The 11S proteasome activator: Isolation from mouse brain and the influence on peptide substrate hydrolysis of the 20S and 26S proteasomes

Abstract We measured phospholipase A2 (PLA2; EC 3.1.1.4) activity in normal and uremic plasma, using [1-14C]oleate-labeled autoclaved Escherichia coli as substrate. Hydrolysis of bacterial phospholipid by crude plasma from both groups was optimal at pH 5.5, was specific for the 2-acyl position of phospholipids, and had an absolute requirement for calcium. Activity was greatest in the presence of added Ca2+, 5 mmol/L, but this increase was inhibited by several divalent cations (Mg2+, Zn2+, Cu2+, Ba2+, Co2+, Pb2+, Fe2+) and by Fe3+. PLA2 activity was also inhibited by heparin at acid and alkaline pH, normal plasma being more sensitive than uremic plasma to this inhibition. Enzyme activity in undiluted plasma was eightfold greater in uremic than in normal plasma. Dilution of plasma by two to fourfold increased the total activity of both normal and uremic plasma. However, the relative differences in total activity between the groups remained constant (eight- to 11-fold). The cause and consequences of the increased PLA2 activity in uremia remain to be established.

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Characterization of Canine Hepatic and Renal Esterases

Canadian Journal of Biochemistry ◽

10.1139/o73-062 ◽

1973 ◽

Vol 51 (5) ◽

pp. 506-513 ◽

Cited By ~ 9

Author(s):

D. J. Ecobichon

Keyword(s):

Cholinesterase Activity ◽

Kinetic Characteristics ◽

Migration Patterns ◽

Tissue Extracts ◽

Liver And Kidney ◽

Inhibition Studies ◽

Hydrolysis Of ◽

Naphthyl Acetate ◽

Substrate Hydrolysis

The esterases of canine liver and kidney were separated electrophoretically into nine bands with identical migration patterns in both tissues. An additional pair of rapidly migrating anodic bands were observed in hepatic extracts. Based on substrate specificity, the predominant tissue esterases were identified as nonspecific carboxylesterases (aliesterases). No cholinesterase activity was detected in the tissue extracts. Kinetic characteristics determined for the hepatic and renal esterases included (1) optimal pH; (2) Km values for esters of α-naphthyl and p-nitrophenol; (3) average rates of hydrolysis of α-naphthyl acetate and p-nitrophenyl acetate by the tissue extracts. Inhibition studies revealed the presence of two types of esterase activity in each tissue: one type being sensitive to organophosphorus esters, the second being resistant. A study of preferential substrate hydrolysis in the presence of known characteristic activators and inhibitors of esterases revealed approximately 5% and 20% arylesterase activity in liver and kidney, respectively. The presence of arylesterase activity in these tissues was confirmed by the hydrolysis of paraoxon (E600).

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Novel activity of endothelin-converting enzyme: hydrolysis of bradykinin

Biochemical Journal ◽

10.1042/bj3270023 ◽

1997 ◽

Vol 327 (1) ◽

pp. 23-26 ◽

Cited By ~ 74

Author(s):

Mien V. HOANG ◽

Anthony J. TURNER

Keyword(s):

Chinese Hamster ◽

Enzyme Hydrolysis ◽

Converting Enzyme ◽

Peptide Substrate ◽

Neprilysin Inhibitor ◽

Endothelin Converting Enzyme ◽

Key Enzyme ◽

Potent Vasoconstrictor ◽

Hydrolysis Of ◽

Physiological Substrates

Endothelin-converting enzyme (ECE) is the key enzyme in the production of the potent vasoconstrictor endothelin from its inactive precursor big endothelin. To date, no other physiological peptide substrate has been identified for ECE. Here, by using Chinese hamster ovary (CHO) cells transfected with rat ECE-1 cDNA, we have established that ECE can hydrolyse the vasodilator bradykinin. The hydrolysis of bradykinin by ECE is exclusively at the Pro7–Phe8 bond, producing bradykinin-(1–7) and bradykinin-(8–9). Hydrolysis is completely inhibited by 100 μM phosphoramidon and 200 μM EDTA, but only slightly by the specific neprilysin inhibitor thiorphan (100 μM). The ability of ECE to act as a peptidyl dipeptidase rather than an endopeptidase in hydrolysing bradykinin suggests a much broader specificity for the enzyme than previously recognized, which may lead to the design of new and specific inhibitors of ECE and to the identification of other potential physiological substrates.

