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At least 757 records · Page 42Linked to original sources
PubMed1980

Surface antigenic characteristics of human melanoma cells defined by xenoantiserum raised against papain-solubilized melanoma-associated antigens.

Xenoantiserum to human malignant melanoma was prepared by immunizing rabbits with melanoma associated antigens (MAA) solubilized from melanoma cell membrane by limited papain digestion. The antiserum was absorbed extensively with red cells, leukemia cells and cultured lymphoid cell lines, and was assayed for its reactivity with different human cell types by the indirect membrane immunofluorescence and radioimmunoprecipitation techniques. The data obtained suggest that there are at least two different MAA on human melanoma cells. The first is melanoma-group specific and can be detected commonly on different melanoma cell lines. The second is oncofetal and is shared by melanoma, carcinoma, and fetal cells tested thus far. Immunoprecipitated material from melanoma cell membrane that had been radioiodinated with lactoperoxidase and solubilized with a non-ionic detergent was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed two major peaks with estimated molecular weights of 90,000 and 120,000 daltons. The 90,000 molecular weight component appears to be oncofetal, as it disappeared when the antiserum was absorbed with either melanoma or carcinoma cell lines.

Animals↗
PubMed1982

Low molecular weight trypsin-plasmin inhibitors isolated from papain treated urinary trypsin inhibitor.

Papain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chrymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotryspin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10(-7) - 2.12 X 10(-6) M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors of plasmin was thought to be different from that to trypsin or chymotrypsin.

Amino Acids↗
PubMed1982

The reaction of cartilage and osteophyte formation after the intraarticular injection of papain.

A histological and autoradiographical study on the reaction of chondrocytes and the growing processes of osteophyte after the intraarticular injection of papain was carried out. The degenerating processes--fibrillation, death of chondrocytes and erosion--were observed as early as 1 day after the injection, and acid mucopolysaccharide staining with safranin-O was completely lost throughout the articular cartilage 1 week after the injection. The regenerating or proliferating figures--chondrocytes mitosis, thickening of fibrous tissue--were observed as early as 3 days after the injection and chondrocyte clusters were noted after 1 week of injection. Four weeks after injection, proliferating processes were more obvious than degenerating processes, safranin-O stainability had recovered and the proliferative figures of cartilage and fibrous tissue became more obvious. As blood vessels invaded into the cartilage and thickened fibrous tissue, osteophyte began to form in these tissues. Four types of growing processes of osteophyte were observed. Hypertrophy of articular cartilage was observed, and new hyaline articular cartilage appeared to be formed through metaplasia from fibrous tissue. Including the osteophyte formation, tremendous regenerating or repairing efforts of chondrocytes and fibroblasts were observed. These results appear to indicate that cartilage has some ability to regenerate and to repair itself if it could avoid further damage.

Animals↗
PubMed1981

Transplantation in miniature swine. IX. Swine histocompatibility antigens: isolation and purification of papain-solubilized SLA antigens.

Histocompatibility antigens have been purified by papain treatment of crude cellular membranes obtained from spleen and mesenteric lymph nodes of individual miniature swine that are homozygous at the major histocompatibility complex. The purification protocol included ultracentrifugation, gel filtration, ion-exchange chromatography, and in some instances preparative isoelectric focusing. This procedure was applied to the preparation of sLA antigens from the 3 haplotypes available in our partially inbred miniature swine herd. The purity of the SLAdd molecules was evidenced by: 1) a single activity peak of approximately 50,000 daltons on gel-permeation chromatography; 2) the antigen elution as a symmetrical peak from ion-exchange resins and from isoelectric focusing columns; 3) the presence of 2 predominant polypeptide chains on SDS-PAGE at apparent m.w. 43,000 and 13,000 daltons; and 4) specific immunoprecipitations by alloantisera and by anti-beta 1 microglobulin antisera. The antigenic activity of SLA products was analyzed by inhibition of complement-mediated cytotoxicity and by removal on anti-beta 2-microglobulin affinity columns. Heavy and light chains were separated by gel-filtration on Sephacryl S-200 in 6 molar guanidine. These SLA products, which have thus been shown to be pure by a variety of criteria, are now readily available for structural analysis and for analysis of biologic activity.

