Chemical Modification of Human Lysozyme. Acetylation and Nitration of Tyrosine

1971 ◽  
Vol 49 (7) ◽  
pp. 816-821 ◽  
Author(s):  
R. L. Fawcett ◽  
T. J. Limbird ◽  
Sandra L. Oliver ◽  
C. L. Borders Jr.
Keyword(s):  
Enzyme Activity ◽  
Chemical Modification ◽  
Gel Filtration ◽  
Degree Of Polymerization ◽  
Tyrosine Residue ◽  
Amino Groups ◽  
Human Lysozyme ◽  
Tyrosine Residues ◽  
Typical Experiment ◽  
High Degree

When human lysozyme is reacted with a 60 M excess of N-acetylimidazole, only one of six tyrosine residues and two amino groups are acetylated. The acetylated lysozyme is 1.2 times as active towards M. lysodeikticus as the unmodified enzyme. When human lysozyme is reacted with a 4 M excess of tetranitromethane, approximately one tyrosine is nitrated. The tetranitromethane also simultaneously induces a high degree of polymerization of the lysozyme. In a typical experiment, nitration leads to a polymerized product that has only 25% of the activity of unmodified enzyme towards M. lysodeikticus. The polymerized lysozyme can be separated into several components by gel filtration on Sephadex G-75. Enzyme activity analyses of the chromatographed lysozyme oligomers indicate that tetranitromethane reduces the activity of human lysozyme primarily by polymerization, since the lysozyme monomer, which contains one nitrotyrosine per molecule, has 65% activity while the trimer has only 5% activity. N-Acetylglucosamine, N,N′-diacetylchitobiose, and N,N',N″-triacetylchitotriose, all inhibitors or substrates of human lysozyme, prevent neither the nitration of the single tyrosine residue nor the polymerization due to tetranitromethane action.

Stimulation of β-(l → 6)-glucanase production by oxidized pustulan

1972 ◽  
Vol 18 (10) ◽  
pp. 1543-1550 ◽  
Author(s):  
Robert G. Brown
Keyword(s):  
Enzyme Activity ◽  
Isoelectric Focusing ◽  
Gel Filtration ◽  
Tween 80 ◽  
Sole Source ◽  
Cellulase Production ◽  
Low Degree ◽  
Degree Of Oxidation ◽  
High Degree ◽  
Stimulation Of

A strain of Penicillium lilacinum, isolated from soil, produced pustulanase, β-(1 → 3)-glucanase, (EC. 3.2.1.6) and cellulase (EC.3.2.1.4) when cultivated on a medium containing pustulan as the sole source of carbon. If pustulan was replaced by ketopustulan, the production of pustulanase was stimulated about 10-fold although the amount of stimulation was dependent on the degree of oxidation of pustulan. β-(1 → 3)-Glucanase production was stimulated slightly by ketopustulan; however, the degree of oxidation did not affect significantly the yield of this enzyme. Cellulase production was either unaffected by the oxidized polymer, or at higher degrees of oxidation, decreased. Tween 80 stimulated the production of the three enzymes in media containing ketopustulan with a low degree of oxidation but was inhibitory to pustulanase and cellulase production in media containing ketopustulan with a high degree of oxidation. A combination of gel filtration and isoelectric focusing revealed that each enzyme activity was attributable to at least two proteins.

scholarly journals 307 Characterization of Amylolytic Activities of Tulip Bulb Scales

HortScience ◽  
1999 ◽  
Vol 34 (3) ◽  
pp. 495D-495
Author(s):  
Anil P. Ranwala ◽  
William B. Miller
Keyword(s):  
Enzyme Activity ◽  
Amylase Activity ◽  
Gel Filtration ◽  
Soluble Starch ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Product Analysis ◽  
Ph Optimum ◽  
Tulip Bulb ◽  
Tulipa Gesneriana

