A sulphydryl-dependent and chloride-activated peptidase (cathepsin C) that hydrolyses alanyl-alanine naphthylamide

Synopsis A peptidase, capable of hydrolysing naphthylamides of alanyl-alanine, leucyl-leucine and glycyl-phenylalanine, as well as amides of glycyl-phenylalanine and glycyl-tyrosine, with a dipeptide and naphthylamine or ammonia as reaction products, was partially purified from hog kidney tissue. The enzyme preparation did not hydrolyse amino acid naphthylamides. The purification procedure included acid precipitation of particles, ammonium sulphate fractionation, gel filtration on Sephadex G 200 and chromatography on DEAE-cellulose. The enzyme was shown to be optimally active at pH 5.0–6.0, dependent on sulphydryl groups, and activated by chloride ions. It was heat stable at 60°C, its isoelectric point was between 4.8 and 6.0, and its molecular weight was 134,000 as determined on gel filtration. The enzyme was suggested as being identical to cathepsin C (E.C. 34. 4. 9).     

Studies on the histochemical „Leucine aminopeptidase“ reaction

Summary Since it was recently observed that purified cathepsin B will hydrolyse the chromogenic substrate LNA the obvious possibility of demonstrating the activity of this enzyme in tissue sections was explored. Chemical and histochemical experiments suggested that all aminopeptidase activity could be excluded by choosing a sufficiently acid pH for incubation, whereby the selective demonstration of cathepsin B activity was possible. This conclusion was further supported by different attempts to influence the histochemical reaction by preincubation with suitable activating and/or inhibiting reagents. This more specific procedure was used on sections from liver, kidney and granulomas rich in active macrophages. The suggested cathepsin B activity was demonstrated in autophagic vacuoles, and in other lysosome-like enlarged vacuoles or granules, appearing as a consequence of cellular injury as well as in actively phagocytosing macrophages.The following Abbreviations have been used AM Pase(s) aminopeptidase(s) in general - NA naphthylamides in general. — Substrates - LA Leucine amide - GPA Gly-Phe-amide - LNA L-leucyl--naphthylamide - Arg-NA L-arginyl--naphthylamide - BANA Benzoyl-dl-arginine--naphthylamide     

Esterolytic and proteolytic enzymes of the rat adenohypophysis

Summary Characteristics of the hydrolysis of histochemical substrates 5-bromoindoxyl acetate, naphthyl acetate, proprionate, butyrate, caprylate, laurate, myristate, and palmitate, acetyl and butyryl thiocholhie, chloroacetyl and trifluoroacetyl -naphthylamide, benzoyl-arginine -naphthylamide and proteinase substrates human hemoglobin and glycyl-phenylalanine amide by the rat pituitary tissue homogenate and DEAE-cellulose chromatography fractions were determined.In DEAE-cellulose chromatography fractions four separate activities were found splitting short and long chain carboxylic esters. The activity hydrolysing most rapidly 5-bromoindoxyl acetate was resistant to E 600 and was identified as C esterase. Three of the remaining esterase activities were sensitive to E 600 and two of them hydrolysed more rapidly short-chain fatty acid esters while one preferred long-chain fatty acid esters as substrate.One peak of activity was identified as nonspecific cholinesterase on the basis of inhibition studies and hydrolysis of thiocholine substrates. Chloroacetyl -naphthylamide was hydrolysed minimally. Hydrolysis of trifluoroacetyl -naphthylamide was ascribed to E 600 resistant enzyme with pH-optimum at 8.3 hydrolysing also the thiocholine substrates and slowly long-chain fatty acid esters.Five different proteinases hydrolysing human hemoglobin were separated, three of them with pH-optima on the acid and two on the alkaline side of pH. The activities hydrolysing benzoyl-arginine naphthylamide were cysteine activated and had pH-optima around 5.3. None of the peaks of the proteinase activities appeared to coinside with the hydrolysis peaks of any of the histochemical ester substrates in the DEAE fractions.     