Animals↗
PubMed1980

Papain hydrolysis products in four M-IgG subclasses.

Four different classes of monoclonal IgG were submitted to papain digestion activated by cysteine. After digestion of subclasses G-1, 3 and 4 about 80% of the initial amount of IgG is converted to fragments Fab and Fc (less in subclass IgG2). In the initial time of digestion subfragments: Fc', Facb-like and nonidentified product of digestion (fraction IV) are also obtained. Additional products (besides Fab and Fc) corresponded to 11-17% of the initial amount of IgG. In contrast to fragments Fab and Fc the amounts of additional fragments did not increase during the time of digestion and the amount of fragment Facb-like in G-1 and G-3 subclasses even decreased at the end of hydrolysis. It is possible that fragment Facb-like is an intermediate product of immunoglobulin digestion. IgG2 was resistant in 60% to 18 h--hydrolysis performed under conditions described. Only 20% of IgG2 was digested to Fab and Fc fragments and 20% to other products of digestion.

Chromatography, Gel↗
PubMed1975

[Fatty acids from papain fragments of normal and myeloma immunoglobulins G].

Studying the lipids bound on normal and myelomatous IgG, we have compared, using gas-liquid chromatography, the fatty acids extracted from untreated proteins, from papain hydrolysis and from Fab and Fc fragments. These last have been purified with affinity chromatography. We have been able, using mass spectrometry, to identify one of the various unknown compounds: it is di-octyl-phthlate.

Chromatography, Gas↗
PubMed1994

Papain digestion of crude Trichoderma reesei cellulase: purification and properties of cellobiohydrolase I and II core proteins.

The major cellulase components produced by Trichoderma reesei are composed of distinct catalytic and cellulose-binding domains. A simple two-step procedure is described for the purification of the catalytic domains, also termed core proteins (cp), of the major components, cellobiohydrolase (CBH) I and II. The novel aspect of this procedure is that native CBH I and II do not have to be purified initially. Papain digestion of a commercial T. reesei cellulase preparation followed by gel filtration on a Superdex 75 column resulted in the separation of fractions containing CBH I cp and CBH II cp; chromatofocusing purified the latter to homogeneity. N-terminal protein sequencing of CBH II cp provided good evidence for its identity. A comparison of the catalytic activity and cellulose-binding ability of these cp was made. A major difference between them was that CBH II cp bound to microcrystalline cellulose, unlike CBH I cp. CBH I cp readily hydrolysed the bond between the aglycone and cellobiose in p-nitrophenyl cellobioside unlike the CBH II cp preparation. Neither CBH I cp nor CBH II cp had activity toward carboxymethylcellulose, but both were able to hydrolyse barley beta-glucan. It was also shown that incubation of cellulose fibres with native CBH I, CBH I cp or CBH II cp resulted in a smoothing of the fibre surface.

Adsorption↗
PubMed1997

Crystallization and preliminary crystallographic studies of a new crystal form of papain from Carica papaya.

A new crystal form of papain from the latex of Carica papaya, complexed with an inhibitor (Z-Arg-Leu-Val-Gly-CHN2) was obtained by the vapor-diffusion method using a methanol/ethanol mixture as a precipitant. The slat-like crystals are monoclinic, space group P2(1), with unit cell parameters a = 42.6 A, b = 49.8 A, c = 50.5 A, A = 111.9 degrees, and contain one molecule in the asymmetric unit. The crystals are stable in the X-ray beam and diffract beyond 1.8 A. A molecular model has been placed in the unit cell by molecular replacement.

Crystallography, X-Ray↗
PubMed1996

Chemoselective Removal of Protecting Groups from O-Glycosyl Amino Acid and Peptide (Methoxyethoxy)ethyl Esters Using Lipases and Papain.

