Simplified assay for transamidinase activities of rat kidney homogenates

Pilsum, John F. Van; Taylor, Dorris; Zakis, Baiba; McCormick, Patrick N.

doi:10.1016/0003-2697(70)90035-7

Cited by 54 publications

(25 citation statements)

References 6 publications

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“…Creatinine was measured by Clinical Laboratories, Health Sciences Center (St. John's, NL, Canada). Renal AGAT activity was measured as described by Van Pilsum et al (25).…”

Section: Methodsmentioning

confidence: 99%

Creatine synthesis: production of guanidinoacetate by the rat and human kidney in vivo

Edison¹,

Brosnan²,

Meyer³

et al. 2007

American Journal of Physiology-Renal Physiology

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Edison EE, Brosnan ME, Meyer C, Brosnan JT. Creatine synthesis: production of guanidinoacetate by the rat and human kidney in vivo. Am J Physiol Renal Physiol 293: F1799-F1804, 2007. First published October 10, 2007; doi:10.1152/ajprenal.00356.2007.-A fraction of the body's creatine and creatine phosphate spontaneously degrades to creatinine, which is excreted by the kidneys. In humans, this amounts to ϳ1-2 g/day and demands a comparable rate of de novo creatine synthesis. This is a two-step process in which L-arginine:glycine amidinotransferase (AGAT) catalyzes the conversion of glycine and arginine to ornithine and guanidinoacetate (GAA); guanidinoacetate methyltransferase (GAMT) then catalyzes the S-adenosylmethioninedependent methylation of GAA to creatine. AGAT is found in the kidney and GAMT in the liver, which implies an interorgan movement of GAA from the kidney to the liver. We studied the renal production of this metabolite in both rats and humans. In control rats, [GAA] was 5.9 M in arterial plasma and 10.9 M in renal venous plasma for a renal arteriovenous (A-V) difference of Ϫ5.0 M. In the rat, infusion of arginine or citrulline markedly increased renal GAA production but infusion of glycine did not. Rats fed 0.4% creatine in their diet had decreased renal AGAT activity and mRNA, an arterial plasma [GAA] of 1.5 M, and a decreased renal A-V difference for GAA of Ϫ0.9 M. In humans, [GAA] was 2.4 M in arterial plasma, with a renal A-V difference of Ϫ1.1 M. These studies show, for the first time, that GAA is produced by both rat and human kidneys in vivo. L-arginine:glycine amidinotransferase; transamidinase; amino acid metabolism CREATINE AND CREATINE PHOSPHATE act to buffer the cytosolic ATP/ADP ratio in tissues that have high and variable rates of ATP usage (e.g., skeletal and cardiac muscle). Creatine kinase catalyzes the reversible transfer of the ␥-phosphate group of ATP to the guanidino group of creatine to yield ADP and creatine phosphate.ATP ϩ Creatine 7 ADP ϩ Creatine Phosphate (Equation 1) In skeletal muscle, creatine kinase activity is high, keeping its reaction at near-equilibrium. This keeps the ADP and ATP concentrations fairly constant and buffers the cytosolic phosphorylation potential (18,27,28). Energy storage and transmission by the creatine kinase system are hypothesized to work in two ways: as a temporal buffer and as a spatial buffer. The temporal energy buffer theory is best exemplified during episodes of high-energy use, such as muscle contraction. As soon as ATP is hydrolyzed, it must be replenished. The high-energy phosphate of creatine phosphate is transferred to ADP to regenerate ATP. This leads to an accumulation of creatine that must be rephosphorylated during recovery from exercise. The spatial energy buffer theory implies that creatine phosphate acts as an energy carrier, working to transport high-energy phosphate from sites of synthesis (mitochondria) to sites of ATP utilization in the cytosol (27

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“…Creatinine was measured by Clinical Laboratories, Health Sciences Center (St. John's, NL, Canada). Renal AGAT activity was measured as described by Van Pilsum et al (25).…”

Section: Methodsmentioning

confidence: 99%

Creatine synthesis: production of guanidinoacetate by the rat and human kidney in vivo

Edison¹,

Brosnan²,

Meyer³

et al. 2007

American Journal of Physiology-Renal Physiology

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“…All purification steps were carried out in the cold room, except gel filtration. Each step was monitored by determining the enzymatic activity [12] and the protein concentration [13]. SDS-PAGE was as in [14].…”

Section: Isolation Of Porcine Amidinorransferasementioning

confidence: 99%

The amino acid sequences of human and pig l‐arginine:glycine amidinotransferase

1994

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We have isolated and sequenced the L-arginine:glycine amidinotransferase of pig kidney mitochondria. Due to endogenous proteolysis, the purified molecules showed some heterogeneity at the N terminus. The longest form recovered had 386 amino acids. Part of the pig amidinotransferase sequence information was used to isolate cDNA clones coding for the human enzyme. The deduced amino acid of the human amidinotransferase was 37 amino acids longer due to the presence of a signal sequence. The mature proteins were 94% identical to each other and 36% identical to the sequences of bacterial L-arginine:inosamine phosphate amidinotransferases.

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“…cm-l as derived from amino acid sequencing. The activity of AT was measured by monitoring the formed L-ornithine by means of a ninhydrin color reaction according to the method of Van Pilsum et al (1970), with slight modifications, e.g. a heating block and polypropylene reaction tubes with a total sample volume of 1 ml were used instead of a boiling water bath and test tubes with a sample volume of 4 ml.…”

Section: Methodsmentioning

confidence: 99%

Substrate Binding and Catalysis by L‐arginine: Glycine Amidinotransferase — A Mutagenesis and Crystallographic Study

Fritsche

Humm

Huber

1997

European Journal of Biochemistry

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L-Arginine:glycine amidinotransferase catalyzes the committed step in the biosynthesis of creatine. Eight active-site mutants, D170N, D254N, H303V, D305A, R322E, S355A, C407S, and C410A of recombinant human L-arginine :glycine amidinotransferase were prepared by site-directed mutagenesis and enzymatically characterized. The crystal structures of the three mutants D170N, D254N, and C407S have been determined at 0.28-nm, 0.29-nm and 0.236-nm resolution, respectively. The mutation of active-site residues which are involved in substrate-binding yielded inactive mutants. Substitution of Asp254, which is not directly involved in substrate binding but is thought to transfer protons in concert with the His303 imidazole group, results in a strongly (2000-fold) reduced activity. However, the substitution of Cys410, a residue near the active site but not involved in catalysis or substrate binding, by Ala does not change the kinetic properties with respect to the wild-type enzyme. The loss of enzymatic activity of the D170N, D254N, C407S and likely all other mutants is solely due to the inserted point mutations, affecting substrate binding or transition-state stabilization, and not due to major conformational rearrangements of the protein.These results show that a His-Asp pair on one side of the substrate and a Cys on the other side are key residues for activity and are part of a disjoint triad. The imidazole ring of the His is proposed to act as a general acid/general base during catalysis whereas the Cys acts as a nucleophile analogous to Cys25 of papain-like cysteine proteinases.

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Simplified assay for transamidinase activities of rat kidney homogenates

Cited by 54 publications

References 6 publications

Creatine synthesis: production of guanidinoacetate by the rat and human kidney in vivo

Creatine synthesis: production of guanidinoacetate by the rat and human kidney in vivo

The amino acid sequences of human and pig l‐arginine:glycine amidinotransferase

Substrate Binding and Catalysis by L‐arginine: Glycine Amidinotransferase — A Mutagenesis and Crystallographic Study

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