Purification and characterization of a detergent‐requiring membrane‐bound metalloendopeptidase from porcine brain

Jeohn, Gwang‐Ho; Matsuzaki, Hidemi; Takahashi, Kenji

doi:10.1046/j.1432-1327.1999.00151.x

Cited by 4 publications

(3 citation statements)

References 17 publications

(15 reference statements)

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“…Additionally, a very significant activity change for free enzyme, at pH 8, was observed depending on buffer composition (Figure A ). This effect of buffer was also described by Jeohn et al for a metalloendopeptidase, obtaining about 30% of activity increase by varying the buffer. Perrin and Dempsey suggested that buffer composition can affect enzyme activity in different ways: ionic strength, interaction with enzyme conformation or active site; interaction with substrate, inhibitors, or cofactors and/or complexing with metals.…”

Section: Resultssupporting

confidence: 76%

Neutrase Immobilization on Alginate−Glutaraldehyde Beads by Covalent Attachment

Ortega

Pérez-Mateos

Pilar

et al. 2008

J. Agric. Food Chem.

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Neutrase, a commercial preparation of Bacillus subtilis , was covalently immobilized on alginate-glutaraldehyde beads. Immobilization conditions and characterization of the immobilized enzyme were investigated. Central composite design and response surface methods were employed to evaluate the effects of immobilization parameters, such as glutaraldehyde concentration, enzyme loading, immobilization pH, and immobilization time. Under optimized working conditions (2% alginate, 6.2% glutaraldehyde, 61.84 U mL(-1) Neutrase, pH 6.2, and 60 min) the immobilization yield was about 50%. The immobilized enzyme exhibited higher K(m) compared to the soluble enzyme. The pH-activity profile was widened upon immobilization. The optimum temperature was shifted from 50 to 60 degrees C, and the apparent activation energy was decreased from 47.7 to 22.0 kJ mol(-1) by immobilization. The immobilized enzyme also showed significantly enhanced thermal stability.

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Section: Resultssupporting

confidence: 76%

Neutrase Immobilization on Alginate−Glutaraldehyde Beads by Covalent Attachment

Ortega

Pérez-Mateos

Pilar

et al. 2008

J. Agric. Food Chem.

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“…5 A and B). All visible proteins were eluted from the gel by passive diffusion (31) (Fig. 1C, bands 1-15) and tested for proteolytic activity with m-fxn (Fig.…”

Section: Resultsmentioning

confidence: 99%

Cryptic proteolytic activity of dihydrolipoamide dehydrogenase

Babady¹,

Pang

Elpeleg

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

110

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The mitochondrial enzyme, dihydrolipoamide dehydrogenase (DLD), is essential for energy metabolism across eukaryotes. Here, conditions known to destabilize the DLD homodimer enabled the mouse, pig, or human enzyme to function as a protease. A catalytic dyad (S456 -E431) buried at the homodimer interface was identified. Serine protease inhibitors and an S456A or an E431A point mutation abolished the proteolytic activity, whereas other point mutations at the homodimer interface domain enhanced the proteolytic activity, causing partial or complete loss of DLD activity. In humans, mutations in the DLD homodimer interface have been linked to an atypical form of DLD deficiency. These findings reveal a previously unrecognized mechanism by which certain DLD mutations can simultaneously induce the loss of a primary metabolic activity and the gain of a moonlighting proteolytic activity. The latter could contribute to the metabolic derangement associated with DLD deficiency and represent a target for therapies of this condition.is a f lavindependent oxidoreductase required for the complete reaction of at least five different multienzyme complexes. In the mitochondrial matrix, the DLD homodimer functions as the E3 component of the pyruvate, ␣-ketoglutarate, and branchedchain amino acid-dehydrogenase complexes and the glycine cleavage system. In the context of these four multienzyme complexes, DLD utilizes dihydrolipoic acid and NAD ϩ to generate lipoic acid and NADH (1). The opposite reaction, from lipoic acid and NADH to dihydrolipoic acid and NAD ϩ , is catalyzed by the DLD homodimer in the context of the NAD ϩ -dependent peroxidase-peroxinitrate reductase of Mycobacterium tuberculosis (2). In addition, DLD has diaphorase activity, being able to catalyze the oxidation of NADH to NAD ϩ by using different electron acceptors such as O 2 , labile ferric iron (3), nitric oxide (4), and ubiquinone (5, 6). In this capacity, DLD is believed to primarily have a prooxidant role, achieved by reducing O 2 to a superoxide radical or ferric to ferrous iron, which in turn catalyzes production of hydroxyl radical through Fenton chemistry (3). However, the ability to scavenge nitric oxide and to reduce ubiquinone to ubiquinol suggests that the diaphorase activity of DLD may also have an antioxidant role (4-6). The oligomeric state of DLD can change from dimeric to monomeric or tetrameric depending on the pH in the mitochondrial matrix, and changes in the oligomeric state of the protein have been shown to correlate with a shift from DDL activity (only present in dimeric and tetrameric DLD) to diaphorase activity (present in both oligomeric and monomeric forms of the protein) (7,8). Thus, DLD represents a highly versatile oxidoreductase with multiple critical roles in energy metabolism and redox balance (2,7,(9)(10)(11)(12).Here, we found that DLD can also function as a moonlighting protease. Moonlighting enzymes are a growing category of molecules that can accomplish multiple functions through a variety of mechanisms including, among other...

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“…To characterize the enzymatic activity of the purified protease nsP2pro and nsP2proDel, we investigated their ability to cleave peptidyl-7-amino-4-methylcoumarine-labelled substrates; this kind of substrate had been previously used to study the activ- ity of a wide range of proteases, such as eukaryotic and viral trypsin-like proteases (Jeohn et al, 1999;Kato et al, 1998;Bessaud et al, 2006a;Yusof et al, 2000) and cysteine proteases (Choe et al, 2006;Melo et al, 2001;Gosalia et al, 2005). Such short substrates are useful to assay the catalytic activity of proteases and to map their specificity at non-prime sites.…”

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confidence: 99%

Expression and biochemical characterization of nsP2 cysteine protease of Chikungunya virus

et al. 2008

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Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes epidemic fever, rash and polyarthralgia in Africa and Asia. Although it is known since the 1950s, new epidemiological and clinical features reported during the recent outbreak in the Indian Ocean can be regarded as the emergence of a new disease. Numerous severe forms of the infection have been described that put emphasis on the lack of efficient antiviral therapy. Among the virus-encoded enzymes, nsP2 constitutes an attractive target for the development of antiviral drugs. It is a multifunctional protein of approximately 90 kDa with a helicase motif in the N-terminal portion of the protein while the papain-like protease activity resides in the C-terminal portion. The nsP2 proteinase is an essential enzyme whose proteolytic activity is critical for virus replication. In this work, a recombinant CHIKV nsP2pro and a C-terminally truncated variant were expressed in Escherichia coli and purified by metal-chelate chromatography. The enzymatic properties of the proteinase were then determined using specific synthetic fluorogenic substrates. This study constitutes the first characterization of a recombinant CHIKV nsP2 cysteine protease, which may be useful for future drug screening.

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Purification and characterization of a detergent‐requiring membrane‐bound metalloendopeptidase from porcine brain

Cited by 4 publications

References 17 publications

Neutrase Immobilization on Alginate−Glutaraldehyde Beads by Covalent Attachment

Neutrase Immobilization on Alginate−Glutaraldehyde Beads by Covalent Attachment

Cryptic proteolytic activity of dihydrolipoamide dehydrogenase

Expression and biochemical characterization of nsP2 cysteine protease of Chikungunya virus

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