Abstract
Creatine kinase (CK) catalyzes the reversible transfer of the gamma-terminal phosphate of MgATP to the guanidine creatine (Cr) forming MgADP and phosphocreatine (PCr). The CK reaction plays a central role in both temporal and spatial ATP buffering in cells displaying high and variable rates of ATP turnover. There is a constant, non-enzymatic conversion of Cr to creatinine that must be compensated for by biosynthesis and/or dietary uptake. In all true vertebrate craniates, there is a capacity for de novo biosynthesis of Cr as evidenced by the presence of the two enzymes of the biosynthetic pathway- arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT). In these organisms the bulk of Cr biosynthesis is partitioned between the pancreas, kidney and liver for subsequent transport to muscle fibers and other cells expressing CK. Many protochordates and a broad spectrum of invertebrates, including the basal metazoan sponges, express CK and contain significant pools of Cr/PCr, particularly in primitive-type spermatozoa. However, repeated attempts at demonstrating the enzymes of Cr biosynthesis in these organisms have failed and it has been suggested that Cr is derived from the diet and/or by direct uptake from seawater. I show in this thesis that the protochordate tunicate Ciona intestinalis expresses what appear to be three unique, but very similar to one another, transcripts for GAMT. To validate that these transcripts code for GAMT, the full length cDNA for one of these was generated by PCR amplification and ligated into an expression vector. Expression resulted in the production of a large amount of soluble protein that was purified to homogeneity by low pressure chromatography. The resulting recombinant enzyme had an experimentally determined N-terminal amino acid sequence and relative molecular mass as predicted by the deduced amino acid sequence from the cDNA. Catalytic studies of this recombinant GAMT showed that it indeed had the capacity to methylate guanidinocetate to Cr with an apparent Km and maximal velocity comparable to GAMTs from vertebrates. Real-time PCR showed that this GAMT is primarily expressed in the stomach and gonad but also is expressed in two other tissue complexes. Analysis of the C. intestinalis genome and EST sequencing projects showed that the AGAT gene is present and is expressed demonstrating that this species has the complete Cr biosynthetic pathway. Perusal of other EST and genome sequencing projects reveal that true GAMTs are present in the lancelet Branchiostoma, the sea urchin Strongylocentrotus and the hydroid Hydractinia and AGAT genes are present in both Branchiostoma and Strongylocentrotus. Given the present experimental results and the emerging EST/genome sequencing data, it is clear that the capacity for de novo Cr biosynthesis is widespread in organisms expressing CK and that the genes for GAMT/AGAT evolved coincident with CK.
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