The selective C-terminal deprotection of O-glycopeptide (methoxyethoxy)ethyl esters is achieved under mild conditions (pH 6.6, 37 degrees C) by enzymatic hydrolysis using papain or lipase M from Mucor javanicus to give building blocks useful for chain-extending glycopeptide synthesis. On the other hand, the selective removal of acetyl protecting groups from the saccharide portion of glycopeptides is accomplished by alternative enzymatic hydrolysis with lipase WG from wheat germ to furnish model substrates for enzymatic glycosyl transfer reactions in order to extend the carbohydrate side chain of these conjugates.

Journal Article↗
PubMed1984

Natural structural variation in enzymes as a tool in the study of mechanism exemplified by a comparison of the catalytic-site structure and characteristics of cathepsin B and papain. pH-dependent kinetics of the reactions of cathepsin B from bovine spleen and from rat liver with a thiol-specific two-protonic-state probe (2,2'-dipyridyl disulphide) and with a specific synthetic substrate (N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide).

Cathepsin B (EC 3.4.22.1) from bovine spleen and the analogous enzyme from rat liver were investigated at 25 degrees C at I0.1 in acidic media by kinetic study of (a) the reactions of their catalytic-site thiol groups towards the two-protonic-state reactivity probe 2,2'-dipyridyl disulphide and (b) their catalysis of the hydrolysis of N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide. Reactivity-probe kinetics showed that nucleophilic character is generated in the sulphur atom of cathepsin B by protonic dissociation with pKa 3.4, presumably to form an S-/ImH+ ion-pair. Substrate-catalysis kinetics showed that ion-pair formation is not sufficient to generate catalytic competence in cathepsin B, because catalytic activity is not generated as the pH is raised across pKa 3.4 but rather as it is raised across pKa 5-6 (5.1 for kcat; 5.6 for kcat./Km for the bovine spleen enzyme and 5.8 for kcat./Km for the rat liver enzyme). The implications of these results and of known structural differences between the catalytic sites of the rat liver enzyme and papain (EC 3.4.22.2) for the mechanism of cysteine-proteinase-catalysed hydrolysis are discussed.

2,2'-Dipyridyl↗
PubMed2006

Synergism between a mycoreovirus and a hypovirus mediated by the papain-like protease p29 of the prototypic hypovirus CHV1-EP713.

Infection of the chestnut blight fungus, Cryphonectria parasitica, by the prototypic hypovirus Cryphonectria hypovirus 1-EP713 (CHV1-EP713) or by the type member, Mycoreovirus 1-Cp9B21 (MyRV1-Cp9B21), of a novel genus (Mycoreovirus) of the family Reoviridae results in hypovirulence, but with a different spectrum of phenotypic changes. The former virus depresses pigmentation and conidiation dramatically, whilst the latter virus has little effect on these processes. This study showed that double infection by the two viruses resulted in a phenotype similar to that of CHV1-EP713 singly infected colonies, but with further decreased levels of host conidiation and vegetative growth and increased levels of MyRV1-Cp9B21 genomic dsRNA accumulation (twofold) and vertical transmission (sixfold). In contrast, CHV1-EP713 RNA accumulation was not altered by MyRV1-Cp9B21 infection. It was also found that the papain-like cysteine protease p29, encoded by CHV1-EP713 ORF A, contributes to the phenotypic alterations and transactivation of MyRV1-Cp9B21 replication and transmission. Chromosomally expressed p29 was able to increase MyRV1-Cp9B21 vertical transmission by more than twofold and genomic RNA accumulation by 80 %. Transactivation was abolished by Cys-->Gly mutations at p29 residues 70 and 72 located within the previously identified symptom-determinant domain required for suppression of host pigmentation and sporulation and p29-mediated in trans enhancement of homologous Deltap29 mutant virus RNA replication. Transactivation was not altered by Ser substitutions at the p29 protease catalytic residue Cys(162). These results indicated a link between p29-mediated enhancement of heterologous virus accumulation and transmission and p29-mediated host symptom expression. The role of p29 as a suppressor of RNA silencing is discussed.

