The primary structure of ovine hypothalamic hypophysiotropic luteinizing hormone-releasing factor, LRF, has been established as pGlu-His-Trp-SerTyr-Gly-Leu-Arg-Pro-Gly-NH2 by hydrolysis of the peptide with chymotrypsin or pyrrolidone-earboxylylpeptidase and by analysis of the products by an Edman-dansylation sequencing technique, as well as by mass spectrometry of the derived phenylthiohydantoins. A decapeptide with the proposed primary structure, prepared by total synthesis, gave the same result on sequencing. The synthetic decapeptide possesses the same biological activities as the native ovine LRF. The amino-acid sequence of ovine LRF is identical to that already published for porcine LRF.Various areas of the central nervous system participate in the fine regulation of the secretion of all adenohypophysial hormones. The ultimate integrator of information originating in the central nervous system is the hypothalamus. The final information from the hypothalamus to the adenohypophysis is not transmitted in the form of nerve impulses, but is carried in the form of specific hypothalamic hypophysiotropic substances, the hypothalamic releasing factors, that are carried through the hypothalamo-hypophysial portal system of capillaries from the median eminence region of the ventral hypothalamus to the cells of the adenohypophysis. There is good physiological evidence that such a hypothalamic control is involved in the secretion of the gonadotropin, luteinizing hormone. In the early 1960s, several investigators reported experimental results that were best explained by proposing the existence of substances that specifically stimulated the secretion of luteinizing hormone, and that were probably polypeptides, in crude aqueous extracts of hypothalamic tissues of various mammalian species (1-3). Preparations of LRF, active at 1 /Ag per dose in animal bioassays, were obtained by gel filtration and ion-exchange chromatography on carboxymethylcellulose (4), an observation that was confirmed by similar methods by several investigators (5, 6). In spite of the vagaries of the various bioassay methods available, several laboratories reported preparations of LRF of increased potency (5, 6). Several of these early publications led to contradictory statements regarding purification and separation of LH-releasing factor (LRF), from a follicle-stimulating hormone releasing factor (5, 7). Two laboratories independently reported the isolation of porcine LRF (8) and ovine LRF (9), both groups concluding that LRF from either species was a nonapeptide containing, on the basis of acid hydrolysis, 1 His, 1 Arg, 1 Ser, 1 Glu, 1 Pro, 2 Gly, 1 Leu, 1 Tyr. Earlier results with the pyrrolidone-carboxylylpeptidase prepared by Fellows and Mudge (10) had led us to conclude (11) that the Nterminal residue of LRF was Glu in its cyclized pyroglutamic (pGlu) form, as in the case of hypothalamic TRF, (pGluHis-Pro-NH2).While our own studies on the amino-acid sequence of ovine LRF were in progress, Matsuo et al. (12) reported that porcine LRF contai...

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Two analogs of the hypothalamic luteinizing hormone releasing factor modified at the histidine-2 position were tested for biological activity (secretion of luteinizing hormone) in cultures of dispersed rat anterior pituitary cells. The analog in which glycine was substituted for histidine at position 2, [Gly(2)]LRF, behaves as a partial agonist releasing less than 50 percent of the luteinizing hormone secreted at maximum concentrations of the releasing factor, while the analog in which histidine at position 2 is deleted has no significant agonist activity at any of the doses tested. When added to the cultured cells at molar ratios 10(3) to 10(4) times that of the luteinizing hormone releasing factor, both analogs decrease the amount of luteinizing hormone secreted in response to the releasing factor.

