The stimulatory effect of the novel gastric-derived hormone, ghrelin, on growth hormone (GH) secretion has been reported in domestic animals as well as in humans and rats. The octanoyl modification on the Ser3 residue of ghrelin appears to be essential for its endocrine activity. A major portion of circulatory ghrelin lacks acylation but possesses some biological activities other than GH stimulation; therefore, both types of acylated and des-acyl ghrelin are supposed to be important for energy homeostasis. The effects of pharmacological doses of rat and/or human ghrelin on GH secretion have been reported recently in ruminants; however, the physiological effect of exogenous bovine ghrelin on its own plasma level and on GH secretion is still unknown. Moreover, the RIA systems for the measurement of bovine active ghrelin and for bovine total ghrelin including acylated ghrelin, des-acyl ghrelin and all ghrelin peptides with an intact bovine C-terminal have not yet been validated. In this study, we established the RIA system for bovine ghrelin, and the dose-dependent effects of synthesized acylated bovine ghrelin(1-27) on plasma active and total ghrelin, GH, insulin and metabolites were measured in Holstein heifers. Six animals were intravenously injected with synthesized acylated bovine ghrelin (0, 0·1, 0·5, 1·0, 5·0, 10·0 µg/kg body weight (BW)) and plasma hormone concentrations were measured from serially collected samples. Bovine ghrelin RIA showed that the basal level of total ghrelin is approximately 16 times higher than that of active ghrelin in bovine plasma. Both forms of ghrelin were increased in a dose-dependent manner in response to bovine ghrelin injections, peak values were reached at 5 min after administration and returned to pre-injected values within 15 min. Plasma GH was responsive to all doses of bovine ghrelin in a dose-dependent manner, peaked as early as at 5-10 min after injection and returned to the basal value within 60 min. The GH area under curve 1 h after injection of the smallest dose of ghrelin used in this experiment (0·1 µg/kg BW) was significantly higher than that of the vehicle (0·1% BSA saline)-injected control group (P<0·05). The GH response to the highest dose of ghrelin (10·0 µg/kg BW) was greater than the response to 5·0 µg/kg BW ghrelin (P<0·001). Plasma glucose concentrations were not significantly altered by the administration of bovine ghrelin while plasma insulin levels were transiently stimulated by the higher doses of ghrelin (1·0, 5·0, 10·0 µg/kg BW). Plasma non-esterified fatty acid levels also increased following ghrelin administration. Our study indicates that a considerable quantity of both acylated and des-acyl ghrelin is circulating in the bloodstream, and also confirms that ghrelin is not only a potent stimulator of GH secretion but also plays a considerable role in energy homeostasis in Holstein heifers.

Two proteins with myosin light chain kinase activity and electrophoretic molecular weights of 155,000 and 130,000 were each isolated from bovine stomach smooth muscle [Kuwayama, H., Suzuki, M., Koga, R., & Ebashi, S. (1988) J. Biochem. 104, 862-866]. The 155 kDa component showed a much higher superprecipitation-inducing activity than the 130 kDa component, when compared on the basis of equivalent myosin light chain kinase activity. In this study, we isolated a cDNA for the entire coding region of the 155 kDa protein. The deduced amino acid sequence revealed a high degree of similarity to those of chicken and rabbit smooth muscle myosin light chain kinases. Multiple motifs, such as three repeats of an immunoglobulin C2-like domain, a fibronectin type III domain, and unusual 20 repeats of 12 amino acids were detected in the sequence. Part of the amino-terminal sequence was similar to that of the actin- and calmodulin-binding domain of smooth muscle caldesmon. These observations suggest that the 155 kDa protein has additional functions other than its enzymatic activity. Two mRNAs of 6.0 and 2.6 kb in length in the bovine stomach smooth muscle RNAs were hybridized with cDNA probes. The 2.6-kb RNA probably encodes telokin, which is the carboxyl terminus of smooth muscle myosin light chain kinase. mRNAs with identical lengths were also detected in bovine aorta.

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The purine binding site of ATP on skeletal muscle myosin has been photoaffinity labeled with 2-azidoadenosine diphosphate (2-N3ADP). 2-N3ADP was stably trapped at the active site (t1/2 approximately 5 days, 0 degree C) by complexation of the two heavy chain reactive thiols (Cys-697 and Cys-707) with Co(III)phenanthroline. Photoincorporation occurred only in the 23-kDa NH2-terminal tryptic fragment of the heavy chain. Extensive serial digestion of photolabeled subfragment 1 of myosin by trypsin and subtilisin yielded a series of labeled peptides which were purified by HPLC. Sequence and radiolabeling analysis of eight photolabeled peptides all indicated that tryptophan-130 was the only labeled residue. This site of labeling confirms earlier photolabeling studies with the non-nucleotide ADP analogue, 2[(4-azido-2-nitrophenyl)-amino]ethyl diphosphate (NANDP), which also labeled Trp-130 [Okamoto, Y., & Yount, R. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1575-1579]. Comparison of the structures of 2-N3ADP and NANDP indicate that their azido groups can be superimposed if both analogues bind to the active site in an extended conformation in a manner analogous to the anti conformation of ATP.