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Selective inhibition of Zn2+-glycerophosphocholine cholinephosphodiesterase by tellurium tetrachloride

Biochemical Journal ◽

10.1042/bj2840641 ◽

1992 ◽

Vol 284 (3) ◽

pp. 641-643 ◽

Cited By ~ 6

Author(s):

D E Sok ◽

M R Kim

Keyword(s):

Metal Ions ◽

Mouse Brain ◽

Selective Inhibition ◽

Inhibitory Effect ◽

Physiological Conditions ◽

Neutral Ph ◽

Tellurium Tetrachloride ◽

Hydrolysis Of ◽

Competitive Pattern

A Zn(2+)-glycerophosphocholine cholinephosphodiesterase (EC 3.1.4.38) purified from mouse brain was found to be reversibly inhibited by tellurium tetrachloride. This effect was characterized by a competitive pattern of inhibition, with apparent Ki values of 0.7 microM and 1.5 microM for the hydrolysis of p-nitrophenylphosphocholine and glycerophosphocholine respectively. Interestingly, the inhibitory effect of tellurium tetrachloride was found to be greatly potentiated by tetramethylammonium salt, indicative of a synergistic interaction between the two compounds. Additionally, it was observed that the effect of tellurium tetrachloride was not affected by a number of other metal ions, and was more pronounced at neutral pH, suggesting that the inhibitory role of the tellurium tetrachloride may be of importance under physiological conditions. Thus Zn(2+)-glycerophosphocholine cholinephosphodiesterase is proposed to be one of the target enzymes which is susceptible to the inhibitory effect of tellurium tetrachloride.

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Alteration of the Kinetics of Thrombin-catalyzed Hydrolysis of Amino Acid Ester Substrates by Sodium Cholate and Other Steroids

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647680 ◽

1974 ◽

Vol 32 (01) ◽

pp. 132-148 ◽

Cited By ~ 2

Author(s):

Edmond R. Cole

Keyword(s):

Sodium Cholate ◽

Amino Acid Ester ◽

Zero Order ◽

Substituent Group ◽

Zero Order Kinetics ◽

Rate Of Hydrolysis ◽

Kinetics Of ◽

Hydrolysis Of ◽

Temperature Optima ◽

Substrate Hydrolysis

SummaryThrombin-catalyzed hydrolysis of TAME proceeds by an initial zero-order phase which later falls off into an apparent first order reaction as substrate becomes limiting. Optimum amounts of sodium cholate not only accelerated TAME hydrolysis but also altered its kinetics to apparent zero-order to complete substrate hydrolysis. As this implies, the rate of hydrolysis in the presence of cholate was found to be independent of substrate concentration, provided concentrations of TAME and cholate were low enough to prevent precipitation of some TAME-cholate as an insoluble complex. The formation of a soluble complex composed of polymeric molecules of TAME and cholate may explain both the acceleration and the change in reaction order. Although the pH and temperature optima for TAME hydrolysis by thrombin were not altered by the presence of cholate, the degree of acceleration increased with rising pH and temperature on the ascending portion of the curves. This is believed to be due to the greater solubility of the TAME-cholate complex. The effects of cholate on thrombin-catalyzed hydrolysis of other arginine esters as well as esters of lysine, histidine and phenylalanine were also studied.Solutions of sodium desoxycholate and androsterone-3-sulfate accelerated TAME hydrolysis as did supensions of testosterone, etiocholanolone, androsterone, androsterone-3-hemisuccinate and pregnandiol-3-glucuronidate. However, isoandrosterone, progesterone, pregnandiol, estradiol, estrone, estriol, estrone-3-sulfate, cholesterol, corticosterone, hydrocortisone and hydrocortisone-3-phosphate had no significant effect on TAME hydrolysis by thrombin. The ability of the androgenic hormones to accelerate hydrolysis appeared to depend to some extent on the configuration of the substituent group at C3 and the hydrogen at C5. Androsterone-3-hemisuccinate was, like cholate, able to accelerate the hydrolysis of TAME at apparent zero-order kinetics to complete substrate hydrolysis.

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Kinetics of hydrolysis of a new peptide substrate containing p-guanidino-L-phenylalanine by trypsin and thrombin.

