Proteases of germinating winged-bean (Psophocarpus tetragonolobus) seeds: purification and characterization of an acidic protease

Two major classes of protease are shown to occur in germinating winged-bean (Psophocarpus tetragonolobus) seeds, by assaying extracts at pH 8.0 and pH 5.1 with [14C]gelatin as substrate. At pH 8.0, the activity profile of the enzyme shows a steady rise throughout the period of germination, whereas the activity at the acidic pH is very low up to day 5 and then increases sharply reaching a peak on day 11, followed by an equally sharp decline. The winged-bean acidic protease (WbAP) has been purified to apparent homogeneity, as attested by a single protein band on both PAGE and SDS/PAGE. WbAP is a monomeric enzyme with a molecular mass of 35 kDa and a pH optimum of 6.0. It is a thiol protease that does not belong to the papain family and it has tightly bound Ca2+ as shown by 45Ca2+-exchange studies. Besides gelatin and casein, it hydrolyses a 29 kDa winged-bean protein, indicating a prospective physiological role for it in storage-protein mobilization. Immunoblot analysis shows that it occurs only in the seeds and sprouting tubers of this plant and also that it is synthesized in developing seeds just before desiccation. It appears that the newly synthesized enzyme is inactive, and activation takes place around day 6 of germination. However, neither the mechanism of activation nor the signal that triggers it is clearly understood.

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Characterization of a winged bean (Psophocarpus tetragonolobus) protein kinase with calmodulin-like domain: regulation by autophosphorylation

Biochemical Journal ◽

10.1042/bj3050205 ◽

1995 ◽

Vol 305 (1) ◽

pp. 205-210 ◽

Cited By ~ 24

Author(s):

P Saha ◽

M Singh

Keyword(s):

Protein Kinase ◽

Histone H1 ◽

Winged Bean ◽

Psophocarpus Tetragonolobus ◽

Serine Residue ◽

Sds Page ◽

Phosphorylation Reaction ◽

Tissue Homogenates ◽

Substrate Phosphorylation

A soluble protein kinase purified from winged bean (Psophocarpus tetragonolobus) shoots, has been assessed as a monomeric enzyme with an approximate M(r) of 60,000 in spite of the presence of two polypeptides of 61 and 58 kDa determined by SDS/PAGE. Immunoblot analyses using either of the two antisera raised individually against the polypeptides, detect both of them in purified preparations and a single larger polypeptide (62 kDa) in freshly prepared tissue homogenates, clearly indicating the likelihood of the doublet being formed from the larger one by proteolysis. Histone H1, syntide 2 and a synthetic myosin light chain-related peptide (MLC-peptide) have been identified as exogenous substrates of the enzyme. Complete Ca(2+)-dependence for substrate phosphorylation, a drastic inhibition of the reaction by a calmodulin (CaM) antagonist which can be partially reversed by a heterologous CaM and direct 45Ca(2+)-binding on blot, form compelling evidence in favour of a CaM-like domain of the enzyme. Both the polypeptides of the purified enzyme undergo intramolecular autophosphorylation on serine residue(s). Unlike the substrate phosphorylation reaction, autophosphorylation is Ca(2+)-independent and is not inhibited by the CaM antagonist. Down-regulation of substrate phosphorylation by auto-phosphorylation, and stimulation of the autophosphorylation by histone H1 and MLC-peptide, are novel regulatory features of the enzyme.

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Purification and characterization of cysteine-S-conjugate N-acetyltransferase from pig kidney

Biochemical Journal ◽

10.1042/bj3170213 ◽

1996 ◽

Vol 317 (1) ◽

pp. 213-218 ◽

Cited By ~ 13

Author(s):

Achim AIGNER ◽

Martina JÄGER ◽

Ralf PASTERNACK ◽

Peter WEBER ◽

Dirk WIENKE ◽

...

