Dinuclear Zinc(II) Complexes of Linear and Cyclic Peptides Containing Two Bis(2-pyridylmethyl)amino Groups as Catalysts for Hydrolysis of RNA Model Substrate

SummaryA simple, rapid and sensitive spectrophotometric assay was developed and evaluated for monitoring proteolysis during cheese ripening, based on the fact that α-amino groups released by hydrolysis of cheese proteins react with trinitrobenzenesulphonic acid to form products that absorb strongly at 420 nm. A linear relationship was shown to exist between A420 and concentration of free α amino groups up to 0·5 HIM (r = 0·999, 38 df, P < 0·001). Repeatability of the method was satisfactory. The coefficient of variance was 0·53% for amino acid solutions and 1·19% for cheese extracts. Average recovery of glycine added to the cheese was 104 ± 2·9%. A comparison of the above method with that of determination of water-soluble N to total N ratio showed that there was good agreement between these two methods of assessment of proteolysis in cheese (r = 0·857, 32 df, P < 0·001). Mainly Feta and Teleme cheese were examined, but a similar correlation was obtained with hard Greek cheeses. Analytical conditions of the procedure are discussed.

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Biodegradation of low molecular weight polyacrylamide under aerobic and anaerobic conditions: effect of the molecular weight

Water Science & Technology ◽

10.2166/wst.2020.109 ◽

2020 ◽

Vol 81 (2) ◽

pp. 301-308 ◽

Cited By ~ 1

Author(s):

Wenzhe Song ◽

Yu Zhang ◽

Amir Hossein Hamidian ◽

Min Yang

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Carbon Chain ◽

Anaerobic Treatment ◽

Low Molecular Weight ◽

Amino Groups ◽

Molecular Weights ◽

Weight One ◽

Hydrolysis Of ◽

Aerobic And Anaerobic

Abstract The biodegradation of polyacrylamide (PAM) includes the hydrolysis of amino groups and cleavage of the carbon chain; however, the effect of molecular weight on the biodegradation needs further investigations. In this study, biodegradation of low molecular weight PAM (1.6 × 106 Da) was evaluated in two aerobic (25 °C and 40 °C) and two anaerobic (35 °C and 55 °C) reactors over 100 days. The removal of the low molecular weight PAM (52.0–52.6%) through the hydrolysis of amino groups by anaerobic treatment (35 °C and 55 °C) was much higher than that of the high molecular weight (2.2 × 107 Da, 11.2–17.0%) observed under the same conditions. The molecular weight was reduced from 1.6 × 106 to 6.45–7.42 × 105 Da for the low molecular weight PAM, while the high molecular weight PAM declined from 2.2 × 107 to 3.76–5.87 × 106 Da. The results showed that the amino hydrolysis of low molecular weight PAM is easier than that of the high molecular weight one, while the cleavage of its carbon chain is still difficult. The molecular weights of PAM in the effluents from the two aerobic reactors (25 °C and 40 °C) were further reduced to 4.31 × 105 and 5.68 × 105 Da by the biofilm treatment, respectively. The results would be useful for the management of wastewater containing PAM.

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THE ACETYLATION OF THROMBIN

Canadian Journal of Biochemistry and Physiology ◽

10.1139/y59-150 ◽

1959 ◽

Vol 37 (1) ◽

pp. 1361-1366 ◽

Cited By ~ 4

Author(s):

Ricardo H. Landaburu ◽

Walter H. Seegers

Keyword(s):

Methyl Ester ◽

Proteolytic Activity ◽

Basic Structure ◽

Amino Groups ◽

Spontaneous Hydrolysis ◽

Optimum Ph ◽

Hydrolysis Of

Purified thrombin-C loses its clotting power upon acetylation. The thrombin-E which is produced during the acetylation has approximately twice the proteolytic activity as the original thrombin-C. Evidently amino groups are not necessary to have thrombin-E activity, but if o-acyl groups are also produced the enzyme does not hydrolyze p-toluenesulphonylarginine methyl ester (TAMe). The activity can be recovered by spontaneous hydrolysis of the o-acyl groups at pH 8.5. Thrombin-E does not activate fibrinogen, but does lyse fibrin. The optimum pH with TAMe as substrate is 8.8. It may be that thrombin-C is a dimer of the basic structure in thrombin-E.

