Beef Pituitaries Research Articles

A pituitary endorphin with novel properties

We describe the further purification of an opioid peptide from a porcine pituitary concentrate. The peptide has typical naloxone-reversible opioid activity in the guinea pig ileum myenteric-plexus preparation and mouse vas deferens, and it inhibits stereospecific binding at opiate receptors. It is distinguished from β-endorphin and the enkephalins by its apparent molecular weight, its slow reversal with washing in the guinea pig ileum preparation, and the resistance of its biologic activity to cyanogen bromide treatment. In beef pituitary, slow-reversing, cyanogen bromide resistant activity is found principally in neurointermediate lobe.

Opioid peptides (endorphins) in pituitary and brain

(1) There are at least three types of endorphin in pituitary and brain. (2) β-lipotropin (βLPH) is a prohormone for one type. All fragments of the type β-LPH-(61-n) are approximately equipotent in the myenteric plexus bioassay. Met-enkephalin [β-LPH-(61–65)] is of this type. In the bioassay and in the receptor binding assay, these peptides are somewhat less potent than morphine. (3) The second type appears to have about the same molecular weight as β-LPH-(61–91). Endorphins of this type are more potent, and their activity is destroyed readily by trypsin. Like the β-LPH type, their opioid activity is destroyed by cyanogen bromide (i.e. they have a critically placed Met residue). Much of the endorphin activity of porcine pituitary is of this type. (4) The third type also has about the same molecular weight as β-LPH-(61–91), but exists as well in a smaller form (1200–1400 daltons). The opioid activity of these endorphins is destroyed by trypsin; but it is insensitive to cyanogen bromide and therefore the peptide could possibly contain Leu-enkephalin. These endorphins are found in beef brain and pars nervosa of beef pituitary. (5) Endorphin activity in beef and rat brain has been found associated with peptides of about the same molecular weight as the native endorphins in pituitary (approx 3000 daltons) as well as in smaller peptides.

In vitro biosynthesis of gamma-lipotropic hormone.

Sheep gamma-lipotropic hormone (gamma-LPH) is a pituitary polypeptide made of 58 amino acids and is formed of the first 58 residues of beta-lipotropic hormone (beta-LPH). The C-terminal portion (41-58) of gamma-LPH is identical with the structure of beta-melanophore-stimulating hormone (beta-MSH). We hypothetized in 1967 that beta-LPH could be the biological precursor of beta-MSH and that gamma-LPH could be an intermediate compound. We demonstrated in 1974 that beta-LPH is actively synthesized in the bovine pituitaries. We now studied the biosynthesis of gamma-LPH by monitoring the incorporation of radioactive amino acids in beef pituitary slices. We separated gamma-LPH from the other radioactive proteins with a method previously described. We characterized the radioactive proteins by ion-exchange chromatography, gel filtration and polyacrylamide gel electrophoresis. Our results show that radioactive gamma-LPH was actively synthesized. This gamma-LPH has all the chemical characteristics of nonradioactive gamma-LPH. However, in the conditions used, we were unable to demonstrate biosynthesis of beta-MSH. These results suggest that gamma-LPH is biosynthesized more slowly than beta-LPH and that the conversion into beta-MSH, if it exists, is a slow or subactive process in the species studied.

Cyclophosphates VI cyclophosphates as substrates and effectors of phosphodiesterase

Summary The synthesis and the chemical and physical properties of a major number of 3′,5′-cyclophosphates is described. These compounds are derivatives of 3′,5′-AMP, 3′,5′-IMP or 3′,5′-GMP, substituted in the 6-, 8-, 2- or 2′-position. Furthermore, the partial purification of phosphodiesterase from beef pituitary is described. The cyclophosphate analogues were tested for their splitting behavior at mM substrate concentration with several different phosphodiesterases. Due to the heterogenicity of this enzyme, the selection of the proper reference compound for calculating the relative splitting rates is of importance. The patterns of splitting rate changes caused by different substituents are discussed. Based on additional measurements of the mutual inhibition behavior, some conclusions on the relationship of splitting rates and inhibition effects are drawn.

Biosynthèse "In Vitro" de l'Hormone Béta-Lipotropique de Boeuf

Sheep beta-lipotropic hormone (β-LPH) is a pituitary hormone made of 90 amino acids and having a portion of its sequence (41–58) identical with the structure of beta-melanocyte-stimulating hormone (β-MSH). We hypothetized that β-LPH could be the biological precursor of β-MSH. We studied the biosynthesis of these two molecules by monitoring the incorporation of radioactive amino acids in beef pituitary slices. We separated β-LPH from the other radioactive proteins with the usual method of purification described previously and we characterized the proteins by ion-exchange chromatography, gel filtration, and polyacrylamide gel electrophoresis. Our results show that the pituitary slices synthesized a radioactive β-LPH which has all the characteristics of non-radioactive β-LPH. However, in the conditions used, we could not demonstrate any biosynthesis of β-MSH after 4 h incubation. These results suggest that the conversion of β-LPH into β-MSH, if it exists, is a slow process and should be studied in more prolonged incubations.

Polyprenols of beef and human pituitary glands.

