The reactivation of phosphorylated chymotrypsin

Green, A. L.; Nicholls, J.

doi:10.1042/bj0720070

Cited by 23 publications

(15 citation statements)

References 18 publications

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“…- Green and Nicholls (1959) showed that Sarininactivated chymotrypsin slowly regained activity when incubated in the abeence of added madiva-. .…”

Section: Resultsmentioning

confidence: 98%

“…The apparent rate constant can therefore be calculated from formula (1) (cf. Green and Nicholls, 1959), where A is the concentration of the reactivator and t i s the time of incubation. Calculation yielded an apparent rate constant for the reactivation of DEPBiochemistry Dissociation constants not previously reported were determined with a Radiometer Type SBRZC/SBUlaiTTTlb; 0.15 mmole of acid in 10 ml of 0.2 M NaCl was titrated with 0.4 N NaOH under a n atmosphere of nitrogen.…”

Section: Resultsmentioning

confidence: 99%

“…This is in accord with the data reported by Stewart and Ouellet (1959 j for the deacetylation of monoacetyltrypsin and also agrees with the findings of Bendei et al (1961) for the deacylation of cinnamoyl-a-chymotrypsin. On the other hand, the rate of spontaneous reactivation of Sarin-inactivated chymotrypsin was reported to occur more rapidly as the pH was lowered (Green and Nicholls, 1959). In order to determine whether this discrepancy was due to the presence of a different organophosphorous functional group, an attempt was made to ascertain the effect of pH upon the reactivation of DEP-chymotrypsin.…”

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confidence: 99%

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The Reactivation of Diethylphosphoryltrypsin^*

Cohen¹,

Lache²,

Erlanger³

1962

Biochemistry

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The process of reactivation of diethylphosphoryltrypsin (DEP-trypsin) was studied in order to determine its relationship to the deacylation step of the catalytic mechanism of trypsin. It was found that DEP-trypsin could be completely reactivated by hydroxylamine and by formohydroxamic acid even after storage for 1 year. In both processes the data indicated the participation of a functional group on the enzyme with a pK, near neutrality. Other hydroxamic acids and oximes were also tested as reactivators, enhanced activities being shown by L-tyrosinehydroxamic acid, phenylaceto-hydroxamic acid, and anti-phenylglyoxaldoxime. DEP-trypsin slowly regained activity in the absence of added reactivator, the rate of this spontaneous reactivation increasing with increasing pH. The rate of reactivation, whether spontaneous or mediated by hydroxylamine or formohydroxamic acid, was not increased by the addition of the competitive inhibitor D-lysine methyl ester hydrochloride. Furthermore, the rate of reactivation of DEP-chymotrypsin, whether spontaneous or in the presence of nucleophilic agents, was not influenced by the presence of indole. It was concluded that, although many elements of similarity exist between dephosphorylation and the deacylation step of the enzyme mechanism, many,discrepancies remain to be explained.Trypsin, like a number of other esterases, can be inactivated by certain organophosphorous compounds (Jansen et al., 1949) which have been shown to cause phosphorylation of a single serine residue present a t the active center of the enzyme (Oosterbaan et al., 1955). Cholinesterase behaves similarly and, moreover, the studies of Wilson (1959) and of Green and Smith (1958) have shown that nucleophilic agents such as oximes and hydroxamic acids can dephosphorylate and thereby reactivate the enzyme. Wilson (1959) was able to demonstrate a correlation between the structure of an efficient nucleophilic agent and the conformation of the active surface of the enzyme. Experiments performed in this laboratory (Cohen and Erlanger, 1960) indicated that this relationship also applied to the reactivation of diethylphosphorylchymotrypsin.This paper reports an investigation of the reactivation of diethylphosphoryltrypsin (DEPtrypsin) by a number of nucleophilic agents. It will be shown that DEP-trypsin is capable of complete reactivation. and that the process is more rapid than that of DEP-chymotrypsin under comparable conditions. The data will be discussed with a view toward elucidating certain aspects of the catalytic action of trypsin. EXPERIMENTALThe trypsin used in this work was Worthington crystalline trypsin, lyophilized.Preparation of DEP-trypsin.-One-tenth ml of tetraethylpyrophosphate (TEPP) (50% pure) * Supported by grants from the National Institutes of Health (E-1672) and the O 5 c e of Naval Research 1. dissolved in 10 ml of 0.05 M Tris-maleate buffer, pH 8.0, containing 0.05 M CaC12, was added in one portion, with swirling, to 1 g of trypsin in 50 ml of the same buffer. The resulting clear solution was allow...

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“…- Green and Nicholls (1959) showed that Sarininactivated chymotrypsin slowly regained activity when incubated in the abeence of added madiva-. .…”

Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

The Reactivation of Diethylphosphoryltrypsin^*

Cohen¹,

Lache²,

Erlanger³

1962

Biochemistry

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“…Use of 32 P- or 33 P-DFP for the first label in double-label assays would be preferable since the P-label would not be lost by ‘aging’, but unfortunately phosphate-labeled DFP was not commercially available. Evaluation of the proteins labeled by DFP was beyond the scope of the present study, but previous studies have shown that ‘aging’ varies among serine hydrolases (‘aging’ is low for chymotrypsin, trypsin, and liver ali-esterase compared with pseudocholinesterase), and with pH, temperature, and labeling agent ( e.g., (Jansz et al 1959; Clothier and Johnson 1979; Coult et al 1966; Green and Nicholls 1959) and references cited therein). Second, clearance of label from DFP-target proteins only assays a specific class of proteins, and overall protein degradation and non-lysosomal proteolysis need to be examined.…”

Section: Discussionmentioning

confidence: 99%

Reduced clearance of proteins labeled with diisopropylfluorophosphate in portacaval-shunted rats

Dienel

Cruz

2013

Metab Brain Dis

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Portacaval shunting is a model for hepatic encephalopathy that causes chronic hyperammonemia, disruption of metabolic, signaling, and neurotransmitter systems, and progressive morphological changes. Exposure of cultured cells to ammonia raises intralysosomal pH and inhibits proteolysis, and the present study tested the hypothesis that proteolytic capacity is diminished in portacaval-shunted rats. Proteins were labeled in vivo with tracer doses of diisopropylfluorophosphate (DFP) and clearance of label was assayed. This approach labeled proteins independent of protein synthesis, which is reported to be altered in shunted rats, and avoided complications arising from re-utilization of labeled amino acids that causes underestimation of degradation rate. Characterization of DFP labeling showed that protein labeling was fast, about 50% of the label was released during a 24h interval, labeling by DFP metabolites was negligible, inhibition of brain acetylcholinesterase was not detectable, and labeling by [3H]- and [14C]DFP was equivalent. To assay degradative capacity, proteins were first labeled with [3H]DFP, followed by labeling with [14C]DFP that was given 24 or 72h later. The 3H/14C ratio in each animal was used as a relative measure of removal of 3H-labeled proteins. 3H/14C ratios were generally significantly higher in portacaval-shunted rats than in controls, consistent with reduced proteolytic capacity. Assays of amino acid incorporation into brain protein generally replicated literature reports, supporting the conclusion that protein synthesis unlikely to be markedly inhibited and amino acid recycling influences calculated protein synthesis rates in shunted rats. Therapeutic strategies to reduce ammonia level would help normalize lysosomal functions and protein and lipid turnover.

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“…The most remarkable effect is their ability to reactivate phosphorylated acetyl cholinesterase; proteolytic enzymes with esterase activities, such as chymotrypsin (Green & Nicholls, 1959) and trypsin (Edery, to be published), can also be reactivated by high concentrations of the oximes.…”

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confidence: 99%