Purification of glutamate dehydrogenase from ox brain and liver. Evidence that commercially available preparations of the enzyme from ox liver have suffered proteolytic cleavage

McCarthy, Alun D.; Walker, J M; Tipton, Keith F.

doi:10.1042/bj1910605

Cited by 62 publications

(28 citation statements)

References 21 publications

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“…The final specific activities (167 units/mg and 104 units/mg for GDH I and GDH [I, respectively) were similar to those reported for the human cerebellum enzyme purified by Hussain et al I51 but higher than that (40 unitdmg) reported for the bovine brain enzyme purified by McCarthy et al [8]. It should be mentioned that the initial velocity studies of GDH isoproteins were performed in the presence of 1 mM ADP, unless otherwise indicated.…”

Section: I1supporting

confidence: 61%

“…This is probably due to the differences in the compositions, assay temperature and the pH of the buffer used for assaying GDH, and the use of the activator ADP by Colon et al [6] but not by McCarthy et al [8]. The results, however, also strongly suggest that both GDH I and GDH I1 are different proteins from the enzyme reported by McCarthy et al 181.…”

Section: I1contrasting

confidence: 53%

“…Previously, McCarthy et al [8] purified a GDH from bovine brain by a combination of ammonium sulfate fractionation and chromatography on DEAE-cellulose and GTP-Sepharose. There was, however, no evidence for the existence of isoproteins.…”

Section: I1mentioning

confidence: 99%

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Two Soluble forms of Glutamate Dehydrogenase Isoproteins from Bovine Brain

Cho

Lee

Choi

1995

European Journal of Biochemistry

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Two soluble forms of novel glutamate dehydrogenase isoproteins, designated GDH I and GDH 11, have been purified from bovine brain. GDH I and GDH I1 were separated on a hydroxyapatite column and eluted by a step gradient at different phosphate concentrations (30 mM and 50 rnM for GDH I and GDH 11, respectively). The preparations were homogeneous on SDS/PAGE. GDH I and GDH II showed similarity in their molecular sizes and are composed of six identical subunits having a molecular size of 57500 Da. Differences between the biochemical properties of GDH I and GDH 11, such as N-terminal amino acid sequences of intact and tryptic-digested enzymes, kinetic parameters, optimum pH and heat stability, were extensively examined in both reductive amination of a-oxoglutarate and oxidative deamination of glutamate. The different effects of ADP on GDH isoproteins were also studied under various conditions. These results indicate that GDH I and GDH 11, isolated from bovine brain, are novel and distinct polypeptides.

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Section: I1supporting

confidence: 61%

Section: I1contrasting

confidence: 53%

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Two Soluble forms of Glutamate Dehydrogenase Isoproteins from Bovine Brain

Cho

Lee

Choi

1995

European Journal of Biochemistry

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“…Enzymes and other markers were assayed as described in the quoted references: 5'-Nucleotidase [24], glutamate dehydrogenase [25], monoamine oxidase A and B using 5-hydr~xy['~C]tryptamine (100 pM, 0.2 Ci/mol) and [14C]phenethylamine (100 pM, 0.5 Ci/mol) respectively [26], adenylate kinase [27], succinate dehydrogenase [28], arylsulphatase [29], glucose-6-phosphatase [30] and aminopeptidase N [31]. DNA was measured by the Burton method [32], modified as in [28] and protein as described in [33].…”

Section: Enzyme Assaysmentioning

confidence: 99%

The subcellular location in rat kidney of the peripheral benzodiazepine acceptor

O'beirne

Williams

1988

European Journal of Biochemistry

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In rat kidney high-affinity binding sites for [3H]Ro-5-4864 and [3H]PK-11195 with the properties of the peripheral-type acceptor were found enriched in mitochondrial (M) and light-mitochondrial-lysosomal (L) fractions on differential centrifugation. When the combined M and L fractions were subjected to sucrose density gradient centrifugation, these binding sites were found enriched at a density of 1.155 g/ml coincident with a population of light mitochondria, whereas a population of heavier mitochondria ( Q = 1.175 g/ml) had few or no binding sites. Transmission electron microscopy showed that whereas the heavier mitochondria appeared highly pure and intact, the lighter mitochondria appeared less intact and to be contaminated with vesicular structures. After fractionation of the light mitochondria and vesicles by centrifugation, both fractions showed the same ratio of [3H]Ro-4864 binding sites to monoamine oxidase activity consistent with the vesicles being of mitochondrial outer-membrane origin.Digitonin pre-treatment had no effect on the density of acceptor-rich fractions on sucrose density gradient centrifugation. However, pretreatment with succinate/iodophenylnitrophenylphenyltetrazolium (INT) perturbed equally the density of acceptor-rich fractions and mitochondrial marker enzymes.When mitochondrial fractions were subjected to sonication prior to density gradient centrifugation the binding sites were now found highly enriched in a much lighter fraction coincident with the monoamine oxidase activity and thus consistent with being outer-membrane vesicles.When a mitochondrial fraction was subjected to hypotonic treatment before assay no evidence for activation/ unmasking of binding sites was found. The hypotonic treatment did not release any inhibitor of the binding sites.These results are consistent with the peripheral benzodiazepine acceptor having an outer-membrane location on a sub-population of rat kidney mitochondria. Those mitochondria showing high levels of the acceptor are either light mitochondria or appear more susceptible to osmotic damage than those mitochondria in which the acceptor is absent or at low levels.The benzodiazepines have anxiolytic, sedative and anticonvulsant actions probably mediated through allosteric modulation of neuronal post-synaptic plasma membrane 4-aminobutyrate-gated chloride channels [l , 21. Whilst the structural analysis of these channel proteins is well advanced [3], the properties and function of a second-class of benzodiazepine-binding sites are still unclear. These nonneuronal or peripheral-type benzodiazepine acceptors [4] appear not to be linked to 4-aminobutyrate-gated chloride channels [5,6], show a different binding profile for benzodiazepines than for the 4-aminobutyrate receptor [4-61 and appear to have different polypeptide subunit M , values depending on the nature of photoaffinity labelling reagents [7, 81. Evidence from various laboratories has suggested that benzodiazepines showing preference for the peripheral-type

