R. Scandurra

Oxalate, phosphate and sulphate determination in serum and urine by ion chromatography

A rapid method for the determination of phosphate, sulphate and oxalate in serum by ion chromatography is described. Serum is deproteinized through a Centrifree filter by centrifugation and the ultrafiltrate directly injected into an ion chromatograph equipped with an anion exchange column and a conductivity detector. By this procedure the sample is not diluted and even small amounts of oxalate in biological fluids can be detected. Mean serum concentrations found in healthy individuals are: phosphate 1.07 mmol/l; sulphate 0.35 mmol/l; oxalate 21.02 mumols/l. Phosphate, sulphate and oxalate contents were also determined in urine from healthy individuals. Values found in serum and urine are in good agreement with those previously reported.

Peptidyl and azapeptidyl methylketones as substrate analog inhibitors of papain and cathepsin B

Summary Peptidyl methylketones containing Phe, Tyr, Tyr(I), Tyr(I 2 ), Leu and Ile in P 2 were synthesized and tested as substrate analog reversible inhibitors of papain and bovine spleen cathepsin B. The most effective cathepsin B inhibitor contained Tyr(I 2 ) and displayed an inhibition constant of 4.7 μM at pH 6.8 and 25°C, while Leu or Ile gave practically inert analogs. Replacement of the amino acids in P 2 with the analogous α-azaamino acids, as well as the glycine in P 1 with α-azaglycine, led to complete loss of inhibiting activity. Introducing alkoxy substituents at the methyl adjacent to the ketone group generally resulted in more effective inhibitors, with inhibition constants in the micromolar range for both papain and cathepsin B.

Irreversible inactivation of papain and cathepsin B by epoxidic substrate analogues

Abstract Epoxidic substrate analogues related to allylamine (4a–4e) and allyl alcohol (5a–5f) were synthesized and tested as models of cysteine-protease inhibitors. They proved to be irreversible inhibitors of papain and cathepsin B with pseudo-first-order inactivation rates ranging from 0.3 to 33 M−1 min−1. The most active of the studied oxiranes 4a bears N-acetyl- l -Phe as peptidyl unity. Most of the inhibitory activity was retained when the recognising moiety was extensively modified, provided that a phenyl group ∂ or the trapping epoxide e was present. Specificity of peptidylepoxides for cysteine-proteases was confirmed, since no inhibitory activity was displayed toward serine or metallo-proteases.

AMBIGUITIES IN THE ENZYMOLOGY OF SULFUR-CONTAINING COMPOUNDS

In the course of this meeting we have occasionally met reactions involving a variety of sulfur-containing compounds carried out by systems or enzymes also used for different purposes. In this final lecture we should like to point out how this phenomenon is much more general than believed, thus indicating the occurrence of a real ambiguity in the enzymology of sulfur-containing compounds. This fact became to the attention of one of us early in 1956, when cystamine (I) and lanthionamine (II) were assayed as substrates for diamine oxidase (1). It was known at that time that diamine oxidase was an enzyme of broad specificity, being able to use as substrates diamines having different carbon chain lengths and also diamines with one of the amino groups substituted with an imidazole or a guanido group (2). However it was not known whether a substantial change of the chemical composition would have impaired the enzymatic attack on the substrate. It was therefore a surprise to find an enzyme unable to recognize the substitution of a methylene carbon of the substrate with one or two atoms of sulfur. Diamine oxidase uses not only I and II as substrates but also lanthionamine sulfone, although at a lower rate than I and II (1). The interest of this finding was increased by the observation that the rate of the oxidation of I and II was in the range of that of the traditional substrates and that the oxidation was followed by the cleavage of the intermediate cystaldimine (IV) leading to the formation of compounds of biological interest like thiocysteamine (VI), hypotau- rine (VII), thiotaurine (VIII) and taurine (IX).

Covalently bound pyruvate in phosphopantothenoylcysteine decarboxylase from horse liver

Horse liver phosphopantothenoylcysteine decarboxylase (EC 4.1.1.36) incorporates nonexchangeable tritium from borotritide with a decrease of the activity. Substrate prevents both tritium incorporation and the decrease in activity. Acid and base hydrolysis of the tritiated protein releases labeled lactate identified by highvoltage paper electrophoresis, paper chromatography and silicic acid chromatography. These results indicate the presence of pyruvate covalently bound through an ester linkage to phosphopantothenoylcysteine decarboxylase which is then another example of a mammalian enzyme in which pyruvate is involved in a catalytic activity.

