Periodate oxidation of carbohydrate moiety of stem bromelain without much alteration in enzymic activity

Biochemistry ◽  
1971 ◽  
Vol 10 (13) ◽  
pp. 2624-2630 ◽  
Author(s):  
Takashi Murachi ◽  
Yoko Yasuda ◽  
Noriko Takahashi
Keyword(s):  
Periodate Oxidation ◽  
Enzymic Activity ◽  
Carbohydrate Moiety ◽  
Stem Bromelain

The structure and mechanism of action of papain

1970 ◽  
Vol 257 (813) ◽  
pp. 237-248 ◽  
Keyword(s):  
Mechanism Of Action ◽  
Catalytic Mechanism ◽  
Proteolytic Enzymes ◽  
Cysteine Proteinase ◽  
Preceding Paper ◽  
Cysteine Residue ◽  
Enzymic Activity ◽  
Critical Survey ◽  
Cysteine Proteinases ◽  
Stem Bromelain

The cysteine proteinases form a group of enzymes which depend for their enzymic activity on the thiol group of a cysteine residue. Several which occur in plants have been investigated extensively and include papain, ficin and stem bromelain (Smith & Kimmel i960). Although the term papain, introduced last century to describe the proteolytic principle in papaya latex (Wurtz & Bouchut 1879) is still used to describe crude dried latex, the crystalline enzyme is readily obtained (Kimmel & Smith 1954). Ficin is known to consist of several closely related enzymes which have been resolved (Sgarbieri, Gupte, Kramer & Whitaker 1964), but for most structural and mechanistic studies the unresolved mixture of enzymes has been used. Stem bromelain also appears to be a mixture of at least two proteolytic enzymes which have not yet been resolved (Ota, Moore & Stein 1962; Murachi 1964). In spite of the recognized heterogeneity of ficin and stem bromelain, it does seem that both structurally and mechanistically they are similar to papain. Only one bacterial cysteine proteinase has received a detailed study, namely, streptococcal proteinase, and it appears to have little or no relation in its amino acid sequence with the plant enzymes (Liu, Stein, Moore & Elliott 1965). The functional groups involved in the catalytic mechanism are apparently the same as in the plant proteinases (Gerwin, Stein & Moore 1966; Liu 1967; Husain & Lowe 1968 a , c ), but the mechanism of action has not been extensively studied. It may well be however that the plant and bacterial cysteine proteinases have converged onto a similar mechanism of action by two independent evolutionary pathways, as now seems apparent for the animal and bacterial serine proteinases (Alden, Wright & Kraut, this volume, p. 119). Because the tertiary crystal structure of papain (Drenth, Jansonius, Koekoek, Swen & Wolthers 1968; see also the preceding paper, p. 231) is now known, a critical survey of this enzyme is apposite.

Composition and structure of carbohydrate moiety of stem bromelain

Biochemistry ◽  
1970 ◽  
Vol 9 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Yoko Yasuda ◽  
Noriko Takahashi ◽  
Takashi Murachi
Keyword(s):  
Carbohydrate Moiety ◽  
Composition And Structure ◽  
Stem Bromelain

Changes in conformation and enzymic activity of stem bromelain in alkaline media

Biochemistry ◽  
1970 ◽  
Vol 9 (9) ◽  
pp. 1935-1938 ◽  
Author(s):  
Takashi Murachi ◽  
Mitsue Yamazaki
Keyword(s):  
Enzymic Activity ◽  
Alkaline Media ◽  
Stem Bromelain

scholarly journals A non-glycosylated extracellular superoxide dismutase variant

1992 ◽  
Vol 288 (2) ◽  
pp. 451-456 ◽  
Author(s):  
A Edlund ◽  
T Edlund ◽  
K Hjalmarsson ◽  
S L Marklund ◽  
J Sandström ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Culture Media ◽  
Periodic Acid ◽  
Chinese Hamster ◽  
Glycosylation Site ◽  
Enzymic Activity ◽  
Carbohydrate Moiety ◽  
Size Exclusion ◽  
Extracellular Superoxide Dismutase ◽  
Periodic Acid Schiff