Keyword(s):

Proteinase Inhibitors ◽

Cysteine Proteinase ◽

Gel Filtration ◽

Protein Band ◽

Exchange Chromatography ◽

Polyclonal Antiserum ◽

Partial Purification ◽

Ph Optimum ◽

Sds Page

Microsomal cysteine-S-conjugate N-acetyltransferase catalyses the N-acetylation of various S-substituted cysteines in liver and kidney. We describe here the purification and more detailed characterization of this enzyme catalysing the final reaction of mercapturic acid biosynthesis, and thus playing a crucial role in the detoxicating metabolism of many xenobiotics. The solubilization of cysteine-S-conjugate N-acetyltransferase by deoxy-BIGCHAP [N,N´-bis-(3-d-gluconamidopropyl)deoxycholamide] was the prerequisite for partial purification by means of anion-exchange chromatography. The molecular mass of the enzyme was determined by gel filtration. A polyclonal antiserum was raised against the excised protein band from SDS/PAGE and purified antibodies were used for the complete purification of native cysteine-S-conjugate N-acetyltransferase by immunoaffinity chromatography. A dimeric form of the enzyme was sometimes detected on SDS/PAGE, depending on the degree of purification. For further characterization of cysteine-S-conjugate N-acetyltransferase, the stability of catalytic activity, the pH optimum and Km values were determined. The inhibitory effects of various agents were tested, revealing a substantial, yet not complete, loss of cysteine-S-conjugate N-acetyltransferase activity after treatment with cysteine proteinase inhibitors or probenecid under various conditions.

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Purification and characterization of cytosolic pyruvate kinase from banana fruit

Biochemical Journal ◽

10.1042/bj3520875 ◽

2000 ◽

Vol 352 (3) ◽

pp. 875-882 ◽

Cited By ~ 11

Author(s):

William L. TURNER ◽

William C. PLAXTON

Keyword(s):

Pyruvate Kinase ◽

Specific Activity ◽

Gel Filtration ◽

Banana Fruit ◽

Ph Optimum ◽

Cytosolic Ph ◽

Pep Carboxylase ◽

Sds Page ◽

Optimal Efficiency

Cytosolic pyruvate kinase (PKc) from ripened banana (Musa cavendishii L.) fruits has been purified 543-fold to electrophoretic homogeneity and a final specific activity of 59.7µmol of pyruvate produced/min per mg of protein. SDS/PAGE and gel-filtration FPLC of the final preparation indicated that this enzyme exists as a 240kDa homotetramer composed of subunits of 57kDa. Although the enzyme displayed a pH optimum of 6.9, optimal efficiency in substrate utilization [in terms of Vmax/Km for phosphoenolpyruvate (PEP) or ADP] was equivalent at pH6.9 and 7.5. PKc activity was absolutely dependent upon the presence of a bivalent and a univalent cation, with Mg2+ and K+ respectively fulfilling this requirement. Hyperbolic saturation kinetics were observed for the binding of PEP, ADP, Mg2+ and K+ (Km values of 0.098, 0.12, 0.27 and 0.91mM respectively). Although the enzyme utilized UDP, IDP, GDP and CDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate and MgATP were the most effective inhibitors, whereas L-aspartate functioned as an activator by reversing the inhibition of PKc by L-glutamate. The allosteric features of banana PKc are compared with those of banana PEP carboxylase [Law and Plaxton (1995) Biochem. J. 307, 807Ő816]. A model is presented which highlights the roles of cytosolic pH, MgATP, L-glutamate and L-aspartate in the co-ordinate control of the PEP branchpoint in ripening bananas.

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Characterization of the soluble, secreted form of urinary meprin

Biochemical Journal ◽

10.1042/bj3150461 ◽

1996 ◽

Vol 315 (2) ◽

pp. 461-465 ◽

Cited By ~ 27

Author(s):

Robert J. BEYNON ◽

Simon OLIVER ◽

Duncan H. L. ROBERTSON

Keyword(s):

