroquine increased procathepsin D secretion in a dose-dependent manner, diverting the majority of newly synthesized procathepsin D from the intracellular protease(s) responsible for proteolytic processing. These results suggest that cysteine proteases participate in the proteolytic maturation of procathepsin D during the transport of newly synthesized enzyme to lysosomes, but cysteine protease-mediated proteolytic processing is not required for cathepsin D activation or lysosomal translocation.

scholarly journals Degradation of myofibrillar proteins by cathepsins B and D

1977 ◽  
Vol 167 (3) ◽  
pp. 811-820 ◽  
Author(s):  
William N. Schwartz ◽  
John W. C. Bird
Keyword(s):  
Skeletal Muscle ◽  
Rat Liver ◽  
Cathepsin B ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Iodoacetic Acid ◽  
Myofibrillar Proteins ◽  
Ph Optimum ◽  
Molecular Weights

1. The procedure of Barrett [(1973) Biochem. J.131, 809–822] for isolating cathepsins B and D from human liver was modified for use with rat liver and skeletal muscle. The purified enzymes appeared to be similar to those reported in other species. 2. Sephadex G-75 chromatography of concentrated muscle extract resolved two peaks of cathepsin B inhibitory activity, corresponding to molecular weights of 12500 and 62000. 3. The degradation of purified myofibrillar proteins by cathepsins B and D was clearly demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. After incubation with enzyme, the polypeptide bands representing the substrates decreased in intensity and lower molecular weight products appeared. 4. Cathepsins B and D, purified from either rat liver or skeletal muscle, were shown to degrade myosin, purified from either rabbit or rat muscle. Soluble denatured myosin was degraded more extensively than insoluble native myosin. Degradation by cathepsin B was inhibited by lack of reducing agent, or by myoglobin, iodoacetic acid and leupeptin, but not by pepstatin. The same potential modifiers were applied to cathepsin D, and only pepstatin produced inhibition. 5. Rat liver cathepsin B had a pH optimum of 5.2 on native rabbit myosin. The pH optimum of cathepsin D was 4.0, with a shoulder of activity about 1pH unit above the optimum. 6. Rat liver cathepsins B and D were demonstrated to degrade rabbit F-actin at pH5.0, and were inhibited by leupeptin and pepstain, respectively. 7. The degradation of myosin and actin by cathepsin D was more extensive than that by cathepsin B.

scholarly journals Inhibition of cysteine proteinases and dipeptidyl peptidase I by egg-white cystatin

1984 ◽  
Vol 223 (1) ◽  
pp. 245-253 ◽  
Author(s):  
M J H Nicklin ◽  
A J Barrett
Keyword(s):  
Cathepsin B ◽  
Bond Cleavage ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Dipeptidyl Peptidase ◽  
Egg White ◽  
Reversible Inhibitor ◽  
Cysteine Proteinases ◽  
Peptide Bond Cleavage ◽  
Specific Oxidation

The interactions between egg-white cystatin and the cysteine proteinases papain, human cathepsin B and bovine dipeptidyl peptidase I were studied. Cystatin was shown to be a competitive reversible inhibitor of cathepsin B (Ki 1.7 nM, k-1 about 2.3×10(-3) s-1). The inhibition of dipeptidyl peptidase I was shown to be reversible (Ki(app.) 0.22 nM, k-1 about 2.2×10(-3) s-1). Cystatin bound papain too tightly for Ki to be determined, but an upper limit of 5 pM was estimated. The association was a second-order process, with k+1 1.0×10(7) M-1×s-1. Papain was shown to form equimolar complexes with cystatin. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of complexes formed between papain or cathepsin B and an excess of cystatin showed no peptide bond cleavage after incubation for 72 h. The reaction of the active-site thiol group of papain with 5,5′-dithiobis-(2-nitrobenzoic acid) at pH 8 and 2,2′-dithiobispyridine at pH 4 was blocked by complex-formation. Dipeptidyl peptidase I and papain were found to compete for binding to cystatin, contrary to a previous report. The two major isoelectric forms of cystatin were found to have similar specific inhibitory activities for papain, and similar affinities for papain, cathepsin B and dipeptidyl peptidase I. This, together with specific oxidation of the N-terminal serine residue with periodate, showed the N-terminal amino group of cystatin 1 to be unimportant for inhibition. General citraconylation of amino groups resulted in a large decrease in the affinity of cystatin for dipeptidyl peptidase I. It is concluded that the interaction of cystatin with cysteine proteinases has many characteristics similar to those of an inhibitor such as aprotinin with serine proteinases.