Amino Acid Substitution↗
PubMed2004

Activity profiling of papain-like cysteine proteases in plants.

Transcriptomic and proteomic technologies are generating a wealth of data that are frequently used by scientists to predict the function of proteins based on their expression or presence. However, activity of many proteins, such as transcription factors, kinases, and proteases, depends on posttranslational modifications that frequently are not detected by these technologies. Therefore, to monitor activity of proteases rather than their abundance, we introduce protease activity profiling in plants. This technology is based on the use of biotinylated, irreversible protease inhibitors that react with active proteases in a mechanism-based manner. Using a biotinylated derivative of the Cys protease inhibitor E-64, we display simultaneous activities of many papain-like Cys proteases in extracts from various tissues and from different plant species. Labeling is pH dependent, stimulated with reducing agents, and inhibited specifically by Cys protease inhibitors but not by inhibitors of other protease classes. Using one-step affinity capture of biotinylated proteases followed by sequencing mass spectrometry, we identified proteases that include xylem-specific XCP2, desiccation-induced RD21, and cathepsin B- and aleurain-like proteases. Together, these results demonstrate that this technology can identify differentially activated proteases and/or characterize the activity of a particular protease within complex mixtures.

Amino Acid Sequence↗
PubMed2006

Hypovirus papain-like protease p29 suppresses RNA silencing in the natural fungal host and in a heterologous plant system.

Virulence-attenuating hypoviruses of the species Cryphonectria hypovirus 1 (CHV1) encode a papain-like protease, p29, that shares similarities with the potyvirus-encoded suppressor of RNA silencing HC-Pro. We now report that hypovirus CHV1-EP713-encoded p29 can suppress RNA silencing in the natural host, the chestnut blight fungus Cryphonectria parasitica. Hairpin RNA-triggered silencing was suppressed in C. parasitica strains expressing p29, and transformation of a transgenic green fluorescent protein (GFP)-silenced strain with p29 resulted in an increased number of transformants with elevated GFP expression levels. The CHV1-EP713 p29 protein was also shown to suppress both virus-induced and agroinfiltration-induced RNA silencing and systemic spread of silencing in GFP-expressing transgenic Nicotiana benthamiana line 16c plants. The demonstration that a mycovirus encodes a suppressor of RNA silencing provides circumstantial evidence that RNA silencing in fungi may serve as an antiviral defense mechanism. The observation that a phylogenetically conserved protein of related plant and fungal viruses functions as a suppressor of RNA silencing in both fungi and plants indicates a level of conservation of the mechanisms underlying RNA silencing in these two groups of organisms.

Ascomycota↗
PubMed1997

Characterization of a second cleavage site and demonstration of activity in trans by the papain-like proteinase of the murine coronavirus mouse hepatitis virus strain A59.

The 21.7-kb replicase locus of mouse hepatitis virus strain A59 (MHV-A59) encodes several putative functional domains, including three proteinase domains. Encoded closest to the 5' terminus of this locus is the first papain-like proteinase (PLP-1) (S. C. Baker et al., J. Virol. 67:6056-6063, 1993; H.-J. Lee et al., Virology 180:567-582, 1991). This cysteine proteinase is responsible for the in vitro cleavage of p28, a polypeptide that is also present in MHV-A59-infected cells. Cleavage at a second site was recently reported for this proteinase (P. J. Bonilla et al., Virology 209:489-497, 1995). This new cleavage site maps to the same region as the predicted site of the C terminus of p65, a viral polypeptide detected in infected cells. In this study, microsequencing analysis of the radiolabeled downstream cleavage product and deletion mutagenesis analysis were used to identify the scissile bond of the second cleavage site to between Ala832 and Gly833. The effects of mutations between the P5 and P2' positions on the processing at the second cleavage site were analyzed. Most substitutions at the P4, P3, P2, and P2' positions were permissive for cleavage. With the exceptions of a conservative P1 mutation, Ala832Gly, and a conservative P5 mutation, Arg828Lys, substitutions at the P5, P1, and P1' positions severely diminished second-site proteolysis. Mutants in which the p28 cleavage site (Gly247 / Val248) was replaced by the Ala832 / Gly833 cleavage site and vice versa were found to retain processing activity. Contrary to previous reports, we determined that the PLP-1 has the ability to process in trans at either the p28 site or both cleavage sites, depending on the choice of substrate. The results from this study suggest a greater role by the PLP-1 in the processing of the replicase locus in vivo.

