Digestive Enzymes of the American Lobster (Homarus americanus)

1970 ◽  
Vol 27 (8) ◽  
pp. 1357-1370 ◽  
Author(s):  
H. Brockerhoff ◽  
R. J. Hoyle ◽  
P. C. Hwang
Keyword(s):  
Gastric Juice ◽  
Proteolytic Activity ◽  
Deae Cellulose ◽  
Homarus Americanus ◽  
Enzymic Activity ◽  
Exchange Chromatography ◽  
Ribonuclease P ◽  
Carboxypeptidase A ◽  
Molecular Weights ◽  
Nitrophenyl Phosphate

Gastric juice of lobster hydrolyzed the following substrates: triolein (enzymic activity: lipase), tributyrin ("lipase"), Azocoll(R) (proteinase), TAME and BAEE ("trypsin"), ATEE ("chymotrypsin"), HPLA ("carboxypeptidase A"), DNA (deoxyribonuclease), RNA (ribonuclease), p-nitrophenyl-phosphate (phosphatase), and p-nitrophenyl-N-acetyl-β-glucosaminide (chitobiase). (The quotation marks signify that the substrate specificities are typical of the quoted enzymes of mammalian or microbial origin. The spectra of specificities, however, of these enzymes and the corresponding enzymes of lobster are not necessarily identical.) Very low activities were found against RBB-starch (amylase) p-nitro-phenyl-α-(and β)-glucopyranoside(α- and β-glucosidase), p-nitrophenyl-β-galactopyranoside (β-galactosidase), and chitin azure (chitinase). No activities corresponding to phospholipase (lecithin), carboxypeptidase B (benzoylglycyl-L-arginine), elastase, glycylglycine dipeptidase, or leucine aminopeptidase were found.Gel filtration of gastric juice proteins on Sephadex(R) G-100 yielded estimates of molecular weights, among them: chitobiase 100,000; phosphatase 80,000; lipase 43,000; DNase 33,000; "trypsin" and ribonuclease 25,000; and two proteinases with optima around pH 4 and 8 and the low molecular weight of 12,500; proteolytic activity at pH 4 was also associated with the molecular weights 25,000 and 50,000.In ion-exchange chromatography, enzymes were eluted from DEAE-cellulose in the following sequence of increasing anionic character: chitobiase, proteinase (pH 4), proteinase (pH 8), lipase, "carboxypeptidase A," DNase, phosphatase, and "trypsin" and "chymotrypsin."Of the principal enzymes, only one (chitobiase) had its optimum at the pH 5 normal for lobster gastric juice. The lipase had an optimal pH around 7, phosphatase near 9, and "trypsin" near 8; proteolytic activity shows a maximum at pH 7–8 and another maximum, unusual for Crustacea, at pH 4.Much of the protein of gastric juice could not be connected with any enzymic activity. The proportion of nonactive protein seemed to increase with starvation of the animals. We found no evidence for the occurrence of zymogens.

scholarly journals Characterization of human liver α-D-mannosidase purified by affinity chromatography

1976 ◽  
Vol 153 (3) ◽  
pp. 579-587 ◽  
Author(s):  
N C Phillips ◽  
D Robinson ◽  
B G Winchester
Keyword(s):  
Affinity Chromatography ◽  
Concanavalin A ◽  
Human Liver ◽  
Early Stage ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Exchange Chromatography ◽  
Lysosomal Storage ◽  
Sepharose 4B

Human liver acidic α-D-mannosidase was purified 1400-fold by a relatively short procedure incorporating chromatography on concanavalin A-Sepharose and affinity chromatography on Sepharose 4B-epsilon-aminohexanoylmannosylamine. In contrast with the acidic enzymic activity the neutral α-mannosidase did not bind to the concanavalin A-Sepharose so the two types of α-mannosidase could be separated at an early stage in the purification. The only significant glycosidase contaminant after affinity chromatography on the mannosylamine ligand was α-L-fucosidase, which was selectively removed by affinity chromatography on the corresponding fucosylamine ligand. The final preparation was free of other glycosidase activities. The pI of the purified enzyme was increased from 6.0 to 6.45 on treatment with neuraminidase. Although the pI and the mol.wt. (220 000) suggested that α-mannosidase A had been purified selectively, ion-exchange chromatography on DEAE-cellulose indicated that the preparation consisted predominantly of α-mannosidase B. This discrepancy is discussed in relation to the basis of the multiple forms of human α-mannosidase. The purified enzyme completely removed the α-linked non-reducing terminal mannose from a trisaccharide isolated from the urine of a patient with mannosidosis. A comparison of the activity of the pure enzyme towards the natural substrate and synthetic substrates suggests that the same enzymic activity is responsible for hydrolysing all the substrates. These results validate the use of synthetic substrates for determining the mannosidosis genotype. They are also further evidence that mannosidosis is a lysosomal storage disease resulting from a deficiency of acidic α-mannosidase.