The ovarial enzymes hydrolysing l-leucyl- and dl-alanyl-β-naphthylamide

Summary Fractionation of the rat ovarial tissue homogenate was performed using gel filtration on Sephadex G 200 and by starch gel electrophoresis. The activities hydrolysing l-leucyl--naphthylamide (Leu--NA) and dl-alanyl--naphthylamide (Ala--NA) were determined and partially characterized. Leu--NA was hydrolysed by four separate enzyme activities separated by both methods. Two of them were thiol-activated, one metal-activated and inhibited by EDTA. One was affected by neither metal chelators nor by sulfhydryl reagents. Ala--NA was hydrolysed by the three first-mentioned activities, but not by the last one. In addition, Ala--NA was hydrolysed by two other activities which were totally inhibited by metal chelators. These were clearly separated only using starch gel electrophoresis. The possibilities for the histochemical demonstration of these activities are discussed.     

β-D-Glycan synthases and the CesA gene family: lessons to be learned from the mixed-linkage (1→3),(1→4)β-D-glucan synthase

Cellulose synthase genes (CesAs) encode a broad range of processive glycosyltransferases that synthesize (14)-D-glycosyl units. The proteins predicted to be encoded by these genes contain up to eight membrane-spanning domains and four `U-motifs' with conserved aspartate residues and a QxxRW motif that are essential for substrate binding and catalysis. In higher plants, the domain structure includes two plant-specific regions, one that is relatively conserved and a second, so-called `hypervariable region' (HVR). Analysis of the phylogenetic relationships among members of the CesA multi-gene families from two grass species,Oryza sativa and Zea mays, with Arabidopsis thaliana and other dicotyledonous species reveals that the CesA genes cluster into several distinct sub-classes. Whereas some sub-classes are populated by CesAs from all species, two sub-classes are populated solely by CesAs from grass species. The sub-class identity is primarily defined by the HVR, and the sequence in this region does not vary substantially among members of the same sub-class. Hence, we suggest that the region is more aptly termed a `class-specific region' (CSR). Several motifs containing cysteine, basic, acidic and aromatic residues indicate that the CSR may function in substrate binding specificity and catalysis. Similar motifs are conserved in bacterial cellulose synthases, the Dictyostelium discoideum cellulose synthase, and other processive glycosyltransferases involved in the synthesis of non-cellulosic polymers with (14)-linked backbones, including chitin, heparan, and hyaluronan. These analyses re-open the question whether all the CesA genes encode cellulose synthases or whether some of the sub-class members may encode other non-cellulosic (14)-glycan synthases in plants. For example, the mixed-linkage (13)(14)-D-glucan synthase is found specifically in grasses and possesses many features more similar to those of cellulose synthase than to those of other -linked cross-linking glycans. In this respect, the enzymatic properties of the mixed-linkage -glucan synthases not only provide special insight into the mechanisms of (14)-glycan synthesis but may also uncover the genes that encode the synthases themselves.     

Isolation and characterization of cathepsin B from bovine brain

Cathepsin B (EC 3.4.22.1) was purified 746-fold with a 21% recovery from bovine brain by autolysis, fractional precipitation with acetone, carboxy-methyl-Sephadex chromatography, affinity chromatography on a cystamine containing column and gel filtration chromatography. The purified cathepsin B eluted on gel filtration with an apparent molecular weight of 27,000 but was resolved into three bands of 30,000, 25,000 and 5,000 molecular weight by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Antibodies to cathepsin B, raised against the 30,000 dalton band, were shown by immunoblots to react with both the 30,000 and 25,000 dalton proteins with results suggesting that the former predominated as the immunoreactive form in bovine brain homogenates. Isoelectric focusing demonstrated multiple bands, ranging from pH 4.75–5.2 with the major band at pH 5.1–5.2, all of which were capable of degrading N-carbobenzoxy-l-arginyl-l-arginine 4-methoxy--naphthylamide. The cathepsin B activity against N-benzoyl-dl-arginine -naphthylamide (BANA) and bovine myelin basic protein (MBP) had a pH optimum of pH 6.0. The Km for the degradation of BANA was 1.0 mM and 5.1 mM when assayed in the presence of 1% and 2.5% dimethylsulfoxide, respectively. Cathepsin B from bovine brain has many properties similar to cathepsin B isolated from other organs. The degradative effect of cathepsin B on MBP suggests a role for this proteinase in inflammatory demyelination.     