Proteolytic Activity ◽

Protein Band ◽

Peptide Sequence ◽

Soluble Form ◽

Α Subunit ◽

Alternative Processing ◽

Sds Page ◽

Processing Pathways ◽

Membrane Bound

A soluble form of the kidney membrane metalloendopeptidase, meprin, is present in urine. Urinary meprin is expressed in BALB/C mice with the Mep-1a/a genotype (high meprin, expressing meprin-α and meprin-β) but not in BALB.K mice of the Mep-1b/b genotype (that only express meprin-β). Western blotting with antisera specific to the meprin-α and the meprin-β subunits established that the only form of meprin present in urine samples was derived from meprin-α. This form of meprin is partially active, and comprises at least three variants by non-reducing SDS/PAGE and by zymography and two protein bands on reducing SDS/PAGE. Sequencing of these two bands established that the N-terminus of the larger protein band begins with the pro-peptide sequence of the α-subunit (VSIKH..), whereas the smaller band possessed the mature meprin N-terminal sequence (NAMRDP..). Trypsin is able to remove the pro-peptide, with a concomitant activation in proteolytic activity. After deglycosylation, the size of the pro- and mature forms of urinary meprin are consistent with cleavage in the region of the X–I boundary. There is a pronounced sexual dimorphism in urinary meprin expression. Females secrete a slightly larger form, and its proteolytic activity is about 50% of that released by males. The urinary meprin is therefore a naturally occurring secreted form of this membrane-bound metalloendopeptidase and is more likely to be generated by alternative processing pathways than by specific release mechanisms.

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Characterization of winged bean (<i>Psophocarpus tetragonolobus</i> (L.) DC.) based on molecular, chemical and physiological parameters

American Journal of Molecular Biology ◽

10.4236/ajmb.2013.34025 ◽

2013 ◽

Vol 03 (04) ◽

pp. 187-197 ◽

Cited By ~ 18

Author(s):

Chandra Sekhar Mohanty ◽

Sushma Verma ◽

Vinayak Singh ◽

Shahina Khan ◽

Priyanka Gaur ◽

...

Keyword(s):

Physiological Parameters ◽

Winged Bean ◽

Psophocarpus Tetragonolobus

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Characterization of glycineN-methyltransferase from rabbit liver

Biochemistry and Cell Biology ◽

10.1139/o04-007 ◽

2004 ◽

Vol 82 (3) ◽

pp. 369-374 ◽

Cited By ~ 7

Author(s):

Doris Kloor ◽

Katrin Karnahl ◽

Jost Kömpf

Keyword(s):

Enzymatic Activity ◽

Kinetic Data ◽

Adenine Nucleotides ◽

The Other ◽

Rabbit Liver ◽

Ph Optimum ◽

Isolation And Purification ◽

Endogenous Adenosine ◽

Sds Page

The enzymatic properties of glycine N-methyltransferase from rabbit liver and the effects of endogenous adenosine nucleosides, nucleotides and methyltransferase inhibitors were investigated using a photometrical assay to detect sarcosine with o-dianisidine as a dye. After isolation and purification the denatured enzyme showed a two-banded pattern by SDS–PAGE. The enzyme was highly specific for its substrates with a pH-optimum at pH 8.6. Glycine N-methyltransferase exhibits Michaelis-Menten kinetics for its substrates, S-adenosylmethionine and glycine, respectively. The apparent Kmand Vmaxvalues were determined for both the substrates, the other substrate being present at saturating concentrations. The enzyme was strongly inhibited in the presence of S-adenosylhomocysteine, 3-deazaadenosine, and 5′-S-isobutylthio-5′-deoxyadenosine. All other inhibitors investigated, adenosine, 2′-deoxyadenosine, aciclovir, and 5′-N-ethylcarboxamidoadenosine were poor inhibitors of the methylation rection. Adenine nucleotides and vidarabin were without effect on the enzymatic activity. Based on the kinetic data glycine N-methyltransferase from rabbit liver exhibits appreciable activity at physiological S-adenosylmethionine and S-adenosylhomocysteine levels.Key words: glycine N-methyltransferase, S-adenosylhomocysteine, S-adenosylmethionine, sarcosine oxidase, peroxidase.

