A critical reexamination of the continous spectrophotometric assay for adenosine deaminase

Murphy, Joe; Baker, David C.; Behling, Cynthia; Turner, Russell A.

doi:10.1016/0003-2697(82)90291-3

Cited by 11 publications

(3 citation statements)

References 27 publications

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“…We were unable to demonstrate inhibition of rabbit heart AMP deaminase by AICA ribotide at concentrations which purportedly inhibited skeletal-muscle AMP deaminase [22]. As discussed elsewhere [43], interference by AICA ribotide in the u.v.-spectrophotometric assay for AMP deamination may have complicated the results of Baggott et al [22]. We did not detect multiple molecular forms ofAMP deaminase in rabbit heart through gradient elution at either the cationexchange (i.e.…”

Section: Enriched Cardiac Amp Deaminase Preparations Have Reportedmentioning

confidence: 60%

Isolation and characterization of AMP deaminase from mammalian (rabbit) myocardium

Thakkar¹,

Janero²,

Yarwood³

et al. 1993

Biochemical Journal

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AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) is a ubiquitous enzyme in eukaryotes, which may play a role in ATP catabolism during myocardial ischaemia. We report isolation of AMP deaminase from rabbit myocardium with a 19% recovery and a 650-fold enrichment, using a newly devised protocol involving sequential cation-exchange, gel-permeation and affinity chromatographies. The cardiac AMP deaminase preparation described was electrophoretically and chromatographically homogeneous and contained one unique N-terminal residue (leucine). The isolated enzyme was sensitive to various cations (K+, Mg2+, Ca2+). The pH optimum of purified cardiac AMP deaminase was 6.8, its pI was 6.5, and it displayed substrate-specificity toward 5'-AMP. The subunit molecular mass of rabbit heart AMP deaminase on SDS/PAGE (81 kDa) and the holoenzyme molecular mass as estimated by non-denaturing size-exclusion h.p.l.c. (330 kDa) indicated that the native enzyme was a tetramer. Cardiac AMP deaminase displayed a sigmoidal substrate-saturation curve in the presence of 100 mM KCl. Apparent Michaelis constants were a Km of 5.8 mM AMP and a Vmax. of 11.1 mumol/min per mg of protein. ATP and ADP were positive allosteric effectors of cardiac AMP deaminase: the apparent Km was decreased to 1.7 mM by 1.0 mM ATP. The enzyme was inhibited by GTP, coformycin, coformycin 5'-phosphate, palmitoyl-CoA, inorganic phosphate compounds, and the metal chelator o-phenanthroline. No inhibition either by product nucleotide (IMP) or by nicotinamide nucleotides was detected when these agents were examined at concentrations up to 2.5 mM. We conclude that this enzyme preparation offers a means by which the kinetic mechanism and regulation of mammalian cardiac AMP deaminase may be directly investigated.

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Section: Enriched Cardiac Amp Deaminase Preparations Have Reportedmentioning

confidence: 60%

Isolation and characterization of AMP deaminase from mammalian (rabbit) myocardium

Thakkar¹,

Janero²,

Yarwood³

et al. 1993

Biochemical Journal

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“…For purified recombinant protein, biochemical activity was assayed using two methods, the first being the coupled GDH assay, and second by following the decrease of absorbance of the substrate adenosine at 265 nm using Infinite M 200 PRO (TECAN) in the presence of 50 mM potassium phosphate, pH 7.6, and 0.045 mM adenosine or adenine. Kinetic parameters were determined at 25°C using 90 nM of enzyme and various concentrations of adenosine (ranging from 0 to 1 mM) [16], [17].…”

Section: Methodsmentioning

confidence: 99%

Biochemical Characterization of Hypothetical Proteins from Helicobacter pylori

Choi

Juárez²,

Ciordia³

et al. 2013

PLoS ONE

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The functional characterization of Open Reading Frames (ORFs) from sequenced genomes remains a bottleneck in our effort to understand microbial biology. In particular, the functional characterization of proteins with only remote sequence homology to known proteins can be challenging, as there may be few clues to guide initial experiments. Affinity enrichment of proteins from cell lysates, and a global perspective of protein function as provided by COMBREX, affords an approach to this problem. We present here the biochemical analysis of six proteins from Helicobacter pylori ATCC 26695, a focus organism in COMBREX. Initial hypotheses were based upon affinity capture of proteins from total cellular lysate using derivatized nano-particles, and subsequent identification by mass spectrometry. Candidate genes encoding these proteins were cloned and expressed in Escherichia coli, and the recombinant proteins were purified and characterized biochemically and their biochemical parameters compared with the native ones. These proteins include a guanosine triphosphate (GTP) cyclohydrolase (HP0959), an ATPase (HP1079), an adenosine deaminase (HP0267), a phosphodiesterase (HP1042), an aminopeptidase (HP1037), and new substrates were characterized for a peptidoglycan deacetylase (HP0310). Generally, characterized enzymes were active at acidic to neutral pH (4.0–7.5) with temperature optima ranging from 35 to 55°C, although some exhibited outstanding characteristics.

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“…The ADA activity of MbADGF recombinant protein was determined using adenosine and 2′‐deoxyadenosine as substrates by measuring the decrease in ADA‐dependent adenosine and 2′‐deoxyadenosine absorbance at 265 nm (continuous spectrophotometric rate determination method) (Murphy et al ., 1982). The assay was carried out at 25 °C and at pH 7.5 in 53.3 m m potassium phosphate buffer, using a cuvette with 1 cm path length.…”

Section: Methodsmentioning

confidence: 99%

Molecular characterization of MbADGF, a novel member of the adenosine deaminase‐related growth factor in the cabbage armyworm, Mamestra brassicae: the functional roles in the midgut cell proliferation

Zhang

Takeda

2007

Insect Molecular Biology

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To clarify the functional mechanism of the adenosine deaminase-related growth factor (ADGF) particularly in the regulation of insect development, the cDNA encoding a homologue of ADGF proteins was cloned from the cabbage armyworm, Mamestra brassicae, named MbADGF. The purified MbADGF recombinant protein stimulated cell proliferation in a dose-dependent manner of SES-MaBr-4 and NIAS-MaBr-93 cell lines that were derived from fat bodies and haemocytes of M. brassicae. The adenosine deaminase activity of MbADGF was detected using adenosine and 2'-deoxyadenosine as substrates. Northern analysis demonstrated that during the larval development the level of MbADGF in the midgut increased. In situ hybridization showed that MbADGF mRNA was expressed in midgut goblet cells and in the apical cytoplasm of columnar cells, which suggests that MbADGF protein may execute its adenosine deaminase activity at the apical cytoplasm of columnar cells to convert adenosine into inosine.

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A critical reexamination of the continous spectrophotometric assay for adenosine deaminase

Cited by 11 publications

References 27 publications

Isolation and characterization of AMP deaminase from mammalian (rabbit) myocardium

Isolation and characterization of AMP deaminase from mammalian (rabbit) myocardium

Biochemical Characterization of Hypothetical Proteins from Helicobacter pylori

Molecular characterization of MbADGF, a novel member of the adenosine deaminase‐related growth factor in the cabbage armyworm, Mamestra brassicae: the functional roles in the midgut cell proliferation

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