The secretory tetrameric extracellular superoxide dismutase (EC-SOD) is the only glycosylated SOD isoenzyme. The importance of the carbohydrate moiety for the properties of the enzyme is unknown. An expression vector defining nonglycosylated EC-SOD (ngEC-SOD) was constructed by mutagenesis of the codon for Asn-89 into a codon for Gln. The vector was transfected into Chinese hamster ovary DXB-11 cells and ngEC-SOD was isolated to 70% purity from the culture media of selected clones. The absence of glycosylation was established by the lack of affinity for various lectins, the absence of staining with the periodic acid-Schiff reagent, the change in mobility and composition of the tryptic peptide containing the mutated glycosylation site, and the reduction in apparent molecular mass upon SDS/PAGE and size-exclusion chromatography. The tetrameric state was retained. The heparin affinity, a fundamental and distinguishing property of EC-SOD, was found to be slightly increased. The enzymic activity was essentially retained. The major difference from native glycosylated enzyme in physical properties was a marked reduction in solubility. Like glycosylated EC-SOD, ngEC-SOD was, after intravenous injection into rabbits, rapidly sequestered by the vessel endothelium, and was promptly released into plasma after injection of heparin. The only difference from glycosylated EC-SOD in this behaviour, was a slightly more rapid elimination of the mutant enzyme from the vasculature. It is concluded that no specific biological role for the EC-SOD carbohydrate moiety could be revealed.

scholarly journals Separation of the bovine colostrum M-1 glycoprotein into two components

1969 ◽  
Vol 115 (4) ◽  
pp. 817-822 ◽  
Author(s):  
Anatoly Bezkorovainy ◽  
Dietmar Grohlich
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Absorption Maximum ◽  
Periodate Oxidation ◽  
Bovine Colostrum ◽  
Carbohydrate Moiety ◽  
Elimination Reaction ◽  
Acid Glycoprotein ◽  
No Absorption

1. Two glycoproteins were isolated from the M-1 acid glycoprotein fraction of bovine colostrum. 2. The lighter glycoprotein had a molecular weight of 7200, contained about 28·4% of carbohydrate, and had an absorption maximum at 275nm. The heavier glycoprotein had a molecular weight of 12000, contained 39·0% of carbohydrate, and had no absorption maxima in the 240–300nm. range of the spectrum. 3. The carbohydrate moiety of both glycoproteins was removable from the polypeptide moiety under the conditions of the β-elimination reaction. 4. Periodate oxidation experiments showed that sialic acid was linked to galactose in both proteins.

scholarly journals The action of glycosylases on dopachrome (2-carboxy-2,3-dihydroindole-5,6-quinone) tautomerase

1992 ◽  
Vol 284 (1) ◽  
pp. 109-113 ◽  
Author(s):  
P Aroca ◽  
J H Martinez-Liarte ◽  
F Solano ◽  
J C García-Borrón ◽  
J A Lozano
Keyword(s):  
Concanavalin A ◽  
Enzymic Activity ◽  
Coated Vesicles ◽  
Carbohydrate Moiety ◽  
Native Enzyme ◽  
Enzyme Treatment ◽  
Strong Affinity ◽  
The Stability ◽  
Beta Galactosidase

It is shown that dopachrome (2-carboxy-2,3-dihydroindole-5,6-quinone) tautomerase (DCT) is a glycoprotein containing N-linked oligosaccharides. The enzymic activity can be stimulated by partial deglycosylation with a number of glycosylases such as neuraminidase, beta-mannosidase and beta-galactosidase. However, the stability of the enzyme after the hydrolytic treatment becomes lower. Thus total deglycosylation with peptide N-glycosidase F directly provokes an inactivation of DCT. The native enzyme also shows a strong affinity for concanavalin A-Sepharose. This affinity decreases after treatment with neuraminidase and/or beta-mannosidase. The DCT associated with coated vesicles seems to be mostly glycosylated, since the action of glycosylases on the enzyme obtained from these vesicles produced a similar stimulation to that with the melanosomal enzyme. Treatment of cultured melanocytes with tunicamycin elicited a decrease in the amount of active DCT inside the cells. All data suggest that the structure of the carbohydrate moiety of DCT should be very similar to, if not identical with, the structure proposed for tyrosinase by Ohkura, Yamashita, Mishima & Kobata (1984) Arch. Biochem. Biophys. 235, 63-77.

Cytochemical Studies of Cell Wall Biosynthesis in Staphylococcus Aureus

1972 ◽  
Vol 30 ◽  
pp. 328-329
Author(s):  
Masaatsu Koike ◽  
Koichi Nakashima ◽  
Kyoko Iida
Keyword(s):  
Staphylococcus Aureus ◽  
Periodate Oxidation ◽  
Early Time ◽  
The Other ◽  
Penicillin G ◽  
Cell Wall Biosynthesis ◽  
Septal Wall ◽  
Peptidoglycan Biosynthesis ◽  
Gram Positive Bacteria ◽  
Cross Linkage

Penicillin exerts the activity to inhibit the peptide cross linkage between each polysaccharide backbone at the final stage of wall-peptidoglycan biosynthesis of bacteria. Morphologically, alterations of the septal wall and mesosome in gram-positive bacteria, which were occurred in early time after treatment with penicillin, have been observed. In this experiment, these alterations were cytochemically investigated by means of silver-methenamine staining after periodate oxidation, which is applied for detection of localization of wall mucopolysaccharide.Staphylococcus aureus strain 209P treated with 100 u/ml of penicillin G was divided into two aliquotes. One was fixed by Kellenberger-Ryter's OSO4 fixative at 30, 60 and 120 min after addition of the antibiotic, dehydrated through alcohol series, and embedded in Epon 812 (Specimen A). The other was fixed by 21 glutaraldehyde, dehydrated through glycolmethacrylate series and embedded in glycolmethacrylate mixture, according to Bernhard's method (Specimen B).

