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

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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Proteins of the kidney microvillar membrane. Purification and properties of the phosphoramidon-insensitive endopeptidase (‘endopeptidase-2’) from rat kidney

Biochemical Journal ◽

10.1042/bj2450515 ◽

1987 ◽

Vol 245 (2) ◽

pp. 515-524 ◽

Cited By ~ 68

Author(s):

A J Kenny ◽

J Ingram

Keyword(s):

Rat Kidney ◽

Polyacrylamide Gel Electrophoresis ◽

Gel Filtration ◽

Staining Intensity ◽

Aminopeptidase Activity ◽

Ph Optimum ◽

Reducing Conditions ◽

Purification And Properties ◽

Relationship Of ◽

Hydrolysis Of

A second endopeptidase is present in the renal microvillar membrane of rats that can be distinguished from endopeptidase-24.11 by its insensitivity to inhibition by phosphoramidon. The purification of this enzyme, referred to as endopeptidase-2, is described. The enzyme was efficiently released from the membrane by treatment with papain. The subsequent four steps depended on ion-exchange and gel-filtration chromatography. These steps were monitored by the hydrolysis of various substrates: 125I-insulin B chain (the normal assay substrate), benzoyl-L-tyrosyl-p-aminobenzoate (Bz-Tyr-pAB), azocasein and benzyloxycarbonyl-L-phenylalanyl-L-arginine 7-amino-4-methylcoumarylamide (Z-Phe-Arg-NMec). All four assays revealed comparable stepwise increases in activity in the main stages of the purification, although it was apparent that the last-named fluorogenic assay depended on traces of aminopeptidase activity present in the preparation. The Km for 125I-insulin B chain was 16 microM and that for Bz-Tyr-pAB was 4.7 mM. Several experimental approaches confirmed that both peptides were hydrolysed by the same enzyme. The pH optimum was 7.3. Phosphate buffers were inhibitory and shifted the optimum to above pH 9. Zinc was detected in the purified enzyme; EDTA and 1,10-phenanthroline were strongly inhibitory. SDS/polyacrylamide-gel electrophoresis revealed polypeptides of equal staining intensity of Mr 80,000 and 74,000 in reducing conditions. In non-reducing conditions a single band of apparent Mr 220,000 was seen. Gel filtration yielded an Mr of 436,000. These results are consistent with an oligomeric structure in which the alpha and beta chains are linked by disulphide bridges. Endopeptidase-2 hydrolysed a number of neuropeptides. Enkephalins resisted attack, only the heptapeptide [Met]enkephalin-Arg6-Phe7 being susceptible to slow hydrolysis. Luliberin (luteinizing-hormone-releasing hormone) and bradykinin were rapidly hydrolysed. Neurotensin was shown to be slowly attacked at the Tyr3-Glu4 bond. Thus the specificity appears to be limited to the hydrolysis of bonds involving the carboxy group of aromatic residues, provided that this P1 residue is extended by additional residues, at least to the P3′ position. The relationship of this membrane metalloendopeptidase to mouse meprin and human ‘PABA peptidase’ is discussed.

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An activator stimulating the enzymic hydrolysis of sphingoglycolipids.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)33814-0 ◽

1976 ◽

Vol 251 (4) ◽

pp. 1159-1163 ◽

Cited By ~ 13

Author(s):

S C Li ◽

Y T Li

Keyword(s):

Enzymic Hydrolysis ◽

Hydrolysis Of

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Variation in Quantity of Methanol Recovered from Raisins

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/46.2.341 ◽

1963 ◽

Vol 46 (2) ◽

pp. 341-343

Author(s):

M Alice Brown ◽

James R Woodward ◽

Floyd DeEds

Keyword(s):

Methyl Ester ◽

Esterase Activity ◽

Enzymic Hydrolysis ◽

Methanol Content ◽

Naturally Occurring ◽

Pectin Esterase ◽

Hydrolysis Of

Abstract The amount of naturally occurring methanol in fruit must be known so that the quantity left as fumigation residue can be determined. In a study of methanol content of raisins, which had given inconsistent results, the raisins were subjected to different conditions of treatment immediately prior to methanol determination. Conditions that favored pectin esterase activity gave higher values for methanol content than conditions known to inactivate enzymes. Evidence was also obtained that both chemical and enzymic hydrolysis of methyl ester groups of pectic materials occur during analysis.