Amylolytic activities extracted from scales of tulip (Tulipa gesneriana L. cv. Apeldoorn) bulbs stored at 4 °C for 6 weeks under moist conditions were characterized. Anion exchange chromatography of enzyme extract on DEAE-Sephacel revealed three peaks of amylolytic activity. Three enzymes showed different electrophoretic mobilties on nondenaturing polyacrylamide gels. The most abundant amylase activity was purified extensively with phenyl-agarose chromatography, gel filtration on Sephacryl S-200, and chromatofocusing on polybuffer exchanger PBE 94. The purified amylase was determined to be an endoamylase based on substrate specificity and end product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55 °C when soluble starch was used as the substrate. The apparent Km value for soluble starch was 1.28 mg/ml. The inclusion of 2 mM CaCl2 in the reaction mixture resulted in a 1.4-fold increase in the enzyme activity. The presence of calcium ions also enhanced the thermo-stability of the enzyme at higher temperatures. The enzyme was able to hydrolyze soluble starch, amylose, amylopectin, and beta-limit dextrin, but it had no activity against pullulan, inulin, maltose, or p-nitrophenyl alpha-glucopyranoside. Only maltooligosaccharides, having a degree of polymerization of 7 or more, were hydrolyzed to a significant extent by the enzyme. Exhaustive hydrolysis of soluble starch with the enzyme yielded a mixture of maltose and matlooligosaccharides. This amylase activity was not inhibited by alpha- or beta-cyclodextrin upto a concentration of 10 mM. Maltose at a 50 mM concentration partially inhibited the enzyme activity, whereas glucose had no effect at that concentration.

scholarly journals The Effects of Different Degrees of Procyanidin Polymerization on the Nutrient Absorption and Digestive Enzyme Activity in Mice

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2916 ◽  
Author(s):  
Huairong Zhong ◽  
Yong Xue ◽  
Xiaoyuan Lu ◽  
Qiang Shao ◽  
Yuelei Cao ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Amylase Activity ◽  
Digestive Enzyme ◽  
Degree Of Polymerization ◽  
Nutrient Absorption ◽  
Digestive Enzyme Activity ◽  
Total Food Intake ◽  
Total Food ◽  
Low Degree ◽  
High Degree

Proanthocyanidins, including polymers with both low and high degrees of polymerization, are the focus of intensive research worldwide due to their high antioxidant activity, medicinal applications, and pharmacological properties. However, the nutritional value of these compounds is limited because they readily form complexes with proteins, polysaccharides, and metal ions when consumed. In this study, we examined the effects of proanthocyanidins with different degrees of polymerization on white mice. Twenty-four male white mice were randomly divided into three groups of eight mice each and fed proanthocyanidins with a low degree of polymerization or a high degree of polymerization or a distilled water control via oral gavage over a 56-day period. We examined the effects of these proanthocyanidins on digestive enzyme activity and nutrient absorption. Compared to the control group, the group fed high-polymer proanthocyanidins exhibited a significant reduction in net body mass, total food intake, food utility rate, amylase activity, protease activity, and major nutrient digestibility (p < 0.05), while the group fed low-polymerization proanthocyanidins only exhibited significant reductions in total food intake, α-amylase activity, and apparent digestibility of calcium and zinc (p < 0.05). Therefore, proanthocyanidins with a high degree of polymerization had a greater effect on digestive enzyme activity and nutrient absorption than did those with a low degree of polymerization. This study lays the foundation for elucidating the relationship between procyanidin polymerization and nutrient uptake, with the aim of reducing or eliminating the antinutritional effects of polyphenols.

scholarly journals Chemical modification of aminopeptidase isolated from Pronase

1994 ◽  
Vol 302 (2) ◽  
pp. 595-600 ◽  
Author(s):  
S H Yang ◽  
C H Wu ◽  
W Y Lin
Keyword(s):  
Enzyme Activity ◽  
Chemical Modification ◽  
Metal Ions ◽  
Conformational Changes ◽  
Enzyme Catalysis ◽  
Competitive Inhibitor ◽  
Amino Groups ◽  
Active Centre ◽  
Modified Enzyme