Fluorescence demonstration of a cathepsin H-like protease in cardiac, skeletal and vascular smooth muscles

Summary A cathepsin H-like enzyme was localized by histochemical techniques in cardiac muscle, extensor digitorum longus and soleus skeletal muscles, and vascular smooth muscle. Using the specific exopeptidase activity of this enzyme against the substrate arg-4-methoxy--naphthylamide, valid histochemical assay conditions were developed. More fluorescent granules were observed in cardiac muscle than in the soleus and about equal amounts in vascular smooth muscle and the extensor digitorum longus. The reaction rate was enhanced by chloride ions and inhibited by 1mm p-chloromercuribenzoate. The maximal activity was observed between pH 5.5 and 6.0. Chemical fixation with periodate-lysine-paraformaldehyde preserved a small amount of enzymatic activity.     

Fluorescent histochemical demonstration of cathepsin B in the rat yolk sac

Summary Fluorescence demonstration of cathepsin B (E.C. 3.4.22.1) activity was performed in the yolk sac of rats near term (18th day of gestation). The enzyme demonstration was performed on freeze-dried and celloidin mounted yolk-sac sections using different substituted -naphthylamide derivatives as substrates and nitrosalicylaldehyde as coupling agent. The discrete reaction products are localized preferentially in the apical part of the visceral yolk-sac epithelium. There is little doubt that cathepsin B is contained here in the well developed lysosomal apparatus of the yolk-sac epithelium.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Distribution of a dipeptide naphthylamidase in rat tissues and its localisation by using diazo coupling and labeled antibody techniques

Summary Several rat tissues (liver, spleen, kidney, pancreas, skin, heart, lung and brain) were shown to contain a peptidase capable of liberating naphthylamine from glycyl-dl-proline naphthylamide (Gly-Pro-NA). A single DEAE-cellulose chromatography of autodigested homogenates of the above tissues produced a partial separation of the peptidase from the enzymes hydrolysing l-leucine -naphthylamide. The Gly-Pro-NA hydrolysing enzyme was localised in tissue sections by using diazo coupling reaction and indirect immunologic techniques. Antibodies were prepared against the enzyme purified from rat liver and kidney in the rabbit. Rabbit -globulin was localized by using goat anti-rabbit -globulin labeled with fluorescein or with peroxidase.     

Cystatins from bovine brain: Purification,some properties,and action on substance P degrading activity

Two cystatins were purified from tissue extract of bovine brain by alkaline treatment, acetone fractionation, gel chromatography on Sephadex G-75, and affinity chromatography on S-carboxymethyl-papain-Sepharose. One of the inhibitors had a relatively high molecular mass, 25 kDa (HMM-cystatin) with pI 4.7, and the other, 11 kDa (LMM-cystatin) with pI 5.23. Both inhibitors showed considerable stability at pH 2 and 80°C. The cystatins inhibited papain, ficin, and cathepsins B and H, but not trypsin, chymotrypsin, thermolysin, nagarse, and cathepsin D. K_i values for the complexes of papain and the inhibitors were estimated to be 2.8×10^–10 M for HMM-cystatin and 1.3×10^–9 M for LMM-cystatin. Both purified cystatins prevented degradation of substance P by soluble fraction and lysosomal extract obtained from synaptosomes, but did not suppress the cleavage of the peptide by synaptosomal plasma membranes.Abbreviations HMM-cystatin high molecular mass inhibitor - LMM-cystatin low molecular mass inhibitor - SP substance P - SPM synaptosomal plasma membranes - p-CMB 4-chloromercuribenzoic acid - BK bradykinin - Bz-Arg-Nap N-benzoyl-dl-arginine--naphthylamide - Arg-Nap dl-arginine--naphthylamide - P-Pxy-Hb hemoglobin initially coupled with pyridoxal-5-phosphate     

The effect of the etched (et) mutation on the amylolytic enzyme activities in germinating kernels and seedlings of Zea mays

