scholarly journals A radiochemical titrant for the determination of the operational molarity of solutions of acid proteinases

1979 ◽  
Vol 183 (2) ◽  
pp. 389-394 ◽  
Author(s):  
G B Irvine ◽  
D T Elmore
Keyword(s):  
Active Site ◽  
Cathepsin D ◽  
Gel Filtration ◽  
British Library ◽  
Excess Reagent ◽  
Irreversible Inhibitors ◽  
Putative Active Site ◽  
Quantitative Separation ◽  
Inhibited Enzyme

N-Diazoacetyl-L-phenylalanine 3-phenyl[2,3-3H]propylamide was synthesized and shown to inhibit pepsin A (EC3,4,23.1) and cathepsin D (EC 3.4.23.5) irreversibly and stoicheiometrically in the presence of Cu2+. Quantitative separation of the inhibited enzyme from excess reagent by gel filtration followed by measurement of the radioactivity of the protein peak provided a method for determining the operational molarity of these enzymes. Several other putative active-site-directed irreversible inhibitors were synthesized, but were inactive. Data on the synthesis of these compounds have been deposited as Supplementary Publication SUP50096 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.

scholarly journals Inhibition of glutathione S-transferase 3-3 by glutathione derivatives that bind covalently to the active site

1991 ◽  
Vol 278 (1) ◽  
pp. 63-68 ◽  
Author(s):  
A E P Adang ◽  
W J Moree ◽  
J Brussee ◽  
G J Mulder ◽  
A van der Gen
Keyword(s):  
Active Site ◽  
Current Knowledge ◽  
Control Level ◽  
Gel Filtration ◽  
Cysteine Residue ◽  
Covalent Modification ◽  
British Library ◽  
Glutathione S Transferase ◽  
Enzyme Active Site ◽  
Active Site Cysteine

In all, 13 GSH derivatives have been synthesized and tested for their potency to inhibit glutathione S-transferase (GST) 3-3. All of these derivatives contained a reactive group that could potentially react with the enzyme active site. Best results were obtained with the phenylthiosulphonate derivative of GSH, GSSO2Ph. Preincubation of GST 3-3 with a 100 microM concentration of this inhibitor resulted in a time-dependent loss of activity: after 30 min at pH 6.5 and 25 degrees C, 51% of the activity was lost. At more alkaline pH, the activity is more rapidly inhibited: at pH 8.0 the 90%-inhibition level is already reached after 10 min preincubation. Separation of enzyme and excess unbound GSSO2Ph after preincubation by gel-filtration chromatography did not result in a reappearance of enzyme activity. If 100 microM-GSH was added to the preincubation mixture at pH 7.4, inhibition was almost completely prevented. Addition of S-(hexyl)glutathione (20 microM) could delay the inhibition but, ultimately, not prevent it. The inhibited enzyme could be re-activated by addition of 10 mM-2-mercaptoethanol: 60 min after this thiol was added, the inhibited GST-3- activity was bacxk to the control level. GSH at the same concentration could not re-activate the enzyme. On the basis of these results, on the known reactivity of thiosulphonate compounds, and on current knowledge about the amino acid residues involved in GST catalysis, a covalent modification of an active-site cysteine residue by mixed-disulphide formation between enzyme and the cosubstrate GSH is postulated. Information on the synthesis and characterization of the GSH derivatives is given in Supplementary Publication SUP 50166 (5 pages) which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5.

scholarly journals Crystal Structure of the Bacterial YhcH Protein Indicates a Role in Sialic Acid Catabolism

2005 ◽  
Vol 187 (16) ◽  
pp. 5520-5527 ◽  
Author(s):  
Alexey Teplyakov ◽  
Galina Obmolova ◽  
John Toedt ◽  
Michael Y. Galperin ◽  
Gary L. Gilliland
Keyword(s):  
Crystal Structure ◽  
Sialic Acid ◽  
Active Site ◽  
Copper Ion ◽  
Protein Fold ◽  
Genomic Context ◽  
Putative Active Site ◽  
Multiple Wavelength ◽  
Β Sheets