Chemical modification of aminopeptidase from pronase has revealed two important histidines in enzyme catalysis. In the absence of metal ions, modification of the readily-modified histidine (pKa 6.9 +/- 0.5) results in a drastic loss of activity, indicating that this residue is indispensible for enzyme activity. In the presence of CaCl2, the modified enzyme still retains approx. 60% of the activity, whereas modification of another histidine (pKa 7.7 +/- 0.2) leads to a dramatic loss of activity. In fact, the enzyme with the first histidine being modified is active only in the presence of metal ions. Moreover, modification of the second histidine is prevented by the presence of Ca(II). These results indicate that the second histidine is serving as a ligand for Ca(II) and the bound Ca(II) is directly involved in enzyme catalysis. The c.d. spectra of the modified and unmodified enzymes in the absence or presence of CaCl2 are all very similar, indicating that no gross conformational changes in protein occur upon modification or by the presence of Ca(II). Modification of both histidines is prevented by the presence of a competitive inhibitor, suggesting that they are located in the active centre. Modification of 11 amino groups, two tyrosines, or four arginines causes no appreciable inactivation of the enzyme, indicating that these residues are not directly involved in enzyme catalysis.

scholarly journals Purification and subunit structure of argininosuccinate lyase from Chlamydomonas reinhardi

1977 ◽  
Vol 167 (1) ◽  
pp. 71-75 ◽  
Author(s):  
R F Matagne ◽  
J P Schlösser
Keyword(s):  
Enzyme Activity ◽  
Crude Extract ◽  
Enzyme Preparation ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Activity Analysis ◽  
Disc Electrophoresis ◽  
Subunit Structure ◽  
Argininosuccinate Lyase

Argininosuccinate lyase (EC 4.3.2.1) was purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The final enzyme preparation was purified 46-fold compared with the crude extract. Electrophoresis of this preparation revealed three bands, the major one having the enzyme activity. Analysis of the enzyme by gel filtration and by disc electrophoresis (in two different concentrations of acrylamide) gave mol.wts. of 200000 (+/- 15000) and 190000 (+/- 20000) respectively. Treatment with sodium dodecyl sulphate and mercaptoethanol dissociated the enzyme into subunits of mol.wt. 39000 (+/-2000). The results are indicative of the multimeric structure of the enzyme, which is composed of five (perhaps four or six) identical subunits.

scholarly journals Partial purification and characterization of an endo-α-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242

1992 ◽  
Vol 288 (2) ◽  
pp. 475-482 ◽  
Author(s):  
I Ishii-Karakasa ◽  
H Iwase ◽  
K Hotta ◽  
Y Tanaka ◽  
S Omura
Keyword(s):  
Enzyme Activity ◽  
Culture Medium ◽  
Gel Filtration ◽  
Partial Purification ◽  
Gastric Mucus ◽  
Purification And Characterization ◽  
Streptomyces Sp ◽  
Optimum Ph ◽  
New Type

For the purification of a new type of endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242 (endo-GalNAc-ase-S) [Iwase, Ishii, Ishihara, Tanaka, Omura & Hotta (1988) Biochem. Biophys. Res. Commun. 151, 422-428], a method for assaying enzyme activity was established. Using purified pig gastric mucus glycoprotein (PGM) as the substrate, oligosaccharides liberated from PGM were pyridylaminated, and the reducing terminal sugars of oligosaccharides larger than Gal beta 1-3GalNAc were analysed by h.p.1.c. The crude enzyme of endo-GalNAc-ase-S was prepared as an 80% (w/v) ammonium sulphate precipitate from the concentrated culture medium. The enzyme was partially purified by gel chromatofocusing and subsequent DEAE-Toyopearl chromatography. Endo-enzyme activity eluted around pI 4.8 on a gel chromatofocusing column and eluted with 0.19-0.25 M-NaCl on a DEAE-Toyopearl column. In the enzyme fraction obtained, no exo-glycosidases or proteases could be detected. The molecular mass of the enzyme was estimated as 105 kDa by gel filtration, and the optimum pH was 5.5. Endo-GalNAc-ase-S hydrolysed the O-glycosidic linkage between GalNAc and Ser (Thr) in 3H-labelled and unlabelled asialofetuin, liberating both the disaccharide (Gal beta 1-3GalNAc) and the tetrasaccharide [Gal beta 1-3 (Gal beta 1-4GlcNAc beta 1-6)GalNAc]. When endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. (endo-GalNac-ase-A) was incubated with 3H-labelled and unlabelled asialofetuin, only the disaccharide (Gal beta 1-3GalNAc) was liberated.

scholarly journals Purification and properties of three (1→3)-β-d-glucanase isoenzymes from young leaves of barley (Hordeum vulgare)