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Kinetic properties of the α-lytic protease of Sorangium sp., a bacterial homologue of the pancreatopeptidases

Canadian Journal of Biochemistry ◽

10.1139/o69-047 ◽

1969 ◽

Vol 47 (3) ◽

pp. 305-316 ◽

Cited By ~ 26

Author(s):

H. Kaplan ◽

D. R. Whitaker

Keyword(s):

Methyl Ester ◽

Catalytic Properties ◽

Substrate Binding ◽

Kinetic Properties ◽

Histidine Residue ◽

Amino Groups ◽

Binding Properties ◽

Histidine Residues ◽

Support Reaction ◽

Hydrolysis Of

The kinetics under consideration are those of a bacterial serine protease with the same "active serine" sequence as chymotrypsin, trypsin, and elastase, and with a single histidine residue in a sequence which closely matches the sequences around histidine-57 of chymotrypsin and the analogous histidine residues of trypsin and elastase. In agreement with previous evidence of an elastase-like specificity, esters of N-substituted, neutral, aliphatic L-amino acids proved to be good to excellent substrates for the α-enzyme; esters of arginine, tyrosine, and tryptophan were not hydrolyzed. The enzyme has a much higher activity than the pancreatopeptidases towards p-nitrophenyl acetate and p-nitrophenyl trimethyl acetate; the catalytic rate coefficient kc for the latter substrate is about fivefold greater than that of elastase.The catalytic properties match those of the pancreatopeptidases in the following respects. As demonstrated with N-acetyl-L-valine methyl ester as substrate, kc is dependent on an ionization with a pKa of 6.7 in water and 7.3 in H22O; Δ log (kc/Km)/ΔpH for this ionization is equal to 1.0; kc is reduced 50% when H2O is replaced by H22O. These findings are consistent with a requirement for a single unprotonated histidine residue and general basic catalysis by that residue. The burst of p-nitrophenol in hydrolyses of p-nitrophenyl trimethyl acetate is proportional to [E]0; the magnitude of the proportionality factor and the rate of attainment of a steady state are consistent with the condition [Formula: see text], as in chymotrypsin kinetics. Thus the purely catalytic properties of the α-enzyme match those of chymotrypsin very closely. These findings do not support reaction mechanisms which require two catalytically functional histidine residues for such catalysis. The substrate-binding properties of the α-enzyme differ from those of chymotrypsin in that substrate binding does not depend on ionization of an N-terminal α-amino group; Km for the hydrolysis of N-acetyl-L-valine methyl ester is constant from pH 5 to pH 10 and enzymatic activity is unaffected by acetylation of the enzyme's α- and ε-amino groups. Ks for the hydrolysis of p-nitrophenyl trimethyl acetate is appreciably greater than the Ks of elastase for this substrate.The chloromethyl ketones of glycine and valine did not inhibit the enzyme or alkylate its histidine residue.

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THE PROTEOLYTIC ENZYMES OF MICROORGANISMS: III. SOME CHARACTERISTICS OF EXTRACELLULAR PROTEASES PRODUCED IN SUBMERGED CULTURE

Canadian Journal of Research ◽

10.1139/cjr50c-036 ◽

1950 ◽

Vol 28c (6) ◽

pp. 600-612 ◽

Cited By ~ 6

Author(s):

W. B. McConnell

Keyword(s):

Low Temperatures ◽

Free Amino ◽

Submerged Culture ◽

Culture Medium ◽

Proteolytic Enzymes ◽

Amino Groups ◽

Extracellular Proteases ◽

Egg Albumin ◽

Rapid Hydrolysis ◽

Hydrolysis Of

Some of the general characteristics of the proteases liberated into the culture medium by molds and actinomycetes grown in submerged culture have been studied. Species of Alternaria, Streptomyces, Mortierella, and Gliocladium were used. The enzymes resemble trypsin in that they are most active at a pH slightly above 7 and are inhibited by a preparation of egg albumin. They are stable at low temperatures but suffer marked losses in activity when stored for 16 hr. above 40 °C. The most rapid hydrolysis of gelatin occurs at temperatures between 40 °C. and 50 °C. The enzymes from different organisms show definite differences with respect to their ability to attack different proteins, gelatin and casein being in general the most readily digested. The protease systems from different organisms also vary with respect to the extent to which they can digest gelatin; some enzymes are able to release about three times as many amino groups from gelatin as others. The limit of the hydrolysis is not dependent upon substrate concentration but is slightly affected by the concentration of enzyme. The enzymes were effective in liberating free amino acids from gelatin.