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Purification and characterization of three extracellular (1→3)-β-d-glucan glucohydrolases from the filamentous fungus Acremonium persicinum

Biochemical Journal ◽

10.1042/bj3080733 ◽

1995 ◽

Vol 308 (3) ◽

pp. 733-741 ◽

Cited By ~ 25

Author(s):

S M Pitson ◽

R J Seviour ◽

B M McDougall ◽

J R Woodward ◽

B A Stone

Keyword(s):

Filamentous Fungus ◽

Gel Filtration ◽

High Specificity ◽

Major Product ◽

Ph Optimum ◽

Sds Page ◽

Molecular Masses ◽

Monomeric Proteins ◽

Ph Range

Three (1-->3)-beta-D-glucanases (GNs) were isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. Homogeneity of the purified proteins was confirmed by SDS/PAGE, isoelectric focusing and N-terminal amino acid sequencing. All three GNs (GN I, II and III) are non-glycosylated, monomeric proteins with apparent molecular masses, estimated by SDS/PAGE, of 81, 85 and 89 kDa respectively. pI values for the three enzymes are 5.3, 5.1, and 4.4 respectively. The pH optimum for GN I is 6.5, and 5.0 for GN II and III. All three purified enzymes displayed stability over the pH range 4.5-10.0. Optimum activities for GN I, II and III were recorded at 65, 55 and 60 degrees C respectively, with both GN II and III having short-term stability up to 50 degrees C and GN I up to 55 degrees C. The purified GNs have high specificity for (1-->3)-beta-linkages and hydrolysed a range of (1-->3)-beta- and (1-->3)(1-->6)-beta-D-glucans, with laminarin from Laminaria digitata being the most rapidly hydrolysed substrate of those tested. K(m) values for GN I, II, and III against L. digitata laminarin were 0.1, 0.23 and 0.22 mg/ml respectively. D-Glucono-1,5-lactone does not inhibit any of the three GNs, some metals ions are mild inhibitors, and N-bromosuccinimide and KMnO4 are strong inhibitors. All three GNs acted in an exo-hydrolytic manner, determined by the release of alpha-glucose as the initial and major product of hydrolysis of (1-->3)-beta-D-glucans, and confirmed by viscometric analysis and the inability to cleave periodate-oxidized laminarin, and may be classified as (1-->3)-beta-D-glucan glucohydrolases (EC 3.2.1.58).

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Purification and characterization of purine nucleoside phosphorylase from developing embryos of Hyalomma dromedarii

Biochemistry and Cell Biology ◽

10.1139/o91-034 ◽

1991 ◽

Vol 69 (4) ◽

pp. 223-231 ◽

Cited By ~ 4

Author(s):

Mamdouh Y. Kamel ◽

Afaf S. Fahmy ◽

Abdel H. Ghazy ◽

Magda A. Mohamed

Keyword(s):

Purine Nucleoside Phosphorylase ◽

Catalytic Mechanism ◽

Kinetic Studies ◽

Purine Nucleoside ◽

Hyalomma Dromedarii ◽

Ph Optimum ◽

Nucleoside Phosphorylase ◽

Apparent Homogeneity ◽

Camel Tick

Purine nucleoside phosphorylase from Hyalomma dromedarii, the camel tick, was purified to apparent homogeneity. A molecular weight of 56 000 – 58 000 was estimated for both the native and denatured enzyme, suggesting that the enzyme is monomeric. Unlike purine nucleoside phosphorylase preparations from other tissues, the H. dromedarii enzyme was unstable in the presence of β-mercaptoethanol. The enzyme had a sharp pH optimum at pH 6.5. It catalyzed the phosphorolysis and arsenolysis of ribo- and deoxyribo-nucleosides of hypoxanthine and guanine, but not of adenine or pyrimidine nucleosides. The Km values of the enzyme at the optimal pH for inosine, deoxyinosine, guanosine, and deoxyguanosine were 0.31, 0.67, 0.55, and 0.33 mM, respectively. Inactivation and kinetic studies suggested that histidine and cysteine residues were essential for activity. The pKa values determined for catalytic ionizable groups were 6–7 and 8–9. The enzyme was completely inactivated by thiol reagents and reactivated by excess β-mercaptoethanol. The enzyme was also susceptible to pH-dependent photooxidation in the presence of methylene blue, implicating histidine. Initial velocity studies showed an intersecting pattern of double-reciprocal plots of the data, consistent with a sequential mechanism.Key words: Acarina, Hyalomma dromedarii, purine nucleoside phosphorylase, kinetics, active site, catalytic mechanism.

