Ramesh C. Kamboj

A selective colorimetric assay for cathepsin L using Z-Phe-Arg-4-methoxy-β-naphthylamide

Among the intracellular proteinases, the thiol proteinases such as cathepsin B (EC 3.4.22.1), cathepsin H (EC 3.4.22.16) and cathepsin L (EC 3.4.22.15) which act at slightly acidic pHs are more likely to play an important role in lysosomal protein catabolism. Out of these, cathepsin L plays a major role primarily because it has high degradative activity on cellular and matrix proteins. However, the studies on cathepsin L in crude homogenates and subcellular fractions have always been hampered by the lack of a specific substrate to exclusively measure the activity of this proteinase. The only synthetic substrate alpha-N-benzyloxycarbonyl-L-Phe-L-Arg-4-methoxy-beta-naphthylamide (Z-Phe-Arg-NNapOMe) which is hydrolysed by cathepsin L is hydrolysed equally well by cathepsin B. This substrate was manipulated to act as a selective substrate for cathepsin L. In presence of 4 M urea at pH 5.0, cathepsin B (the only other cathepsin which also hydrolyses Z-Phe-Arg-NNapOMe) was inactivated and, therefore, under these conditions, the enzyme activity quantitated by using this substrate is only due to cathepsin L. Using this newly-developed colorimetric assay method specific for cathepsin L, the subcellular and regional distribution of this proteinase were established in goat brain tissue. About 80% cathepsin L activity was recovered in the lysosomal fraction thus establishing its lysosomal nature. Among the various brain parts, highest activity was found in cerebrum followed by cerebellum, pituitary body, pons-varolli, thalamus, medulla-oblongata and hypothalamus.

Purification and characterization of cathepsin B from goat brain

Cathepsin B was purified to an apparent homogeneity from goat brain utilizing the techniques of homogenization, autolysis at pH 4, 30–70% (NH4)2SO4 fractionation, Sephadex G-100 column chromatography, organomercurial afinity chromatography and ion-exchange chromatography on CM-Sephadex C-50. The enzyme had a pH optima of 6 with α-N-benzoyl-D, L-arginine-β-naphthIylamide, benzyloxycarbonyl-arginine-arginme-4-methoxy -β-naphthylamide and azocasein as substrates. TheKm values for the hydrolysis of α-N-benzoyl-D, L-arginine-β-naphthylamide and benzyloxycarbonyl-arginine-arginine-4-methoxy -β-naphthylamide were 2.36 and 0.29 mM respectively in 2.5% dimethylsulphoxide. However, the correspondingKm values for these substrates in 1 % dimethylsulphoxide were 0.51 and 0.09 mM. The enzyme was strongly inhibited by thiol inhibitors and tetrapeptidyl chloromethylketones. Leupeptin inhibited the enzyme competitively withKi value of 12.5 × l0−9M. Dithioerythritol was found to be the most potent activator of this sulfhydryl protease. Molecular weight estimations on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and on analytical Sephadex G-75 column were around 27,000 and 29,000 daltons respectively. Cathepsin B was found to reside in the lysosomes of goat brain. The highest percentage of cathepsin B was in cerebrum. However, the specific activity of the enzyme was maximum in pituitary gland.

Photochemistry of 3-alkoxychromones I — Photocyclisation of 6-chloro-3-alkoxy-2-(furan-3-yl)-4H-chromen-4-ones

3-Alkoxy/allyloxy-6-chloro-2-(furan-3-yl)-4H-chromen-4-ones yield angular tetracyclic products involving the 2-furyl group on photoirradiation through the intermediacy of 1,4-biradicals. The nature of the 3-alkoxy/allyloxy group influenced the photoproduct distribution. The stereochemical dispositions of the products have been established using the J/Φ relationship and were corroborated by MM2 calculations.