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CYCLIC PEPTIDES: X. Bitter Taste and Chymotryptic Hydrolysis of Cyclic Depsidipeptides Containing a Tryptophan Residue*

International journal of peptide & protein research ◽

10.1111/j.1399-3011.1981.tb01974.x ◽

2009 ◽

Vol 17 (1) ◽

pp. 118-124 ◽

Cited By ~ 2

Author(s):

KOICHI MIYAZAKI ◽

AKIRA YASUTAKE ◽

NORIKAZU NISHINO ◽

HARUHIKO AOYAGI ◽

TETSUO KATO ◽

...

Keyword(s):

Cyclic Peptides ◽

Bitter Taste ◽

Tryptophan Residue ◽

Hydrolysis Of

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THE ACETYLATION OF PROTHROMBIN

Canadian Journal of Biochemistry and Physiology ◽

10.1139/o60-075 ◽

1960 ◽

Vol 38 (6) ◽

pp. 613-620 ◽

Cited By ~ 12

Author(s):

Ricardo H. Landaburu ◽

Walter H. Seegers

Keyword(s):

Calcium Ions ◽

Sodium Citrate ◽

Brain Extract ◽

Amino Groups ◽

Ph Optimum ◽

Platelet Factor ◽

Alkaline Side ◽

Proportionate Change ◽

Hydrolysis Of ◽

Zero Time

Purified bovine prothrombin was acetylated to a range of 27 to 30% of its amino groups. Except for just significant amounts it lost its power to generate thrombin-C. Upon activation the yield of thrombin-E was 100% on the basis of the thrombin-C yield that was obtained on an aliquot sample of the prothrombin before acetylation. Activation was achieved in 25% sodium citrate solution, with calcium ions plus brain extract thromboplastin, and with calcium ions plus protein-free brain thromboplastin. Evidently no Ac-globulin was needed; however, a concentrate of Ac-globulin accelerated the activation. The pH optimum for activation with brain extract thromboplastin is more to the alkaline side than with prothrombin. Activation with purified platelet factor 3 was quite slow, but was accelerated by adding a small amount of thrombin-E, in the form of acetylated thrombin, at zero time. Thrombin-C did not function in that capacity. A change in prothrombin had to be accompanied by a proportionate change in the thrombin to have the latter serve as a catalyst in the activation. Ac-globulin accelerates the hydrolysis of p-toluenesulphonyl-L-arginine methyl ester by thrombin. Ac-globulin owes its activity to thrombin. The view is expressed that Ac-globulin is measured as accelerated thrombin activity in the activation of prothrombin. In other words Ac-globulin accelerates the activation of acetylated prothrombin by thrombin-E. The amino groups on the N-terminal glutamic acid residues are free in the thrombin-E obtained from acetylated prothrombin. It is possible to acetylate an amino group in prothrombin that is necessary for thrombin-C activity.

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Substrate Specificity of Atrazine Chlorohydrolase and Atrazine-Catabolizing Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.66.10.4247-4252.2000 ◽

2000 ◽

Vol 66 (10) ◽

pp. 4247-4252 ◽

Cited By ~ 48

Author(s):

Jennifer L. Seffernick ◽

Gilbert Johnson ◽

Michael J. Sadowsky ◽

Lawrence P. Wackett

Keyword(s):

Substrate Specificity ◽

Homologous Gene ◽

Clavibacter Michiganensis ◽

Amino Groups ◽

Substrate Analogs ◽

Cell Extracts ◽

Alkyl Groups ◽

Chlorine Substituent ◽

Cyano Groups ◽

Hydrolysis Of

ABSTRACT Bacterial atrazine catabolism is initiated by the enzyme atrazine chlorohydrolase (AtzA) in Pseudomonas sp. strain ADP. Other triazine herbicides are metabolized by bacteria, but the enzymological basis of this is unclear. Here we begin to address this by investigating the catalytic activity of AtzA by using substrate analogs. Purified AtzA from Pseudomonas sp. strain ADP catalyzed the hydrolysis of an atrazine analog that was substituted at the chlorine substituent by fluorine. AtzA did not catalyze the hydrolysis of atrazine analogs containing the pseudohalide azido, methoxy, and cyano groups or thiomethyl and amino groups. Atrazine analogs with a chlorine substituent at carbon 2 and N-alkyl groups, ranging in size from methyl to t-butyl, all underwent dechlorination by AtzA. AtzA catalyzed hydrolytic dechlorination when one nitrogen substituent was alkylated and the other was a free amino group. However, when both amino groups were unalkylated, no reaction occurred. Cell extracts were prepared from five strains capable of atrazine dechlorination and known to containatzA or closely homologous gene sequences:Pseudomonas sp. strain ADP, Rhizobium strain PATR, Alcaligenes strain SG1, Agrobacterium radiobacter J14a, and Ralstonia picketti D. All showed identical substrate specificity to purified AtzA fromPseudomonas sp. strain ADP. Cell extracts fromClavibacter michiganensis ATZ1, which also contains a gene homologous to atzA, were able to transform atrazine analogs containing pseudohalide and thiomethyl groups, in addition to the substrates used by AtzA from Pseudomonas sp. strain ADP. This suggests that either (i) another enzyme(s) is present which confers the broader substrate range or (ii) the AtzA itself has a broader substrate range.