Summary The etched (et) mutation in maize causes distinct depressions and structural gaps in the endosperm and also gives rise to virescent seedlings, - and -Amylase activities were observed to be higher in et ⁺ et ⁺ kernels and seedlings as compared to that of the et et mutant. The total amylase and -amylase trends during germination also differed between normal and mutant kernels and seedlings (it increases in the wildtype and decreases in et et). On the contrary, the overall -amylase trend was found to be similar in both genotypes (slight decrease during germination). The native gel electrophoresis of crude enzyme extracts did not reveal any qualitative differences in and amylases during germination. The germinating et et kernels initially showed lower levels of starch compared with the wild type kernels, whereas no such difference was found at later stages of germination. It is concluded that et gene associated endosperm lesions lead to an impairment of starch degradation in germinating kernels resulting in virescent seedlings.     

Chemoselectivity and enhanced activity of poly(ethylene glycol)-modified lipases acylating hydrophilic aminoacid derivatives in organic solvents

Summary Lipases, when covalently modified with poly(ethylene grycol), catalyse the acylation of hydrophilic substrates in organic solvents with increase in the reaction rate even in the presence of 4% water. As a further result the modified Lipoprotein lipase from Pseudomonas species acylates only amino group in serine--naphthylamide.     

Partial Purification and Characterization of Aminopeptidase II from Chara australis

Aminopeptidase II, one of the two major aminopeptidases in the giant alga Chara australis, was partially purified. Its molecular weight was estimated to be about 80,000 by gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that it is composed of a single polypeptide with a molecular weight of about 85,000. Aminopeptidase II hydrolyzed alanine-2-naphthylamide more efficiently than the naphthylamides of lysine and proline, and only weakly hydrolyzed the naphthylamides of arginine, phenylalanine, valine, and leucine. The optimal pH for the hydrolysis of alanine-2-naphthylamide was near 7.0. The activity of aminopeptidase II was inhibited by the SH-reagents p-chloromercuribenzoic acid and N-ethylmaleimide and by the metal chelator 1,10-phenanthroline.     

Purification and characterization of thermostable β-amylase and pullulanase from high-yielding Clostridium thermosulfurogenes SV2

Thermostable -amylase and pullulanase, secreted by the thermophilic anaerobic bacterium Clostridium thermosulfurogenes strain SV2, were purified by salting out with ammonium sulphate, DEAE-cellulose column chromatography, and gel filtration using Sephadex G-200. Maltose was identified as a major hydrolysis product of starch by -amylase, and maltotriose was identified as a major hydrolysis product of pullulan by pullulanase. The molecular masses of native -amylase and pullulanase were determined to be 180 and 100 kDa by gel filtration, and 210 and 80 kDa by SDS–PAGE, respectively. The temperature optima of purified -amylase and pullulanase were 70 and 75°C, respectively, and both enzymes were completely stable at 70°C for 2h. The presence of starch further increased the stability of both the enzymes to 80°C and both displayed a pH activity optimum of 6.0. The starch hydrolysis products formed by -amylase action had -anomeric form.     

An acetyl esterase of Trichoderma reesei and its role in the hydrolysis of acetyl xylans

Summary An acetyl esterase was purified from Trichoderma reesei by cation and anion exchange chromatography. The enzyme had a molecular weight of 45 000 as determined by SDS-electrophoresis, or 67 000 as determined by gel filtration. In chromatofocusing the enzyme was shown to consist of two isoenzymes with isoelectric points of 6.8 and 6.0. The enzyme showed activity towards naphthyl acetate, triacetin and glucose-and xylose acetates. However, it liberated acetic acid from acetylated xylo-oligomers only to a small extent. The liberation of acetic acid from the oligomeric substrate was enhanced by addition of endoxylanase and -xylosidase.     

Purification and characterization of β-xylosidase from Aureobasidium pullulans

Summary -Xylosidase was obtained from Aureobasidium pullulans CBS 58475 with an activity of 0.35 units/ml culture filtrate. The production of the enzyme was strongly inducible. -Xylosidase was purified in two steps by anion exchange and gel-permeation chromatography to high purity. The enzyme is a glycoprotein with an apparent molecular mass of 224 kDa in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and separates into two subunits of equal molecular mass. After SDS-PAGE -xylosidase could be renatured and stained with methylumbelliferyl--xylopyranoside. The enzyme was able to split substrates of other glycosidases. The maximum activity was reached at pH 4.5 and 80° C. -Xylosidase showed high stability over a broad pH range from pH 2.0 to 9.5 and up to 70° C. Analysis of cleavage patterns revealed that the enzyme was a typical glycosidase. Larger oligosaccharides consisting of xylose were degraded by an exomechanism together with a transxylosylation reaction.     