Properties of cathepsin B immobilized in calcium alginate beads

Cathepsin B (EC 3.4.22.1), purified from goat brain, was immobilized in calcium alginate beads in the presence of bovine serum albumin. The immobilized enzyme retained ∼63% of the original activity and could be used for seven successive batch reactions with retention of 22-30% of the initial activity. Immobilized cathepsin B hydrolysed α-N-benzoyl-D,L-arginine-β-naphthylamide (BANA) maximally at pH 5.5, exhibiting a shift of 0.5 pH unit from that of the soluble enzyme (pH optima 6.0). It showed enhanced stability in acidic as well as alkaline environments in comparison to the free enzyme. The optimal temperature and thermal stability were not altered significantly after immobilization. The K m value for the immobilized enzyme was two-fold higher than for the soluble enzyme.

Dipeptidyl peptidase I from goat brain: Purification, characterization and its action on leu-enkephalin

Brain dipeptidyl peptidase (DPP) I has been purified 2990-fold to apparent homogeneity shown by a single protein band in electrophoreses at pH 4.5, 8.4 and in SDS-PAGE at pH 7.2. The purification techniques included homogenization of brain acetone powder, autolysis at pH 4.2, 30-80% (NH4)2SO4 fractionation. Sephadex G-100 column chromatography, heat treatment at 65 C. organomercurial affinity chromatography. CM-Sephadex cation-exchange chromatographies at pH 5.6 and 5.0 and anion-exchange chromatography on DEAE-Sephadex at pH 6.8. The enzyme hydrolysed synthetic substrate Gly-Arg-4-methoxy-beta-naphthyl-amide maximally at pH 6.0. The Km values for Gly-Arg-beta-naphthylamide and Gly-Arg-4-methoxy-beta-naphthylamide substrates were 0.10 mM and 0.14 mM respectively. The enzyme was inhibited by thiol inhibitors like p-chloromercuribenzoic acid, iodoacetic acid, iodoacetamide and microbial inhibitors leupeptin and antipain. Molecular weight estimations on a calibrated Sephadex G-200 column afforded a value of 180,000 Da while in denaturing conditions on sodium dodecyl sulphate polyacrylamide gel electrophoresis, the subunit molecular weight was 22,000 Da. The subunit structure of the native enzyme was unfolded in presence of different concentrations of urea. In 8 M urea, the enzyme dissociated completely into monomers of 25,000 Da but 6, 5 and 4 M urea concentrations revealed the existence of dimers, tetramers and hexamers. Leu-enkephalin. Tyr-Gly-Gly-Phe-Leu was degraded by DPPI into Tyr-Gly and Gly-Phe-Leu with no further degradation of the newly generated tripeptide.

Photoreorganisation of some bischromones

Abstract Photoreorganisation of 2,2′-dithienyl/diphenyl-3,3′-polymethylene-dioxychromones is described. The product formation has been found to depend upon the length of the intervening alkyl chain.

Photochemistry of 3-alkoxychromones: photocyclisation of 2-aryl-6-chloro-3-{(thiophen-2-yl)methoxy}chromones

1,4-Biradicals generated upon photo-irradiation of 2-aryl-3-{(thiophen-2-yl)methoxy}chromones produced angular tetracyclic products bearing the thienyl group. The dehydrogenation and ring contraction products were also observed depending upon the electron density on the 2-aryl ring.

Synthesis of spiropyrans: H-abstractions in 3-cycloalkenyloxybenzopyrans

A photochemical route for the synthesis of some benzopyronospiropyrans from 2-furyl-3-cycloalkenyloxybenzopyrones involving H-abstraction is reported. How a methyl group on the furyl ring affects the product formation is also investigated.

An economic, simple and convenient synthesis of 2-aryl/heteroaryl/styryl/alkylbenzothiazoles using SiO2–HNO3

The present work was undertaken to develop an economic method for the synthesis of 2-aryl/heteroaryl/styryl/alkylbenzothiazoles mediated by SiO2–HNO3. In this report, we have demonstrated the catalytic potential of SiO2–HNO3 for the oxidative condensation of 2-aminothiophenol and aldehydes. Instant reaction at room temperature under solvent-free condition, high substrate to catalyst ratio 50:1 (by weight), practical application by large-scale synthesis, and excellent yields are the main advantages of the present protocol.Graphical Abstract

Photolysis of xylylbischromones

Photoreorganisation of xylylbischromones occurring through 1,4-biradical is described. In these bischromones, the two chromophores have been found to behave independently.