ISOLATION OF LH, FSH AND TSH FROM HUMAN PITUITARIES AFTER THE REMOVAL OF HGH

1974 ◽  
Vol 77 (3) ◽  
pp. 485-497 ◽  
Author(s):  
P. A. Torjesen ◽  
T. Sand ◽  
N. Norman ◽  
O. Trygstad ◽  
I. Foss
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Molecular Weights ◽  
Pituitary Glands

ABSTRACT Highly purified human LH, FSH and TSH were isolated from batches of 300 frozen pituitary glands (200 g) by pH, acetone and ethanol fractionation, Sephadex gel filtration, ion-exchange chromatography on DEAE-cellulose and CM-Sephadex, and preparative polyacrylamide-gel electrophoresis. Sodium dodecyl-sulphate (SDS) polyacrylamide gel electrophoresis was used in order to check the purity, the identity and the molecular weight of the purified LH, FSH and TSH. This procedure showed that the hormone preparations consisted of two subunits with molecular weights of: LH: 21 300 and 17 900, FSH: 22 100 and 18 300 and TSH: 20 800 and 16 400. The purity of the hormone preparations was also evaluated by analytical disc electrophoresis at pH 8.9. The purified hormone preparations had radioimmunological activity as follows: LH: 20 000 IU/mg, FSH: 16 500 IU/mg and TSH: 5 IU/mg. All preparations had high biological potency.

scholarly journals The purification and properties of a single chicken pepsinogen fraction and the pepsin derived from it

1973 ◽  
Vol 133 (1) ◽  
pp. 105-115 ◽  
Author(s):  
Margaret L. Green ◽  
Joanna M. Llewellin
Keyword(s):  
Amino Acids ◽  
Thiol Group ◽  
Enzymic Activity ◽  
Low Ph ◽  
Carboxypeptidase A ◽  
Molecular Weights ◽  
Ph Values ◽  
Purification And Properties ◽  
Terminal Amino ◽  
Low Ionic Strength

1. Evidence is given for the presence of at least five pepsinogens in a crude extract of mixed chicken stomachs. One of these was purified and could be activated to yield a single pepsin. 2. The molecular weights of the pepsinogen and pepsin were 36000 and 34000 respectively. The pepsin associated at low pH values and low ionic strength. 3. The amino acid analyses of both proteins are given. The pepsin was devoid of phosphate but contained carbohydrate. 4. The N-terminal amino acids of pepsinogen and pepsin were serine and threonine respectively. Five amino acids were released by carboxypeptidase A and it was deduced that serine may be the C-terminal one. 5. Each protein contained one thiol group per molecule as determined by titration with p-chloromercuribenzoate. The rate of the reaction was very rapid with pepsin, but much slower with pepsinogen, although the same group appeared to react in both instances. The enzymic activity of pepsin was unaffected by the modification. 6. The isoionic point of the pepsin was close to pH4.0 and the enzyme was stable for long periods at pH values up to 7.0. 7. The enzyme hydrolysed bisphenyl sulphite almost as rapidly as did pig pepsin A.

scholarly journals Studies on the structure and activity of rabbit C1q (a subcomponent of the first component of complement)

1974 ◽  
Vol 143 (2) ◽  
pp. 265-272 ◽  
Author(s):  
Diane M. Lowe ◽  
Kenneth B. M. Reid
Keyword(s):  
Amino Acids ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Haemolytic Activity ◽  
Subunit Structure ◽  
Carboxypeptidase A ◽  
Rapid Loss ◽  
Collagenase Digestion ◽  
Polypeptide Chains ◽  
Structure And Activity

1. The subunit structure of rabbit subcomponent C1q was examined in a previous publication (Reid et al., 1972). The present paper describes some aspects of the structure of the polypeptide chains derived from the molecule. 2. The three polypeptide chains, produced by performic oxidation, of rabbit subcomponent C1q were isolated by ion-exchange chromatography in 8m-urea on DEAE-cellulose. 3. Each chain was found to contain 15–18% glycine and significant amounts of the amino acids hydroxyproline and hydroxylysine. 4. By means of collagenase digestion it was shown that all three chains of rabbit subcomponent C1q contain collagen-like sequences of amino acids which constitute about 40% of each chain. 5. By use of carboxypeptidase A it was established, indirectly, that the collagen-like sequences, in one of the chains, are probably located near, or at, the N-terminal end of the chain. 6. Collagenase digestion and heating at 52°C (but not at 49°C) caused rapid loss of native rabbit subcomponent C1q haemolytic activity.