AbstractAn unknown lipid previously isolated in our laboratory from beef and human pituitaries was shown to have the same structure as dolichol from pig liver. Beef pituitaries contained ca. 130 mg/kg wet tissue, of which ca. 25% was esterified to fatty acids. Human pituitaries contained ca. 1.4 g/kg, mainly as the free alcohol.

Lipid composition of beef and human pituitary glands

AbstractThe lipid composition of beef and human pituitary was determined by chromatographic and spectrophotometric methods. Beef pituitary lipid contained about 25% nonpolar lipids and 75% phospholipids whereas nonpolar lipids made up approximately 60% of the total in human pituitaries. The main nonpolar (i.e., low polarity) lipids in human pituitary were triglycerides, cholesterol, free fatty acids and an unidentified component in the triglyceride fraction. Cholesterol was the major nonpolar lipid component in freshly collected beef anterior and posterior pituitary, but the amount of free fatty acids appeared to increase during storage. Preliminary investigation of the unknown nonpolar lipid in human pituitaries suggested that it was an unsaturated hydroxy compound with no carbonyl functions. Thin layer chromatography indicated that it was also present in smaller amounts in freshly collected beef pituitaries. The main phospholipids of beef anterior, posterior and human pituitary were phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl inositol, phosphatidyl serine and sphingomyelin. The fatty acid composition of total nonpolar lipids, free fatty acids, total phospholipids, phosphatidyl ethanolamine and phosphatidyl choline of beef anterior and posterior pituitary was determined by gas liquid chromatography. Mixtures of saturated and unsaturated fatty acids ranging from C12 to C22 were present; the main fatty acids were palmitic, stearic, oleic, linoleic and arachidonic.

Fractionnement d'hypophyses de rats et étude des activités thyréotrope, gonadotropes et hétérothyréotrope des produits purifies

A four-step fractionation procedure yields from rat pituitary glands gonadotropic and thyrotropic fractions of relatively high specific activity (about 50 u/mg for TSH, 1.2 u/mg for LH, and 16 u/mg for FSH). Heterothyrotropic activity appears inseparable from gonadotropic (FSH or LH) activity. This result, similar to those of experiments with beef pituitary, conform with a hypothesis according to which mammalian gonadotropins are able to stimulate thyroids of teleost fishes.

Localization and characterization of cholinesterase in subcellular fractions of rat brain and beef pituitary.

Fresh rat brains and fresh anterior and posterior pituitary glands of beef were separated by differential centrifugation into subcellular fractions, characterized on the basis of sedimentation and succinic dehydrogenase activity. Cholinesterase activity was measured by both manometric and colorimetric methods, the results of which were comparable. Cholinesterase activity of rat brain was found mainly in the microsome and supernatant fractions. It was quite uniformly distributed in all subcellular fractions of both anterior and posterior pituitary. Comparisons of the relative rates of hydrolysis of acetylthiocholine and butyrylthiocholine, and of inhibition by eserine, indicated that brain contains a much higher percentage of acetylcholinesterase than do both lobes of the pituitary, which contain relatively low concentrations of the specific enzyme. Total cholinesterase activity and its sensitivity to inhibition by eserine in the posterior pituitary were found to be midway between those of the anterior lobe and of the brain, from which the posterior pituitary was derived during embryological development.

Comparative potency of growth hormone preparations in alkali or acid.

Growth hormone preparations obtained from beef pituitary through alkali extraction (methods of Li and Wilhelmi) were compared for growth activity in hypophysectomized rats with acid extracted porcine growth hormone (method of Raben). The alkali extracted preparations showed reduced activity when assayed in acid media. The reduced potency was shown to result from storage at the acid pH, rather than from inactivity at the time of injection. Hydrochloric acid was shown to be more damaging than acetic acid of comparable pH. Somatotropic preparations obtained from beef or pork pituitary through acid extraction showed no difference when assayed in basic or acidic media. It was concluded that the differential effect of pH on Li or Wilhelmi growth hormone as compared to Raben type preparations is not due to the gland source, but rather to the method of preparation.

Some Effects of Growth Hormone Preparations in Pullets and Mature Hens

GROWTH hormone has been shown to have a growth stimulating effect on mature rats and dogs by Evans and Long (1921); Putnam, Benedict and Tell (1929); Li and Evans (1944); Li and Evans (1948); Gaebler (1951). Very little work has been reported on the effects of growth hormone in poultry. Hulbé (1949) injected dwarf chicks daily with an extract of fresh beef pituitaries for a period of 6 months, in monthly increasing doses of 70–230 rat units. The injected birds showed a more prolonged increase in weight than the controls. Hsieh, Wang and Blumenthal (1952) found that growth hormone exerted a diabetogenic and growth-promoting action when injected into the developing chick embryo. No published reports could be found which describe the effects of purified growth hormone in laying hens.The purpose of this paper is to report the effects of injecting low levels of growth hormone in pullets and …

Growth Hormone from Salmon Pituitary Glands

Injections of an alkaline extract of the anterior lobe of beef pituitary produced a prolonged acceleration of growth in rats and a temporary increase in weight of goldfish. A comparable extract of salmon pituitaries (Oncorhynchus tshawytscha) had no effect on growth of rats but produced a slight temporary increase in growth of goldfish. Negative effects of salmon extracts on rats are considered significant, but all effects on goldfish are questioned since the extracts were not free of toxicity.