Chemical and Pharmaceutical Bulletin ◽

10.1248/cpb.34.1351 ◽

1986 ◽

Vol 34 (3) ◽

pp. 1351-1354 ◽

Cited By ~ 3

Author(s):

HIDEAKI TSUNEMATSU ◽

KOICHI MIZUSAKI ◽

YOSHIHIRO HATANAKA ◽

MASAO KAMAHORI ◽

SATORU MAKISUMI

Keyword(s):

Peptide Substrate ◽

Kinetics Of Hydrolysis ◽

Kinetics Of ◽

Hydrolysis Of

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Characterization of a Zn2+-requiring glycerophosphocholine cholinephosphodiesterase possessing p-nitrophenylphosphocholine phosphodiesterase activity

Biochemical Journal ◽

10.1042/bj2860435 ◽

1992 ◽

Vol 286 (2) ◽

pp. 435-440 ◽

Cited By ~ 12

Author(s):

D E Sok ◽

M R Kim

Keyword(s):

Bacillus Cereus ◽

Molecular Mass ◽

Mouse Brain ◽

Concanavalin A ◽

Heat Denaturation ◽

Phosphodiesterase Activity ◽

Optimum Ph ◽

Hydrolysis Of ◽

Optimal Ph

p-Nitrophenylphosphocholine phosphodiesterase activity was purified 5000-fold from mouse brain by treatment of membranes with Bacillus cereus phospholipase C preparation and sequential chromatographies on concanavalin A-Sepharose and CM-Sephadex columns. The phosphodiesterase (Zn(2+)-requiring) showed Km and Vmax. values of 5.5 microM and 4.2 mumol/min per mg respectively in the hydrolysis of p-nitrophenylphosphocholine, and possessed an optimum pH of 10.5 and a molecular mass of approx. 74 kDa. The purified enzyme was found to convert glycerophosphocholine into glycerol and phosphocholine, with Km and Vmax. of 48 microM and 5 mumol/min per mg respectively. In the hydrolysis of glycerophosphocholine the enzyme also exhibited a Zn2+ requirement and optimal pH at 10.5. Additionally, the p-nitrophenylphosphocholine phosphodiesterase activity was competitively inhibited by glycerophosphocholine, with a Ki value of 50 microM. These observations, together with chromatographic behaviour and heat-denaturation analyses, indicate that both p-nitrophenylphosphocholine phosphodiesterase and glycerophosphocholine cholinephosphodiesterase activities reside in the same protein.

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Cloning, Expression and Characterization of a Novel Cold-adapted β-galactosidase from the Deep-sea Bacterium Alteromonas sp. ML52

Marine Drugs ◽

10.3390/md16120469 ◽

2018 ◽

Vol 16 (12) ◽

pp. 469 ◽

Cited By ~ 6

Author(s):

Jingjing Sun ◽

Congyu Yao ◽

Wei Wang ◽

Zhiwei Zhuang ◽

Junzhong Liu ◽

...

Keyword(s):

Deep Sea ◽

Glycoside Hydrolase ◽

Sea Water ◽

Maximum Activity ◽

Glycoside Hydrolase Family ◽

Cold Adapted ◽

Hydrolysis Of ◽

Hydrolase Family ◽

Substrate Hydrolysis

The bacterium Alteromonas sp. ML52, isolated from deep-sea water, was found to synthesize an intracellular cold-adapted β-galactosidase. A novel β-galactosidase gene from strain ML52, encoding 1058 amino acids residues, was cloned and expressed in Escherichia coli. The enzyme belongs to glycoside hydrolase family 2 and is active as a homotetrameric protein. The recombinant enzyme had maximum activity at 35 °C and pH 8 with a low thermal stability over 30 °C. The enzyme also exhibited a Km of 0.14 mM, a Vmax of 464.7 U/mg and a kcat of 3688.1 S−1 at 35 °C with 2-nitrophenyl-β-d-galactopyranoside as a substrate. Hydrolysis of lactose assay, performed using milk, indicated that over 90% lactose in milk was hydrolyzed after incubation for 5 h at 25 °C or 24 h at 4 °C and 10 °C, respectively. These properties suggest that recombinant Alteromonas sp. ML52 β-galactosidase is a potential biocatalyst for the lactose-reduced dairy industry.

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