Purification and characterization of chymosin and pepsin from kid

2006 ◽  
Vol 73 (1) ◽  
pp. 49-57 ◽  
Author(s):  
Ekaterini E Moschopoulou ◽  
Ioannis G Kandarakis ◽  
Efstathios Alichanidis ◽  
Emmanouil M Anifantakis
Keyword(s):  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Temperature Dependent ◽  
Molecular Weights ◽  
Indigenous Breed ◽  
Increased Sensitivity ◽  
Pepstatin A

The objective of this work was to study the characteristics of the gastric aspartic proteinases chymosin and pepsin which are constituents of the kid rennet. The two enzymes were extracted from abomasal tissue of one kid from a local indigenous breed, separated from each other by DEAE-cellulose chromatography and then were purified by gel filtration and anion-exchange chromatography. The molecular weights of the purified kid chymosin and pepsin as determined by gel filtration were 36 kDa and 40 kDa respectively. The isoelectric point of kid chymosin was as multiple forms of 3–6 zones at pH 4·6–5·1, while that of kid pepsin was at pH [les ]3·0. Kid pepsin contained 0·37 molecules phosphorous per molecule and was totally inhibited by 5 μM pepstatin A, being more sensitive than kid chymosin. Both enzymes were almost equally as proteolytic as calf chymosin on total casein at pH 5·6. Kid pepsin activity was more pH and temperature dependent than kid chymosin activity. In comparison with the calf chymosin temperature sensitivity, the order of increased sensitivity was: calf chymosin <kid chymosin <kid pepsin.

scholarly journals Mannosidosis in Angus cattle. The enzymic defect

1974 ◽  
Vol 137 (2) ◽  
pp. 363-371 ◽  
Author(s):  
N. C. Phillips ◽  
D. Robinson ◽  
B. G. Winchester ◽  
R. D. Jolly
Keyword(s):  
Gel Filtration ◽  
Residual Activity ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Starch Gel Electrophoresis ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Angus Cattle ◽  
Starch Gel ◽  
Component C

Normal calf α-mannosidase activity exists in at least three forms separable by chromatography on DEAE-cellulose and by starch-gel electrophoresis. Two components, A and B, have optimum activity between pH3.75 and 4.75, but component C has an optimum of pH6.6. Components A and B are virtually absent from the tissues of a calf with mannosidosis and the residual activity is due to component C. The acidic and neutral forms of α-mannosidase differ in their molecular weights and sensitivity to EDTA, Zn2+, Co2+ and Mn2+. An acidic α-mannosidase component (pH optimum 4.0) accounts for most of the activity in normal plasma but it is absent from the plasma of a calf with mannosidosis. Although the acidic α-mannosidase component is probably related to tissue components A and B, it can be distinguished from them by ion-exchange chromatography and gel filtration. The optimum pH of the low residual activity in the plasma from a calf with mannosidosis is pH5.5–5.75. The results support the hypothesis that Angus-cattle mannosidosis is a storage disease caused by a deficiency of lysosomal acidic α-mannosidase activity.

scholarly journals Isolation of pathogenesis-related proteins from TMV-infected tobacco and their influence on infectivity of TMV

2010 ◽  
Vol 41 (No. 2) ◽  
pp. 52-57 ◽  
Author(s):  
M. Šindelářová ◽  
L. Šindelář
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Leaf Tissue ◽  
Exchange Chromatography ◽  
Pr Proteins ◽  
Inhibitory Influence ◽  
Pathogenesis Related Proteins ◽  
Molecular Weights ◽  
Pathogenesis Related ◽  
Related Proteins

The composition of pathogenesis-related proteins (PR-proteins) in the intercellular fluid (ICF) and leaf tissue of the hypersensitive tobacco cultivar Xanthi-nc inoculated with Tobacco mosaic virus (TMV), and their inhibitory influence on TMV multiplication were studied. The ICF PR-proteins of infected plants were separated after solubilisation by decreasing gradient of ammonium sulphate, the cell PR-proteins were separated after acidic homogenisation of leaf tissues. The ICF and cell PR-proteins were further purified by ion exchange chromatography on DEAE cellulose. Using discontinuous non-denaturating polyacrylamide gel electrophoresis of DEAE cellulose fractions the PR-proteins were detected. Their molecular weights were estimated by SDS-PAGE. The ICF and cell proteins of infected leaves included PR-proteins of the molecular weights 15–16 kDa (Group 1), 27–28 kDa (Group 3: chitinases) and 36–40 kDa (Group 2a: &#1704-1,3-glucanases). Fractions with different PR-proteins were tested for their effect on infectivity of TMV. Particularly the PR3 and PR2a proteins seem to decrease the infectivity of TMV.

scholarly journals Isolation, Purification, And Characterization Of Some Cystein Proteases From Bovine Mastites Milk