ABSTRACT The yhcH gene is part of the nan operon in bacteria that encodes proteins involved in sialic acid catabolism. Determination of the crystal structure of YhcH from Haemophilus influenzae was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein. The structure was determined at 2.2-Å resolution by multiple-wavelength anomalous diffraction. The protein fold is a variation of the double-stranded β-helix. Two antiparallel β-sheets form a funnel opened at one side, where a putative active site contains a copper ion coordinated to the side chains of two histidine and two carboxylic acid residues. A comparison to other proteins with a similar fold and analysis of the genomic context suggested that YhcH may be a sugar isomerase involved in processing of exogenous sialic acid.

scholarly journals Purification, crystallization and properties of porphobilinogen deaminase from a recombinant strain of Escherichia coli K12

1988 ◽  
Vol 254 (2) ◽  
pp. 427-435 ◽  
Author(s):  
P M Jordan ◽  
S D Thomas ◽  
M J Warren
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Recombinant Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Porphobilinogen Deaminase ◽  
Terminal Sequence ◽  
E Coli ◽  
Human Enzyme

Porphobilinogen deaminase has been purified and crystallized from an overproducing recombinant strain of Escherichia coli harbouring a hemC-containing plasmid which has permitted the purification of milligram quantities of the enzyme. Determination of the Mr of the enzyme by SDS/polyacrylamide-gel electrophoresis (35,000) and gel filtration (32,000) agrees with the gene-derived Mr of 33,857. The enzyme has a Km of 19 +/- 7 microM, an isoelectric point of 4.5 and an N-terminal sequence NH2-MLDNVLRIAT. The substrate, porphobilinogen, binds to the active-site dipyrromethane cofactor to form three intermediate complexes: ES, ES2 and ES3. The gene-derived primary structure of the E. coli deaminase is compared with that derived from the cDNA of the human enzyme.

Turnover of Human Extrinsic (Tissue-Type) Plasminogen Activator in Rabbits

1981 ◽  
Vol 46 (03) ◽  
pp. 658-661 ◽  
Author(s):  
C Korninger ◽  
J M Stassen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Intravenous Injection ◽  
Active Site ◽  
Half Life ◽  
Degradation Products ◽  
Gel Filtration ◽  
Tissue Type ◽  
Organ Distribution ◽  
Small Rise

SummaryThe turnover of highly purified human extrinsic plasminogen activator (EPA) (one- and two-chain form) was studied in rabbits. Following intravenous injection, EPA-activity declined rapidly. The disappearance rate of EPA from the plasma could adequately be described by a single exponential term with a t ½ of approximately 2 min for both the one-chain and two-chain forms of EPA.The clearance and organ distribution of EPA was studied by using 125I-labeled preparations. Following intravenous injection of 125I-1abeled EPA the radioactivity disappeared rapidly from the plasma also with a t ½ of approximately 2 min down to a level of 15 to 20 percent, followed by a small rise of blood radioactivity. Gel filtration of serial samples revealed that the secondary increase of the radioactivity was due to the reappearance of radioactive breakdown products in the blood. Measurement of the organ distribution of 125I at different time intervals revealed that EPA was rapidly accumulated in the liver, followed by a release of degradation products in the blood.Experimental hepatectomy markedly prolonged the half-life of EPA in the blood. Blocking the active site histidine of EPA had no effect on the half-life of EPA in blood nor on the gel filtration patterns of 125I in serial plasma samples.It is concluded that human EPA is rapidly removed from the blood of rabbits by clearance and degradation in the liver. Recognition by the liver does not require a functional active site in the enzyme. Neutralization in plasma by protease inhibitors does not represent a significant pathway of EPA inactivation in vivo.

Sign in / Sign up

Export Citation Format

Share Document