Amino Acid Sequence↗
PubMed2000

Identification of mouse hepatitis virus papain-like proteinase 2 activity.

Mouse hepatitis virus (MHV) is a 31-kb positive-strand RNA virus that is replicated in the cytoplasm of infected cells by a viral RNA-dependent RNA polymerase, termed the replicase. The replicase is encoded in the 5'-most 22 kb of the genomic RNA, which is translated to produce a polyprotein of >800 kDa. The replicase polyprotein is extensively processed by viral and perhaps cellular proteinases to give rise to a functional replicase complex. To date, two of the MHV replicase-encoded proteinases, papain-like proteinase 1 (PLP1) and the poliovirus 3C-like proteinase (3CLpro), have been shown to process the replicase polyprotein. In this report, we describe the cloning, expression, and activity of the third MHV proteinase domain, PLP2. We show that PLP2 cleaves a substrate encoding the first predicted membrane-spanning domain (MP1) of the replicase polyprotein. Cleavage of MP1 and release of a 150-kDa intermediate, p150, are likely to be important for embedding the replicase complex in cellular membranes. Using an antiserum (anti-D11) directed against the C terminus of the MP1 domain, we verified that p150 encompasses the MP1 domain and identified a 44-kDa protein (p44) as a processed product of p150. Pulse-chase experiments showed that p150 is rapidly generated in MHV-infected cells and that p44 is processed from the p150 precursor. Protease inhibitor studies revealed that unlike 3CLpro activity, PLP2 activity is not sensitive to cysteine protease inhibitor E64d. Furthermore, coexpression studies using the PLP2 domain and a substrate encoding the MP1 cleavage site showed that PLP2 acts efficiently in trans. Site-directed mutagenesis studies confirmed the identification of cysteine 1715 as a catalytic residue of PLP2. This study is the first to report enzymatic activity of the PLP2 domain and to demonstrate that three distinct viral proteinase activities process the MHV replicase polyprotein.

Animals↗
PubMed1982

Comparison of a homemade blood culture broth containing a papain digest of liver, with four commercially available media for the isolation of anaerobes from simulated paediatric blood cultures.

The recovery of small inocula of fastidious organisms, mainly non-sporing anaerobes, was studied in simulated paediatric blood culture experiments where only 1.5 ml of blood was added to each broth. A 25 ml homemade Queen Elizabeth Hospital medium (QEH medium) containing a papain digest of liver showed the best overall performance during the first four days of incubation; this medium was also satisfactory for maintenance of the majority of the organisms tested for longer than one week. LAB M Fastidious Anaerobe Broth (75 ml) with thymidine, also showed early isolation and satisfactory survival of most organisms. Difco Thiol broth, 50 ml with Liquoid, yielded early growth of the three strains of Bacteroides fragilis tested and maintained these organisms well; however, variable results were obtained with some other organisms in Difco Thiol media. Southern Group Brewer's thioglycollate broth (80 ml) gave the least satisfactory performance.

Aerobiosis↗
PubMed1987

Papain-induced changes in intracellular lipid levels in cartilage.

Loss of proteoglycans from the cartilage matrix of the ears of young rabbits was induced by intravenous injection of papain so as to permit histochemical observation of lipid levels in chondrocytes during both the depletion process and subsequent proteoglycan synthesis by the chondrocytes. Lipid content was quantified using an image analyser. Between 4 and 24 h after injection, loss of proteoglycans was accompanied by a fall in intracellular fat levels. In the 24- to 120-hour post-injection period, matrix proteoglycan levels and basophilia recovered parallel to the intracellular fat content. The function of intracellular lipids in cartilage is discussed in the light of these results.

Animals↗