2009 ◽  
Vol 3 (1) ◽  
pp. 85-97
Author(s):  
K.S. Doosh
Keyword(s):  
Cathepsin B ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Bovine Mastitis ◽  
Deae Cellulose ◽  
Cysteine Proteases ◽  
Enzyme Characterization ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Molecular Weights

Proteolytic activity of cysteine proteases were studied in bovine mastitis milk, four fractions designated as F1,F2,F3,F4 with cysteine protease activity were separated from leukocytes cell by ion- exchange Chromatography through DEAE-Cellulose The most active fraction F4 was selected for further purification utilizing gel filtration Chromatography on Sephadex G-100 column it has been found that F4 most likely being cathepsin B. purification folds and the enzyme yield was 46.66 and 31.81% respectively . polyacrylamide gel electrophoresis test indicated that the enzyme has been purified to homogeneity by giving a single band . The results of enzyme characterization showed that the molecular weights were 31000 and 30000 Daltons as determined by gel filtration and electrophoresis methods in present of reducing agent SDS- PAGE respectively. The optimum pH for the enzyme activity was 6.0 and it was stable at pH values ranged between 4.5 - 6.5. The enzyme exhibited the maximum activity at 45ºC and the enzyme retained its entire activity over 30 min incubation at 30 -50 C and it retained (50, 20, 10) % of its entire activites over 30 min incubation at (60, 70, 80) C respectively. From this results and results observed from the effect of inhibitor and activator reagents we suggest that enzyme F4 possibly belonged to cathepsin B.

scholarly journals Multiple forms of nuclear deoxyribonucleic acid polymerases and their relationship with the soluble enzyme

1973 ◽  
Vol 131 (2) ◽  
pp. 237-246 ◽  
Author(s):  
R. L. P. Adams ◽  
M. A. L. Henderson ◽  
W. Wood ◽  
J. G. Lindsay
Keyword(s):  
Molecular Weight ◽  
Dna Synthesis ◽  
High Molecular Weight ◽  
Dna Polymerase ◽  
Deae Cellulose ◽  
Polymerase Activity ◽  
Enzymic Activity ◽  
Supernatant Fraction ◽  
Molecular Weights ◽  
Molecular Weight Peak

1. DNA polymerase from nuclear and supernatant fractions of cultured mouse L929 cells was fractionated on columns of Sephadex G-200, Sepharose 4B and of DEAE-cellulose. Several peaks of activity are found on Sephadex chromatography and the distribution of activity between these depends on: (a) the source of the enzyme, i.e. nuclear or supernatant fraction; (b) the mode of extraction of the enzyme from the nucleus; (c) the amount of enzyme applied to the column. 2. The DNA polymerase activity in the lower-molecular-weight peaks (approximate molecular weights are 35000, 70000 and 140000) is firmly bound within the cell nucleus and shows a preference for native DNA as template, whereas the high-molecular-weight peak (peak I, molecular weight 250000 or greater) is found in supernatant fractions and shows greater activity with a denatured DNA template. 3. During periods of DNA synthesis the high-molecular-weight enzyme becomes more firmly bound within the nucleus. 4. Peak I enzymic activity is relatively unstable and is inhibited by thiol-blocking reagents and deoxycholate, but it is stimulated by univalent cations. 5. Very little endonuclease is present in the polymerase preparations, but a very active exonuclease and nucleoside diphosphokinase are present. On Sephadex chromatography, however, it was shown that the immediate precursors for DNA synthesis, at least by peak I enzyme, are the deoxyribonucleoside triphosphates. 6. Attempts to decrease the molecular weight of the peak I enzyme while still retaining activity failed.

Separation of Heparin into Subtractions by DEAE-Cellulose Chromatography

1979 ◽  
Vol 42 (05) ◽  
pp. 1452-1459 ◽  
Author(s):  
Robert H Yue ◽  
Toby Starr ◽  
Menard M Gertler
Keyword(s):  
High Activity ◽  
Uronic Acid ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Net Charge ◽  
Uronic Acid Content ◽  
Successful Separation ◽  
Salt Gradient ◽  
Structure And Activity ◽  
Low Activity

SummaryCommercial porcine heparin can be separated into three distinct subtractions by using DEAE-cellulose chromatography and a stepped salt gradient. Gram quantities of heparin can be fractionated by this technique. All three heparin subtractions can accelerate the inhibition of thrombin by antithrombin III with different efficiency. The specific activities of the high activity heparin, intermediate activity heparin and low activity heparin are 228 units/mg, 142 units/mg and 95 units/mg, respectively. Both the uronic acid content and the quantity of N-SO4 for all three heparin subfractions have been evaluated. The high activity heparin has the lowest uronic acid and N-SO4 content. The successful separation of commercial heparin into three distinct subfractions by means of ion-exchange chromatography suggests that the net charge on these three heparin components will serve as a model system in the elucidation of the structure and activity relationship to the biological function of heparin.

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