1993 ◽  
Vol 289 (2) ◽  
pp. 453-461 ◽  
Author(s):  
M Hrmova ◽  
G B Fincher
Keyword(s):  
Hordeum Vulgare ◽  
Elevated Temperatures ◽  
Gel Filtration ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Purification And Properties ◽  
Young Leaves ◽  
Monomeric Proteins ◽  
Ph Range ◽  
Hydrolysis Of

Three (1->3)-beta-D-glucan glucanohydrolase (EC 3.2.1.39) isoenzymes GI, GII and GIII were purified from young leaves of barley (Hordeum vulgare) using (NH4)2SO4 fractional precipitation, ion-exchange chromatography, chromatofocusing and gel-filtration chromatography. The three (1->3)-beta-D-glucanases are monomeric proteins of apparent M(r)32,000 with pI values in the range 8.8-10.3. N-terminal amino-acid-sequence analyses confirmed that the three isoenzymes represent the products of separate genes. Isoenzymes GI and GII are less stable at elevated temperatures and are active over a narrower pH range than is isoenzyme GIII, which is a glycoprotein containing 20-30 mol of hexose equivalents/mol of enzyme. The preferred substrate for the enzymes is laminarin from the brown alga Laminaria digitata, an essentially linear (1->3)-beta-D-glucan with a low degree of glucosyl substitution at 0-6 and a degree of polymerization of approx. 25. The three enzymes are classified as endohydrolases, because they yield (1->3)-beta-D-oligoglucosides with degrees of polymerization of 3-8 in the initial stages of hydrolysis of laminarin. Kinetic analyses indicate apparent Km values in the range 172-208 microM, kcat. constants of 36-155 s-1 and pH optima of 4.8. Substrate specificity studies show that the three isoenzymes hydrolyse substituted (1->3)-beta-D-glucans with degrees of polymerization of 25-31 and various high-M(r), substituted and side-branched fungal (1->3;1->6)-beta-D-glucans. However, the isoenzymes differ in their rates of hydrolysis of a (1->3;1->6)-beta-D-glucan from baker's yeast and their specific activities against laminarin vary significantly. The enzymes do not hydrolyse (1->3;1->4)-beta-D-glucans, (1->6)-beta-D-glucan, CM-cellulose, insoluble (1->3)-beta-D-glucans or aryl beta-D-glycosides.

scholarly journals Analysis of the forms of acetylcholinesterase from adult mouse brain

1975 ◽  
Vol 147 (2) ◽  
pp. 205-214 ◽  
Author(s):  
E D Adamson ◽  
S E Ayers ◽  
Z A Deussen ◽  
C F Graham
Keyword(s):  
Enzyme Activity ◽  
Mouse Brain ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Acetylcholinesterase Activity ◽  
Total Activity ◽  
Polyacrylamide Gel ◽  
Soluble Extract ◽  
Molecular Sizes

The solubilization of 80% of the acetylcholinesterase activity of mouse brain was performed by repeated 2h incubations of homogenates at 37 degrees C in an aqueous medium. Analysis of the soluble extract by gel filtration on Sephadex G-200 showed that up to 80% of the enzyme activity was eluted in a peak which was estimated to consist of molecules of about 74000mol.wt. This peak was called the monomer form of the enzyme. After 3 days at 4 degrees C, the soluble extract was re-analysed and was eluted from the column in four peaks of about 74000, 155000, 360000 and 720000 mol.wt. Since the total activity of the enzyme in these peaks was the same as that in the predominantly monomer elution profile of fresh enzyme, we concluded that the monomer had aggregated, possibly into dimers, tetramers and octomers. Extracts of the enzyme were analysed by polyacrylamide-gel electrophoresis and the resulting multiple bands of enzyme activity on gels were shown to separate according to their molecular sizes, that is by molecular sieving. All these forms had similar susceptibilities to the inhibitors eserine, tetra-isopropyl pyrophosphoramide and compound BW 284c51 [1,5-bis-(4-allyldimethylammoniumphenyl)pentan-3-one dibromide]. Thus the forms of the enzyme in mouse brain which can be detected by gel filtration and polyacrylamide-gel electrophoresis may all be related to a single low-molecular-weight form which aggregates during storage. This supports similar suggestions made for the enzyme in other locations.

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