Binding of Tissue Plasminogen Activator to Vascular Grafts

1989 ◽  
Vol 61 (01) ◽  
pp. 131-136 ◽  
Author(s):  
Richard A Harvey ◽  
Hugh C Kim ◽  
Jonathan Pincus ◽  
Stanley Z Trooskin ◽  
Josiah N Wilcox ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Polymer Surface ◽  
Enzymic Activity ◽  
Vascular Prostheses ◽  
Chemically Modified ◽  
Insoluble Surfactant ◽  
Tissue Plasminogen

SummaryTissue plasminogen activator labeled with radioactive iodine (125I-tPA) was immobilized on vascular prostheses chemically modified with a thin coating of water-insoluble surfactant, tridodecylmethylammonium chloride (TDM AC). Surfactant- treated Dacron, polytetrafluoroethylene (PTFE), silastic, polyethylene and polyurethane bound appreciable amounts of 125I- tPA (5-30 μg 125I-tPA/cm2). Upon exposure to human plasma, the amount of 125I-tPA bound to the surface shows an initial drop during the first hour of incubation, followed by a slower, roughly exponential release with a t½ of appoximately 75 hours. Prostheses containing bound tPA show fibrinolytic activity as measured both by lysis of clots formed in vitro, and by hydrolysis of a synthetic polypeptide substrate. Prior to incubation in plasma, tPA bound to a polymer surface has an enzymic activity similar, if not identical to that of the native enzyme in buffered solution. However, exposure to plasma causes a decrease in the fibrinolytic activity of both bound tPA and enzyme released from the surface of the polymer. These data demonstrate that surfactant-treated prostheses can bind tPA, and that these chemically modified devices can act as a slow-release drug delivery system with the potential for reducing prosthesis-induced thromboembolism.

Stem Bromelain Proteolytic Machinery: Study of the Effects of its Components on Fibrin (ogen) and Blood Coagulation

2020 ◽  
Vol 27 (11) ◽  
pp. 1159-1170
Author(s):  
Mohamed Azarkan ◽  
Mariana Marta González ◽  
Rafaèle Calvo Esposito ◽  
María Eugenia Errasti
Keyword(s):  
Blood Coagulation ◽  
Proteolytic Activity ◽  
Fibrinolytic Activity ◽  
Cysteine Proteases ◽  
Antithrombotic Agent ◽  
Proteolytic System ◽  
Total Extract ◽  
Low Concentrations ◽  
Fibrin Plate ◽  
Stem Bromelain

Background: Antiplatelet, anticoagulant and fibrinolytic activities of stem bromelain (EC 3.4.22.4) are well described, but more studies are still required to clearly define its usefulness as an antithrombotic agent. Besides, although some effects of bromelain are linked to its proteolytic activity, few studies were performed taking into account this relationship. Objective: We aimed at comparing the effects of stem bromelain total extract (ET) and of its major proteolytic compounds on fibrinogen, fibrin, and blood coagulation considering the proteolytic activity. Methods: Proteolytic fractions chromatographically separated from ET (acidic bromelains, basic bromelains, and ananains) and their irreversibly inhibited counterparts were assayed. Effects on fibrinogen were electrophoretically and spectrophotometrically evaluated. Fibrinolytic activity was measured by the fibrin plate assay. The effect on blood coagulation was evaluated by the prothrombin time (PT) and activated partial thromboplastin time (APTT) tests. Effects were compared with those of thrombin and plasmin. Results: Acidic bromelains and ananains showed thrombin-type activity and low fibrinolytic activity, with acidic bromelains being the least effective as anticoagulants and fibrinolytics; while basic bromelains, without thrombin-like activity, were the best anticoagulant and fibrinolytic proteases present in ET. Procoagulant action was detected for ET and its proteolytic compounds by the APTT test at low concentrations. The measured effects were dependent on proteolytic activity. Conclusion: Two sub-populations of cysteine proteases exhibiting different effects on fibrin (ogen) and blood coagulation are present in ET. Using well characterized stem bromelain regarding its proteolytic system is a prerequisite for a better understanding of the mechanisms underlying the bromelain action.

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