Exosite-dependent regulation of factor VIIIa by activated protein C

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4802-4807 ◽  
Author(s):  
Chandrashekhara Manithody ◽  
Philip J. Fay ◽  
Alireza R. Rezaie
Keyword(s):  
Protein C ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Activated Protein C ◽  
Coagulation Cascade ◽  
Factor Va ◽  
Factor Viiia

AbstractActivated protein C (APC) is a natural anticoagulant serine protease in plasma that down-regulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. Recent results have indicated that basic residues of 2 surface loops known as the 39-loop (Lys37-Lys39) and the Ca2+-binding 70-80–loop (Arg74 and Arg75) are critical for the anticoagulant function of APC. Kinetics of factor Va degradation by APC mutants in purified systems have demonstrated that basic residues of these loops are involved in determination of the cleavage specificity of the Arg506 scissile bond on the A2 domain of factor Va. In this study, we characterized the properties of the same exosite mutants of APC with respect to their ability to interact with factor VIIIa. Time course of the factor VIIIa degradation by APC mutants suggested that the same basic residues of APC are also critical for recognition and degradation of factor VIIIa. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of the factor VIIIa cleavage reactions revealed that these residues are involved in determination of the specificity of both A1 and A2 subunits in factor VIIIa, thus facilitating the cleavages of both Arg336 and Arg562 scissile bonds in the cofactor.

scholarly journals Purification and Characterization of Chitosanase from Bacillus sp. Strain KCTC 0377BP and Its Application for the Production of Chitosan Oligosaccharides

2004 ◽  
Vol 70 (8) ◽  
pp. 4522-4531 ◽  
Author(s):  
Yeon Jin Choi ◽  
Eun Jung Kim ◽  
Zhe Piao ◽  
Young Chul Yun ◽  
Yong Chul Shin
Keyword(s):  
Liquid Chromatography ◽  
Glycoside Hydrolase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Glycoside Hydrolase Family ◽  
Reaction Products ◽  
Enzymatic Production ◽  
Chitosan Oligosaccharides ◽  
Hydrolase Family

ABSTRACT For the enzymatic production of chitosan oligosaccharides from chitosan, a chitosanase-producing bacterium, Bacillus sp. strain KCTC 0377BP, was isolated from soil. The bacterium constitutively produced chitosanase in a culture medium without chitosan as an inducer. The production of chitosanase was increased from 1.2 U/ml in a minimal chitosan medium to 100 U/ml by optimizing the culture conditions. The chitosanase was purified from a culture supernatant by using CM-Toyopearl column chromatography and a Superose 12HR column for fast-performance liquid chromatography and was characterized according to its enzyme properties. The molecular mass of the enzyme was estimated to be 45 kDa by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme demonstrated bifunctional chitosanase-glucanase activities, although it showed very low glucanase activity, with less than 3% of the chitosanase activity. Activity of the enzyme increased with an increase of the degrees of deacetylation (DDA) of the chitosan substrate. However, the enzyme still retained 72% of its relative activity toward the 39% DDA of chitosan, compared with the activity of the 94% DDA of chitosan. The enzyme produced chitosan oligosaccharides from chitosan, ranging mainly from chitotriose to chitooctaose. By controlling the reaction time and by monitoring the reaction products with gel filtration high-performance liquid chromatography, chitosan oligosaccharides with a desired oligosaccharide content and composition were obtained. In addition, the enzyme was efficiently used for the production of low-molecular-weight chitosan and highly acetylated chitosan oligosaccharides. A gene (csn45) encoding chitosanase was cloned, sequenced, and compared with other functionally related genes. The deduced amino acid sequence of csn45 was dissimilar to those of the classical chitosanase belonging to glycoside hydrolase family 46 but was similar to glucanases classified with glycoside hydrolase family 8.

scholarly journals Proteolytic Processing of Sapovirus ORF1 Polyprotein

2005 ◽  
Vol 79 (12) ◽  
pp. 7283-7290 ◽  
Author(s):  
Tomoichiro Oka ◽  
Kazuhiko Katayama ◽  
Satoko Ogawa ◽  
Grant S. Hansman ◽  
Tsutomu Kageyama ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteolytic Processing ◽  
Open Reading Frames ◽  
Rabbit Hemorrhagic Disease Virus ◽  
Translation System ◽  
Hemorrhagic Disease ◽  
Rabbit Hemorrhagic Disease ◽  
Capsid Protein Vp1

ABSTRACT The genome of Sapovirus (SaV), a causative agent of gastroenteritis in humans and swine, contains either two or three open reading frames (ORFs). Functional motifs characteristic to the 2C-like NTPase (NTPase), VPg, 3C-like protease (Pro), 3D-like RNA-dependent RNA polymerase (Pol), and capsid protein (VP1) are encoded in the ORF1 polyprotein, which is afterwards cleaved into the nonstructural and structural proteins. We recently determined the complete genome sequence of a novel human SaV strain, Mc10, which has two ORFs. To investigate the proteolytic cleavage of SaV ORF1 and the function of protease on the cleavage, both full-length and truncated forms of the ORF1 polyprotein either with or without mutation in 1171Cys to Ala of the GDCG motif were expressed in an in vitro coupled transcription-translation system. The translation products were analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by immunoprecipitation with region-specific antibodies. The ORF1 polyprotein was processed into at least 10 major proteins: p11, p28, p35, p32, p14, p70, p60, p66, p46, and p120. Seven of these products were arranged in the following order: NH2-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. p66, p46 and p120 were precursors of p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol), respectively. Mutagenesis in the 3C-like protease motif fully abolished the proteolytic activity. The cleavage map of SaV ORF1 is similar to those of other heretofore known members of the family Caliciviridae, especially to rabbit hemorrhagic disease virus, a member of the genus Lagovirus.

