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TCK, the creatine kinase (ATP:creatine Nphosphotransferase) from sperm flagella of the sea urchin Strongylocentrotus purpuratus, is a Mr 145,000 axonemal protein that is employed in energy transport. Its amino acid sequence was obtained by analysis of fragments from cyanogen bromide digestion and by sequencing cDNA clones from two sea urchin testis libraries. TCK contains three complete but nonidentical creatine kinase segments joined by regions of sequence that are not creatine kinase-like and flanked by unique amino and carboxyl termini. Each creatine kinase segment is homologous to vertebrate creatine kinases of both muscle and brain types, and all three repeats contain the essential active-site cysteine. The sequence differences among repeats suggest an ancient gene triplication, around the time of the chordateechinoderm divergence. The echinoderm, with a unique creatine kinase in sperm, arginine kinase in eggs, and both phosphagen kinases in somatic cells, may represent a preserved branch point in evolution, and TCK may be a relic of this event.Structurally similar creatine kinases (CKs; EC 2.7.3.2) are found in many invertebrates and vertebrates (1), existing as dimers or octamers of subunit Mr -40,000 (2, 3). However, an echinoderm-specific CK is significantly larger: that purified from the flagellum of Strongylocentrotus purpuratus sperm is monomeric, with an estimated Mr of 145,000 (4). This CK participates in an energy shuttle that utilizes phosphocreatine to transfer the energy from ATP generated by the mitochondrion in the sperm head to dynein in the distal portions of the flagellum (5). A mitochondrial CK transphosphorylates creatine and ATP, maintaining the high levels of ADP that permit maximum respiration (5, 6). As phosphocreatine diffuses along the flagellum, the flagellar creatine kinase (TCK) regenerates ATP for use as a substrate by dynein. Specific inhibition of CKs by low levels of 1-fluoro-2,4, dinitrobenzene attenuates flagellar beating (6), indicating that the energy shuttle is essential for normal flagellar motion. TCK specifically associates with the axoneme and may bind directly to polymerized microtubules (7). To ascertain the primary structure of this unusually large CK, and to provide tools to examine its association with the axoneme, we have purified TCK, isolated and sequenced six CNBr peptides, and obtained the sequence of clones from two independent cDNA libraries.t MATERIALS AND METHODS S. purpuratus were obtained at low tide along the northern shore of the Olympic Peninsula in Washington. Restriction endonucleases were purchased from United States Biochemical and Boehringer Mannheim. All other reagents were ofthe highest grade available.Sperm were obtained and TCK was purified as described (4). TCK (46 mg) was dialyzed against 1.44 M Tris (pH 8.6) and S-carboxymethylated (8); the sample was then dialyzed against 70% formic acid and cleaved at methionine residues with CNBr (9). CNBr peptides were lyophilized, dissolved in 6 M guanidinium chloride buffered to pH 6.0...

We measured maximal coronary artery conductance in near-term fetal sheep before and after chronic infusion with adenosine to determine whether an increase in coronary flow without hypoxemia results in increased coronary vascular growth. Adenosine was infused into the circumflex coronary artery for 12 h each day for 4 days. Coronary flow was maintained at double the resting level by regulating the infusion of adenosine via a computerized servocontrol device signaled by a Doppler flow-velocity sensor. Total arterial hemoglobin, oxygen content, and hemodynamics were unchanged. Resting circumflex coronary blood flow increased from control of 250 +/- 111 to 530 +/- 216 ml x min(-1) x 100 g left ventricle(-1) with adenosine on day 1 and from 194 +/- 74 to 878 +/- 210 ml x min(-1) x 100 g left ventricle(-1) with adenosine on the last day (P < 0.01). Coronary conductance, determined during maximal vasodilation, increased from 14.0 +/- 5.0 to 26.9 +/- 3.9 ml x min(-1) x 100 g(-1) x mmHg(-1) over the 4 days (P < 0.001). Coronary flow reserve, the difference between resting and maximal myocardial blood flow interpolated at 40 mmHg, increased from 299 +/- 196 to 672 +/- 266 ml x min(-1) x 100 g(-1) (P < 0.001). Maximal coronary conductance was unchanged in control saline-infused fetuses (18.5 +/- 5.1 vs. 18.5 +/- 8.7 ml x min(-1) x 100 g(-1) x mmHg(-1)). We conclude that chronic intracoronary adenosine administration to the fetal myocardium modulates coronary vascular growth, even in the absence of tissue hypoxia.

The incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) mediate insulin responses that are proportionate to nutrient intake to facilitate glucose tolerance1. The GLP-1 receptor (GLP-1R) is an established drug target for the treatment of diabetes and obesity2, whereas the therapeutic potential of the GIP receptor (GIPR) is a subject of debate. Tirzepatide is an agonist at both the GIPR and GLP-1R and is a highly effective treatment for type 2 diabetes and obesity3,4. However, although tirzepatide activates GIPR in cell lines and mouse models, it is not clear whether or how dual agonism contributes to its therapeutic benefit. Islet beta cells express both the GLP-1R and the GIPR, and insulin secretion is an established mechanism by which incretin agonists improve glycemic control5. Here, we show that in mouse islets, tirzepatide stimulates insulin secretion predominantly through the GLP-1R, owing to reduced potency at the mouse GIPR. However, in human islets, antagonizing GIPR activity consistently decreases the insulin response to tirzepatide. Moreover, tirzepatide enhances glucagon secretion and somatostatin secretion in human islets. These data demonstrate that tirzepatide stimulates islet hormone secretion from human islets through both incretin receptors.