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“…These authors concluded that ''chymotryptic digestion generates a 'superactive' form that is quite stable to further digestion'' (by chymotrypsin), but that the addition of small amounts of trypsin to this stable species results in rapid inactivation. The apparent effect of trypsin was also of interest, given earlier reports that this proteinase was responsible for cleavage of the Nterminal amino acids (Hucho et al 1975), a complication in the purification of GDH (McCarthy et al 1980).…”

mentioning

confidence: 99%

The structural basis of proteolytic activation of bovine glutamate dehydrogenase

Carrigan¹,

Engel²

2008

Protein Science

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In this work, we re-examine the previously reported phenomenon of the creation of a superactive glutamate dehydrogenase by proteolytic modification by chymotrypsin and explore the various discrepancies that came to light during those studies. We find that superactivation is caused by cleavage at the N terminus of the protein and not the C-terminal allosteric site, as has previously been suggested. N-terminal sequencing reveals that TLCK-treated chymotrypsin cleaves bovine glutamate dehydrogenase at phenylalanine 10. We suggest that trypsin contamination in nontreated chymotrypsin may have led to the production of the larger 4-5 kDa digestion product, previously misinterpreted as having caused the activation. In line with some previous studies, we can confirm that GTP inhibition is attenuated to some extent by the proteolysis, while ADP activation is almost abolished. Utilizing the recently solved structures of bovine glutamate dehydrogenase, we illustrate the cleavage points.Keywords: chymotrypsin; glutamate dehydrogenase; activation; guanosine triphosphate; adenosine triphosphateThe glutamate dehydrogenase of bovine liver, readily available commercially in a pure form from the 1950s onward, attracted the interest of enzymologists over many years because of its interesting and complex regulatory behavior (Olson and Anfinsen 1953;Talal and Tomkins 1964;Yielding et al. 1964;Engel and Dalziel 1969;Huang and Frieden 1969) cited by Monod et al. (1963) as one of the striking examples to establish the reality of allosteric control. Specifically, in addition to homotropic allosteric behavior with the oxidized and reduced coenzymes (Olson and Anfinsen 1953;Yielding et al. 1964;Huang and Frieden 1969), the enzyme also exhibited potent inhibition by GTP and activation by ADP (Wolff 1962;Hershko and Kindler 1966). This led to much experimentation and controversy over the number and location of nucleotide sites and the relationship between them (Markau et al. 1971;Koberstein and Sund 1973;Bayley and O'Neill 1980). Chemical modification experiments (Colman and Frieden 1966;Price and Radda 1969;Coffee et al. 1971) uniformly showed that the response to purine nucleotides was associated with the C-terminal 50 amino acids of the protein, a finding that correlated well with the finding that homologous GDHs of prokaryotes and fungi lacked both the 50-amino acid section and the regulation by GTP/ADP (Wootton et al. 1974;Mattaj et al. 1982). Latterly, the recent solution of the threedimensional structure of bovine GDH (Peterson and Smith 1999) has revealed that these 50 amino acids form an ''antenna'' protruding from the main body of the hexameric structure and totally missing in various other solved GDH structures (Rice et al. 1987;Stillman et al. 1995

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Purification of glutamate dehydrogenase from ox brain and liver. Evidence that commercially available preparations of the enzyme from ox liver have suffered proteolytic cleavage

Cited by 62 publications

References 21 publications

Two Soluble forms of Glutamate Dehydrogenase Isoproteins from Bovine Brain

Two Soluble forms of Glutamate Dehydrogenase Isoproteins from Bovine Brain

The subcellular location in rat kidney of the peripheral benzodiazepine acceptor

The structural basis of proteolytic activation of bovine glutamate dehydrogenase

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