Iodo and diiodotyrosine epoxysuccinyl derivatives as selective inhibitors of cathepsin B

Eight new analogs of l-trans-epoxysuccinyl-l-leucylamido(3-methyl)butane (E-64-c) containing Phe, Tyr, Tyr(1) or Tyr(I2) in place of Leu, were synthesized and tested as inhibitors of papain, bovine spleen cathepsin B, calpain I and II from porcine red cells and porcine kidney, respectively. By use of kinetic methods, the new E-64 analogs proved to irreversibly inactivate both papain and cathepsin B via reversible enzyme-inhibitor intermediates EI. Second-order rate constants for inactivation were in the range 3500-55 100 M−1s−1 for papain and 650–105 000 M−1s−1 for cathepsin B. For the inactivation of calpain I and II they ranged between 250 and 2000 M−1s−1 and were similar to those of the known E-64-c. The effectiveness of the amino acid contained in the inhibitors tested increased in the order Tyr(I) ≈ Tyr(I2) < Tyr < Phe < Leu for papain and Phe < Tyr < Tyr(I) < Leu < Tyr(I2) for cathepsin B inactivation. Replacement of the l with the d-trans-epoxysuccinyl unit caused a 10–100-fold decrease in inhibitor potencies.

N-Haloacetyl-amino-acid amides as active-site-directed inhibitors of papain and cathepsin B

Abstract A series of N -haloacetyl-amino-acid amides were synthesized and tested as models of cysteine-protease inhibitors. They irreversibly inactivated papain and cathepsin B via a reversible enzyme-inhibitor intermediate. Apparent second-order rate constants of inactivation ranging from 65 to 16 700 M −1 s −1 were observed. Reactivity against papain, as compared to glutathione, was increased 16 400-fold for N -bromoacetyl-leucine isopentylamide and 25 700-fold for the corresponding iodoacetyl derivative; these increases are probably due to proximity effects. No inhibition of trypsin, chymotrypsin and porcine pancreatic elastase was observed. Haloacetamides represent an interesting class of easily synthesized, efficient, irreversible inhibitors of cysteine proteases, which have low non-specific alkylating properties.

Synthesis and inhibiting activities of 1-peptidyl-2-haloacetyl hydrazines toward cathepsin B and calpains

Abstract Twenty-four 1-peptidyl-2-haloacetyl hydrazines which can be considered azapeptide halomethanes were synthesized and tested as models of cathepsin B, calpain I and calpain II inhibitors. Reagents designed for cathepsin B inactivation include Z-Tyr, Z-Tyr(I) and Z-Leu-Leu attached to an α-azaglycine or α-azaalanine unit in P 1 . By use of kinetic analysis, they proved to irreversibly inactivate cathepsin B via a reversible enzyme-inhibitor intermediate. Second-order rate constants in the range 725-306 000 M −1 s −1 were found for cathepsin B inactivation, with no more than 7 500 M −1 s −1 for calpain II. K 1 for the reversible EI adducts ranged from 11 to 0.06 μM for cathepsin B. Structure of the possible reversible EI complex is proposed and used to discuss the effects of structural variation of the inhibitors on the kinetic parameters of inactivation. 1-Peptidyl-2-haloacetyl hydrazines designed for calpain inactivation include Boc-Val-(N ϵ -carbomethoxy)Lys-Leu, Boc-Val-Lys(N ϵ -Z)-Leu, Boc-Val-Lys(N ϵ -Tos)-Leu and Z-Leu-Leu attached to an α-azatyrosine unit in P 1 . They gave poor results. Title compounds proved to be selective for cysteine proteases, since no inhibiting activity could be detected toward trypsin, chymotrypsin and porcine pancreatic elastase at 0.1 mM concentration. Relatively low aspecific alkylating properties were also demonstrated in tests using glutathione as the nucleophile.

[200] Cysteamine oxygenase (Horse Kidney)

Publisher Summary This chapter discusses the method of preparation of Cysteamine Oxygenase (Horse Kidney). Cysteamine oxygenase oxidizes cysteamine to hypotaurine in the presence of Na 2 S, that acts as a cofactor. Molecular oxygen is used for this oxidation. When the concentration of the substrate is reduced to the level of 10 -5 M, the enzyme is active without any cofactor. Na 2 S may be replaced by other compounds. Traces of taurine and thiotaurine are produced as side reactions from hypotaurine during the enzymatic reaction. This enzyme is present in a number of animal tissues, and was purified from horse kidney. The assay is based on the paper chromatographic separation of hypotaurine from cysteamine. Taurine and thiotaurine when present are added to hypotaurine. Quantitation is obtained by using 35 S-labeled cysteamine as substrate and counting the area of paper corresponding to the labeled products using a chromatogram scanner. The enzyme seems specific for cysteamine. Under the conditions where cysteamine is largely oxidized to hypotaurine, cysteine and glutathione are not oxidized to a level higher than that of a disulfide.

Functional residues at the active site of horse liver phosphopantothenoylcysteine decarboxylase

Horse liver phosphopantothenoylcysteine decarboxylase (EC 4.1.1.36) is rapidly inactivated by N‐acetoacetylation with diketene following a pseudo‐first‐order kinetics: the presence of substrate quantitatively protects against this inactivation. Histidine photo‐oxidation with methylene blue or rose bengal brings about the total loss of activity. These results indicate the presence of functional lysyl and histidyl groups at the active site of the enzyme. The substrate sulphydryl group is essential for enzyme activity. Enzymatic decarboxylation is proposed to result from a combined action of the keto group of the enzyme‐bound pyruvate protonated by an essential histidine and a protonated amino group of a lysine.