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Permeability of muscle capillaries to small heme-peptides. Evidence for the existence of patent transendothelial channels.

The Journal of Cell Biology ◽

10.1083/jcb.64.3.586 ◽

1975 ◽

Vol 64 (3) ◽

pp. 586-607 ◽

Cited By ~ 380

Author(s):

N Simionescu ◽

M Siminoescu ◽

G E Palade

Keyword(s):

Plasma Proteins ◽

Intercellular Junctions ◽

Rat Diaphragm ◽

Enzymic Hydrolysis ◽

Graphic Analysis ◽

Heme Peptides ◽

Future Work ◽

Hydrolysis Of ◽

Muscle Capillaries

Two heme-peptides (HP) of about 20-A diameter (heme-undecapeptide [H11P], mol wt approximately 1900 and heme-octapeptide [H8P], mol wt approximately 1550), obtained by enzymic hydrolysis of cytochrome c, were sued as probe molecules in muscle capillaries (rat diaphragm). They were localized in situ by a perixidase reaction, enhanced by the addition of imidazole to the incubation medium. Chromatography of plasma samples showed that HPs circulate predominantly as monomers for the duration of the experiments and are bound by aldehyde fixatives to plasma proteins to the extent of approximately 50% (H8P) to approximately 95% (H11P). Both tracers cross the endothelium primarily via plasmalemmal vesicles which become progressively labeled (by reaction product) from the blood front to the tissue front of the endothelium, in three successive resolvable phases. By the end of each phase the extent of labeling reaches greater than 90% of the corresponding vesicle population. Labeled vesicles appear as either isolated units or chains which form patent channels across the endothelium. The patency of these channels was checked by specimen tilting and graphic analysis of their images. No evidence was found for early or preferential marking of the intercellular junctions and spaces by reaction product. It is concluded that the channels are the most likely candidate for structural equivalents of the small pores of the capillary wall since they are continuous, water-filled passages, and are provided with one or more strictures of less than 100 A. Their frequency remains to be established by future work.

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Investigation of the products of an enzymic hydrolysis of collagens

Biochemistry ◽

10.1021/bi00840a009 ◽

1969 ◽

Vol 8 (12) ◽

pp. 4716-4723 ◽

Cited By ~ 17

Author(s):

Howard B. Bensusan

Keyword(s):

Enzymic Hydrolysis ◽

Hydrolysis Of

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Constancy of the optimum temperature of an enzyme under varying concentrations of substrate and of enzyme

Proceedings of the Royal Society of London. Series B, Containing Papers of a Biological Character ◽

10.1098/rspb.1914.0073 ◽

1914 ◽

Vol 88 (602) ◽

pp. 258-262

Keyword(s):

Experimental Evidence ◽

Aspergillus Oryzae ◽

Hydrolytic Enzyme ◽

Fixed Duration ◽

Optimum Temperature ◽

Enzymic Hydrolysis ◽

Main Interest ◽

General Law ◽

The Moment ◽

Hydrolysis Of

In a recent paper a new enzymic relation is recorded. For the enzymic hydrolysis of salicin—by the enzyme which Gabriel Bertrand and the author have named salicinase —it is found that, in an action of fixed duration, the temperature of greatest activity of the ferment is always the same, whatever the dilutions of substrate and of enzyme adopted for the determination. In other words, the duration of the action being constant, the optimum temperature of the ferment is independent of the concentration both of the substrate and of the enzyme. The observation is suggestive: if true of one enzyme it may be true of all, and possibly becomes the enunciation of a general law. Herein, for the moment, lies its main interest. In the present paper further experimental evidence for this hypothesis in given, in the case of another hydrolytic enzyme, the maltase of Aspergillus oryzæ (taka-diastase).

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