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The role of the liver nerves in the disposition of peripherally administered glucose was examined in seven hepatic innervated (HI) and nine hepatic denervated (HD) 42-h-fasted conscious dogs. After a 40-min basal period, there was a 4-h experimental period during which the hepatic glucose load was increased twofold via peripheral glucose infusion. Somatostatin was infused to suppress pancreatic endocrine secretion, and insulin and glucagon were infused intraportally to produce a fourfold increase in insulin and a gradual decrease (approximately 25%) in glucagon. The area under the curve of net hepatic glucose uptake (NHGU) during the glucose infusion period totaled 483 +/- 82 and 335 +/- 32 mg/kg in HD and HI, respectively (P < 0.05). The area under the curve of the hepatic fractional extraction of glucose was 27% greater in HD (P < 0.05). Net hepatic lactate output was similar in the two groups, and net hepatic glycogen synthesis was 3.8 +/- 0.8 vs. 2.7 +/- 0.5 mg.kg dog wt-1.min-1 in HD and HI, respectively (P = 0.13). The direct pathway of glycogen synthesis was responsible for 54-58% of net hepatic glycogen synthesis in both HI and HD (n = 6 for both). In summary 1) NHGU in response to peripheral glucose infusion was approximately 44% greater in HD than in HI, 2) net hepatic glycogen synthesis was enhanced by 41% in HD although the probability of this change was 0.13, and 3) the contribution of the direct pathway to glycogen synthesis was the same in HD and HI. These data are consistent with a role for the liver nerves in regulating the magnitude of NHGU in response to glucose administration. They also indicate that the absence of liver nerves may reduce glycogen turnover during glucose infusion.

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The reaction of cyclohexene-1,3,3-4¡ with HC1 in acetic acid yields a mixture of .s>«-HCl adduct SC, anti-HC1 adduct AC, and anti-HOAc adduct AA under conditions of kinetic control. The ratio of AC to AA increases markedly with the HC1 concentration, in the presence of tetramethylammonium chloride, or in the presence of water, while the ratio of SC to AA remains essentially unchanged. The ratio of SC to AA does, however, increase significantly with temperature. No syn-HOAc addition was detected. Cyclohexene-1,3,3-d3 recovered after partial reaction showed no evidence of exchange or rearrangement. Analysis of these results, together with those of the preceding paper, shows that three competing reactions are involved. One involves a termolecular reaction of olefin, HC1, and dissociated chloride ion leading to AC while termolecular reaction of olefin, HC1, and acetic acid forms AA. A bimolecular reaction of HC1 and olefin leads to formation of a carbonium chloride ion pair which collapses primarily to a mixture of SC and AA.A number of years ago we reported results of a study of the stereochemistry of polar HBr addition to cyclohexene-1,3,3-^3 in acetic acid.3 Only anti addition of HBr was observed at temperatures between 15 and 60°as contrasted with an earlier report4 that DBr addition to cyclohexene in acetic acid resulted in syn addition in amounts varying from 26 to 74% with increasing temperature between 10 and 60°. An attempt was made to study the kinetics of HBr addition to cyclohexene under the conditions employed for the stereochemical studies in order to elucidate the mechanism involved in anti-HBr addition, but the kinetics proved to be more complex than expected.We turned then to a study of HC1 addition to cyclohexene in acetic acid which seemed a more suitable system for study. In the preceding paper5 we reported the results of the latter study and contrasted them with results obtained from a similar study of z-butylethylene and styrene.6 It was shown that the addition of HC1 to cyclohexene, but not that to z-butylethylene or styrene, is subject to catalysis by chloride ion. In this paper we report studies of the stereochemistry of addition to cyclohexene-1,3,3-d¡, conducted under conditions identical with those employed in the kinetic and product studies of the preceding paper, and show that the chloride-ion catalysis is associated with stereospecific anti addition to cyclohexene. ResultsCyclohexene-1,3,3-d3 was prepared from cyclohexanone according to the following reaction sequence.3(1) (a) Reported in part at the 155th National Meeting of the Ameri-

SynopsisAn improved method for the preparation of Merrifield resin esters is presented. This method is rapid, is free of racemization, and is not complicated by a quaternization side reaction. Chloromethylated resin beads, t-butoxycarbonyl amino acid, and potassium t-butoxide are heated a t 80°C in dimethylsulfoxide for one-half hour to yield resin esters of suitable substitution for solid-phase peptide synthesis. All twenty of the BOC protected common amino acids were esterified to the resin by this method. Resin substitution values lie in the range of 0.13 meq/g (BOC-Glu(NH,)) to 0.66 meq/g (BOC/ Pro), with most of the amino acids yielding 0.344 meq/g (on a resin containing 0.8 meq Cl/g).