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Fragmentation of proteins by free radicals and its effect on their susceptibility to enzymic hydrolysis

Biochemical Journal ◽

10.1042/bj2340399 ◽

1986 ◽

Vol 234 (2) ◽

pp. 399-403 ◽

Cited By ~ 251

Author(s):

S P Wolff ◽

R T Dean

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Biological Significance ◽

Peroxyl Radicals ◽

Cellular Proteins ◽

Enzymic Hydrolysis ◽

Amino Groups ◽

Radical Species ◽

Reducing Conditions ◽

Hydrolysis Of ◽

Increased Susceptibility

Defined radical species generated radiolytically were allowed to attack proteins in solution. The hydroxyl radical (OH.) in the presence of O2 degraded bovine serum albumin (BSA) to specific fragments detectable by SDS/polyacrylamide-gel electrophoresis; fragmentation was not obvious when the products were analysed by h.p.l.c. In the absence of O2 the OH. cross-linked the protein with bonds stable to SDS and reducing conditions. The superoxide (O2-.) and hydroperoxyl (HO2.) radicals were virtually inactive in these respects, as were several other peroxyl radicals. Fragmentation and cross-linking could also be observed when a mixture of biosynthetically labelled cellular proteins was used as substrate. Carbonyl and amino groups were generated during the reaction of OH. with BSA in the presence of O2. Changes in fluorescence during OH. attack in the absence of O2 revealed both loss of tryptophan and changes in conformation during OH. attack in the presence of O2. Increased susceptibility to enzymic proteolysis was observed when BSA was attacked by most radical systems, with the sole exception of O2-.. The transition-metal cations Cu2+ and Fe3+, in the presence of H2O2, could also fragment BSA. The reactions were inhibited by EDTA, or by desferal and diethylenetriaminepenta-acetic acid (‘DETAPAC’) respectively. The increased susceptibility to enzymic hydrolysis of radical-damaged proteins may have biological significance.

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Chemical modification of a cerebral sodium-plus-potassium ion-stimulated adenosine triphosphatase preparation

Biochemical Journal ◽

10.1042/bj1190377 ◽

1970 ◽

Vol 119 (3) ◽

pp. 377-385 ◽

Cited By ~ 7

Author(s):

I. Pull

Keyword(s):

High Speed ◽

Adenosine Triphosphatase ◽

Total Phospholipid ◽

Amino Groups ◽

Amide Bonds ◽

Adenosine Triphosphatase Activity ◽

Acid Reaction ◽

Acid Anhydrides ◽

Kinetics Of ◽

Hydrolysis Of

1. A particulate Na++K+-stimulated adenosine triphosphatase preparation obtained by treatment of bovine cerebral microsomes with a sodium iodide reagent has been further treated with acid anhydrides likely to convert amino groups into acidic derivatives. 2. The extent of acylation of amino groups was determined by reaction of the remaining amino groups with 2,4,6-trinitrobenzenesulphonic acid. The unmodified preparation contains about 1.2 μequiv. of amino groups/mg of protein of which only about 0.5 μequiv. are accounted for by protein amino groups. Kinetics of the trinitrobenzenesulphonic acid reaction with the unmodified preparation are complex and are altered by ATP or ouabain. 3. The compounds examined cause loss of Na++K+-stimulated adenosine triphosphatase activity when relatively few amino groups are modified but ATP was found to afford partial protection against inactivation by methylmaleic anhydride. Na++K+-stimulated adenosine triphosphatase activity is partly restored to the dimethylmaleylated preparation by hydrolysis of the dimethylmaleyl–amide bonds but not if more than about 20% of the amino groups have been acylated. 4. Supernatants obtained by high-speed centrifugation of the dimethylmaleylated preparation contained up to 45% of the total protein with less than 10% of the total phospholipid. Methylmaleyl and benzenetricarboxylyl derivatives of the enzyme preparation behaved similarly but tetrafluorosuccinylated material was almost entirely deposited by centrifugation.

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