Localization of adrenocorticotropic hormone by histochemical and immunochemical methods.

The fluorescent antibody technique was adapted to the localization of native protein antigens in cells and tissues. This method was applied specifically to the localization of adrenocorticotropic hormone in the pituitary gland. An antiserum to hog ACTH was produced in an adrenalectomized rabbit. The gamma(2)-globulin fraction of the serum was conjugated with fluorescein. After purification, the fluorescent antibody solution stained selectively the cytoplasm of basophil cells of the hog pituitary. No cells of sheep or beef pituitary or of hog kidney were stained. A fluorescent globulin solution prepared from normal rabbit serum gave no selective staining in any of these tissues. Immunochemical tests showed that the fluorescent antibody gave a precipitin reaction with a highly active ACTH preparation of low molecular weight. The supernatant solution from this reaction showed a loss of hormone activity.

Tests of Mammalian Gonad-Stimulating Hormones on Gonads of Fishes

Summary(1) FSH from beef pituitaries, powdered serum of the pregnant mare, and a commercial preparation of human pregnancy urine (Prolan, Elberfeld) were otherwise proved potent and then injected d...

THE EFFECT OF ANTI-GONADOTROPIC SERA UPON GONADOTROPIC SECRETION IN PARABIOTIC RATS1¹

THE ABILITY of anti'gonadotropic serum, non-specific in nature, to in hibitthe effect of the endogenous secretion of an animal's pituitary has bee nreported previously. Parkes and Rowlands (i) have shown that nonspecific anti'gonadotropic sera' prepared by prolonged injections of beef pituitary, can inhibit ovulation in the rabbit. Rowlands (2) also indicated that such anti'gonadotropic serum suppresses ovulation in the pubertal rat but is un' able to prevent corpus luteum formation in the adult animal. Collip (3) was able to make normal cyclic rats acyclic by injections of anti'gonadotropic sera prepared by prolonged administration of sheep pituitary in the lamb. Thompson (4) produced effects similar to those of hypophysectomy in the guineapig thyroid by injections of anti-thyrotropic hormone. Meyer and Hert8(5) have shown that the ovarian hypertrophy which results when an immature female rat is placed in parabiosis with a castrated male or female littermate,

THE INDUCTION OF PREMATURE PUBERTY WITH ANDROGENIC SUBSTANCE

In recent years numerous reports concerning the treatment of cryptorchidism and hypogenitalism with the use of various gonadotropic hormones have appeared in the literature. Most reports have dealt with the use of gonadotropic hormones either from pregnancy urine, placenta, or beef pituitary and have been used chiefly for the treatment of the afore-mentioned conditions in young males. Recently, Thompson (1) and his associates have reported on the production of precocious puberty and hypertrophy of the genitalia in males receiving gonadotropic hormone for the treatment of cryptorchidism. In their cases, however, these conditions were produced only after large doses of hormone had been administered over long periods. In our own experience (2) covering a period of over 6 years, during which time we have treated a considerable number of males with hypogenitalism, some with and some without cryptorchidism, we have not observed the development of premature or precocious puberty or abnormal hypertrophy of the ge...

Glutathione Concentration of Livers and Muscles of Rats Following Injection of Hypophyseal Growth Hormone.

We have previously reported1 that there is an increase in concentration of glutathione in muscles of mature rats after 15 daily injections of a potent growth hormone obtained by alkaline extraction of beef pituitary. In control animals similarly treated with a heat inactivated solution of the hormone we found no such change. Analyses of the livers showed no significant change. However, if animals were sacrificed a few hours after a single injection of the potent hormone we found a decided fall in the glutathione concentration of the liver with only a slight change, if any, in the muscle glutathione. We have no explanation to offer for this sudden change in the liver but believe that it may furnish a method for estimation of the potency of the growth hormone that is more rapid than the usual method by measuring growth after 20 days of injection. It may prove to have a more important significance since by its use the fundamental biochemical reactions induced by the growth hormone may be studied quantitative...

PRODUCTION OF OVULATION BY GONADOTROPHIC EXTRACTS*

The luteinizing and follicular-stimulating fractions of anterior pituitary extracts have rarely been utilized in the ovulation studies which have been made. Furthermore, few studies have been made on immature female animals in which the normal gonadotrophic activity of the animal’s own pituitary is thought to be at a minimum. It was pointed out by Hertz and Hisaw (1934) that ovulation occurred in 45 per cent of the juvenile rabbits to which they administered subcutaneously, first follicular-stimulating and then luteinizing extract, each over a three-day period. Ovulation did not occur in young rabbits treated with unfractionated pituitary extracts, purified follicular-stimulating extracts, or luteinizing extracts alone. In working with beef pituitaries Evans and associates (1933) reported the failure to produce ovulation in immature female rats by injections of crude extracts, but when their preparations were subjected to further purification ovulation was produced by their administration. It has seemed n...