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Purification and characterization of an extracellular (1 → 6)-β-glucanase from the filamentous fungus Acremonium persicinum

Biochemical Journal ◽

10.1042/bj3160841 ◽

1996 ◽

Vol 316 (3) ◽

pp. 841-846 ◽

Cited By ~ 29

Author(s):

Stuart M. PITSON ◽

Robert J. SEVIOUR ◽

Barbara M. McDOUGALL ◽

Bruce A. STONE ◽

Maruse SADEK

Keyword(s):

Molecular Mass ◽

Filamentous Fungus ◽

Gel Filtration ◽

Purification And Characterization ◽

Eisenia Bicyclis ◽

Ph Optimum ◽

Hydrolysis Products ◽

Sds Page ◽

Hydrolysis Of

An endo-(1 → 6)-β-glucanase has been isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. SDS/PAGE of the purified enzyme gave a single band with an apparent molecular mass of 42.7 kDa. The enzyme is a non-glycosylated, monomeric protein with a pI of 4.9 and pH optimum of 5.0. It hydrolysed (1 → 6)-β-glucans (pustulan and lutean), initially yielding a series of (1 → 6)-β-linked oligoglucosides, consistent with endo-hydrolytic action. Final hydrolysis products from these substrates were gentiobiose and gentiotriose, with all products released as β-anomers, indicating that the enzyme acts with retention of configuration. The purified enzyme also hydrolysed Eisenia bicyclis laminarin, liberating glucose, gentiobiose, and a range of larger oligoglucosides, through the apparent hydrolysis of (1 → 6)-β- and some (1 → 3)-β-linkages in this substrate. Km values for pustulan, lutean and laminarin were 1.28, 1.38, and 1.67 mg/ml respectively. The enzyme was inhibited by N-acetylimidazole, N-bromosuccinimide, dicyclohexylcarbodi-imide, Woodward's Regent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4 and some metal ions, whereas D-glucono-1,5-lactone and EDTA had no effect.

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Purification and characterization of glutathione-dependent dehydroascorbate reductase from rat liver

Biochemical Journal ◽

10.1042/bj3010471 ◽

1994 ◽

Vol 301 (2) ◽

pp. 471-476 ◽

Cited By ~ 87

Author(s):

E Maellaro ◽

B Del Bello ◽

L Sugherini ◽

A Santucci ◽

M Comporti ◽

...

Keyword(s):

Rat Liver ◽

Protein Identification ◽

Reductase Activity ◽

Sequence Similarity ◽

Gel Filtration ◽

Dehydroascorbic Acid ◽

Protein Band ◽

Dehydroascorbate Reductase ◽

Apparent Homogeneity ◽

Sds Page

GSH-dependent enzymic reduction of dehydroascorbic acid to ascorbic acid has been studied in rat liver cytosol. After gel filtration of cytosol on Sephadex G-100 SF, dehydroascorbate reductase activity was recovered in two distinct peaks, one corresponding to glutaredoxin (an enzyme already known for its dehydroascorbate reductase activity) and another, much larger one, corresponding to a novel enzyme different from glutaredoxin. The latter was purified to apparent homogeneity. The purification process involved (NH4)2SO4 fractionation, followed by DEAE-Sepharose, Sephadex G-100 SF and Reactive Red chromatography. SDS/PAGE of the purified enzyme in either the presence or absence of 2-mercaptoethanol demonstrated a single protein band of M(r) 31,000. The M(r) determined by both Sephadex G-100 SF chromatography and h.p.l.c. was found to be approx. 48,000. H.p.l.c. of the denatured enzyme gave an M(r) value identical with that obtained by SDS/PAGE (31,000). The apparent Km for dehydroascorbate was 245 microM and the Vmax. was 1.9 mumol/min per mg of protein; for GSH they were 2.8 mM and 4.5 mumol/min per mg of protein respectively. The optimal pH range was 7.5-8.0. Microsequence analysis of the electro-transferred enzyme band showed that the N-terminus is blocked. Data on internal primary structure were obtained from CNBr-and N-chlorosuccinimide-derived fragments. No significative sequence similarity was found to any of the protein sequences contained in the Protein Identification Resource database.

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