scholarly journals Impaired secretion of mutant alpha 2-plasmin inhibitor (alpha 2 PI- Nara) from COS-7 and HepG2 cells: molecular and cellular basis for hereditary deficiency of alpha 2-plasmin inhibitor

Blood ◽  
1990 ◽  
Vol 75 (5) ◽  
pp. 1092-1096 ◽  
Author(s):  
O Miura ◽  
N Aoki
Keyword(s):  
Cell Line ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Hepatoma Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Secretory Process ◽  
Hepatoma Cell Line ◽  
Pi Deficiency ◽  
Plasmin Inhibitor

Abstract The elongated mutant of alpha 2-plasmin inhibitor (alpha 2 PI) designated as alpha 2 PI-Nara is caused by a frameshift mutation found near the 3′ end of the coding region of the alpha 2 PI gene. To elucidate the mechanism by which this molecular abnormality leads to alpha 2 PI deficiency in plasma, we transfected an expression plasmid for alpha 2 PI-Nara into a monkey kidney cell line COS-7 or human hepatoma cell line HepG2 synthesizing alpha 2 PI, and analyzed the secretory process of the expressed alpha 2 PI-Nara by radioimmunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography. The results obtained showed that the recombinant alpha 2 PI-Nara was retained within the cells for prolonged periods as an endoglycosidase H- sensitive precursor form, and only a small portion of the recombinant protein was secreted into the medium as a neuraminidase-sensitive mature form. These results suggest that instead of being secreted from the cells, most of the alpha 2 PI-Nara undergoes degradation within the cells while its transport is retarded in the intracellular secretory pathway; thus, alpha 2 PI-Nara should lead to the alpha 2 PI deficiency primarily by causing a block in the intracellular transport from the endoplasmic reticulum to the Golgi complex.

scholarly journals Simultaneous Ultrasound and Heat Enhance Functional Properties of Glycosylated Lactoferrin

Molecules ◽  
2020 ◽  
Vol 25 (23) ◽  
pp. 5774
Author(s):  
Zhipeng Li ◽  
Dexue Ma ◽  
Yiyang He ◽  
Siqi Guo ◽  
Fuguo Liu ◽  
...  
Keyword(s):  
Functional Properties ◽  
Maillard Reaction ◽  
Spatial Configuration ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Hydrophobicity ◽  
Orthogonal Experimental Design ◽  
Maillard Reaction Products ◽  
Reaction Products ◽  
Ultrasound Assisted

Protein-polysaccharide covalent complexes exhibit better physicochemical and functional properties than single protein or polysaccharide. To promote the formation of the covalent complex from lactoferrin (LF) and beet pectin (BP), we enhanced the Maillard reaction between LF and BP by using an ultrasound-assisted treatment and studied the structure and functional properties of the resulting product. The reaction conditions were optimized by an orthogonal experimental design, and the highest grafting degree of 55.36% was obtained by ultrasonic treatment at 300 W for 20 min and at LF concentration of 20 g/L and BP concentration of 9 g/L. The formation of LF-BP conjugates was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FTIR) spectroscopy. Ultrasound-assisted treatment can increase the surface hydrophobicity, browning index, 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2’-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) free radicals scavenging activity of LF due to the changes in the spatial configuration and formation of Maillard reaction products. The thermal stability, antioxidant activity and emulsifying property of LF were significantly improved after combining with BP. These findings reveal the potential application of modified proteins by ultrasonic and heat treatment.

scholarly journals Two-step affinity-chromatographic purification of cathepsin D from pig myometrium with high yield

1981 ◽  
Vol 197 (2) ◽  
pp. 519-522 ◽  
Author(s):  
E G Afting ◽  
M L Recker
Keyword(s):  
Molecular Weight ◽  
Concanavalin A ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Yield ◽  
Acid Proteinase ◽  
Chromatographic Purification

Cathepsin D was purified by two-step affinity chromatography on concanavalin A- and pepstatin-Sepharose. The main purification was achieved by washing the enzyme bound to the pepstatin-Sepharose column with buffered 6 M-urea. This step separated cathepsin D from all low- and high-molecular-weight impurities. Although the 1700-fold purified acid proteinase was homogeneous on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it still showed microheterogeneity.

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