A comparison of male and female recombination frequency in wheat using RFLP maps of homoeologous group 6 and 7 chromosomes

A novel approach was used to compare male and female recombination rates in wheat. Doubled haploid lines were developed from an F₁ using two distinct approaches: the anther-culture technique and the Hordeum bulbosum system, from which sets of lines were developed from male and female meioses, respectively. The genotype of the lines was established at RFLP and isozyme markers polymorphic on chromosomes of homoeologous groups 6 and 7, and male and female linkage maps were calculated using this information. The markers in one segment of chromosome 6B exhibited disturbed segregation frequencies in the anther-culture population. The male and female maps differed significantly in recombination frequency between some markers on two chromosomes, and these were consistent in direction within chromosomes and inconsistent in direction between chromosomes. In two of the four chromosomes studied the male map was much longer than the female map. These results suggest that significant differences may exist in male and female recombination frequencies in bread wheat which are specific to certain chromosomal segments but are inconsistent in direction between chromosomes. Other factors, such as environmental influences, may also be important in creating differences.     

Purification and properties of an extra cellular xylanase enzyme ofClostridium strain SAIV

An extracellular xylanase enzyme fraction A from a mesophilicClostridium strain SAIV was purified by ammonium sulfate precipitation, Sephadex G-50 gel filtration and DEAE-Sephadex A-50 ion exchange. The xylanase exhibited a molecular weight of 30,000 and it was stable upto 55° C with an optimum temperature of 50° C. It was most stable between pH 5–7, with an optimum pH of around 6. The Km value was 7.0 mg·xylan ml^-1 and V_max was 36 mol·xylose liberated mg^-1 min^-1. Carboxymethyl cellulose, filter paper cellulose and 4-p-nitrophenyl -D-xylopyranoside were not hydrolysed. The specific activity of xylanase fraction A (9.8 U mg^-1) is 2–10 fold higher than the specific activity of xylanase in other mesophilic, xylanolytic, obligate anaerobic bacteria. A minor fraction of xylanase activity designated as xylanase B was also obtained supporting the view that the multiplicity of xylanases is common in microorganisms.     

Evolutionary origin of the class A and class C β-lactamases

Summary The protein sequences of 18 class A -lactamases and 2 class C -lactamases were analyzed to produce a rooted phylogenetic tree using the DD peptidase of Streptomyces R61 as an outgroup. This tree supports the penicillin-binding proteins as the most likely candidate for the ancestoral origin of the class A and class C -lactamases, these proteins diverging from a common evolutionary origin close to the DD peptidase. The actinomycetes are clearly shown as the origin of the class A -lactamases found in other non-actinomycete species. The tree also divides the -lactamases from the Streptomyces into two subgroups. One subgroup is closer to the DD peptidase root. The other Streptomyces subgroup shares a common branch point with the rest of the class A -lactamases, showing this subgroup as the origin of the non-actinomycete class A -lactamases. The non-actinomycete class A -lactamase phylogenetic tree suggests a spread of these -lactamases by horizontal transfer from the Streptomyces into the non-actinomycete gram-positive bacteria and thence into the gram-negative bacteria. The phylogenetic tree of the Streptomyces class A -lactamases supports the possibility that horizontal transfer of class A -lactamases occurred within the Streptomyces.     

Quantitative measurement of starch in very small amounts of leaf tissue

Summary A specific enzyme method is described for the routine estimation of starch in small quantities (10–30 mg) of dried leaf tissue. A -glucanase-free preparation of amyloglucosidase is employed to hydrolyse starch to glucose; this is subsequently estimated by the glucose oxidase technique. The method gives result which agree closely with those obtained by a specific iodine-precipitation method.