Enzymatic O-glycosylation of kappa-caseinomacropeptide by ovine mammary Golgi membranes

The results of subcellular fractionation of sheep mammary gland membranes indicate that N-acetylgalactosaminyl polypeptide transferase and galactosyl-N-acetylgalactosaminyl transferase, which are involved in the assembly of disaccharide units of kappa-casein, are localized chiefly in Golgi membranes. The glycosyltransferase activities incorporating N-acetyl [1-14C] galactosamine and [U-14C] galactose from uridine diphosphate N-acetyl [1-14C] galactosamine and uridine diphosphate [U-14C] galactose, respectively, were measured after membrane solubilization with Triton X-100 either with unglycosylated caseinomacropeptide, or with this polypeptide containing the N-acetylgalactosamine side chain residues (desialylated and degalactosylated caseinomacropeptide). Radioactive N-acetylgalactosamine was incorporated in the unglycosylated acceptor peptide, and the glycosidic bonds in the product were alkali labile, suggesting that they were linked to the hydroxyamino acid residues. In addition radioactive N-acetylgalactosamine was released after alpha N-acetyl-D-galactosaminidase treatment of labelled caseinomacropeptide. [U-14C] galactose was incorporated in the desialylated and degalactosylated acceptor peptide. Reductive alkaline treatment of [U-14C] galactose peptide resulted in the release of a major product, the chromatographic properties of which in TLC were identical with authentic galactosyl (1 leads to 3) N-acetylgalactosaminitol. The structure of the labelled disacchariditol determined after periodate oxidation (two equivalents) by gas liquid chromatography-mass spectrometry revealed that the [U-14C] galactose was linked to position C-3 on the N-acetylgalactosaminyl-residue. The anomery of the galactose, as determined by a chemical method, indicates unambiguously a beta configuration.     

Affinity Purification of Synenkephalin-Containing Peptides Including a Novel 23.3-Kilodalton Species

Abstract: Affinity chromatography has been used for rapid and high-yield purification of synenkephalin (proenkephalin 1 -70) containing peptides present in bovine adrenal medulla (BAM) chromaffin granular lysate. A column of CN-Br-activated Sepharose 4B coupled to synenkephalin antiserum bound synenkephalin immunoreactivity which was eluted by a stepwise gradient of 50 mM ammonium acetate containing 20% (vol/vol) acetonitrile over the pH range 7–3. Synenkephalin immunoreactivity emerged as two peaks, eluting at pH 5.5 and 4.5. Characterization of the two peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting indicated that the pH 5.5 peak contained principally low-molecular-weight proenkephalin species (8.6 and 12.6 kilodaltons), whereas the pH 4.5 peak contained, in addition, high-molecular-weight proenkephalin species (18.2 and 23.3 kilodaltons). The 8.6- and 12.6- kilodalton species were isolated from the pH 5.5 peak by TSK gel filtration HPLC, whereas the pH 4.5 peak was further purified by passage over successive affinity columns coupled to antiserum against BAM 22P (proenkephalin 182–203) and [Met⁵]-enkephalin-Arg⁶-Gly⁷-Leu⁸. The former column retains the 23.3-kilodalton species, whereas the latter column retains the 18.2-kilodalton species. The 23.3- kilodalton peptide represents a novel putative proenkephalin intermediate (proenkephalin-1–206), containing [Leu⁵]- enkephalin at the C-terminus.     

Amino Acid Sequences of Neuropeptides in the Sinus Gland of the Land Crab Cardisoma carnifex: A Novel Neuropeptide Proteolysis Site

The sinus gland is a major neurosecretory structure in Crustacea. Five peptides, labeled C, D, E, F, and I, isolated from the sinus gland of the land crab have been hypothesized to arise from the incomplete proteolysis at two internal sites on a single biosynthetic intermediate peptide "H", based on amino acid composition additivities and pulse-chase radiolabeling studies. The presence of only a single major precursor for the sinus gland peptides implies that peptide H may be synthesized on a common precursor with crustacean hyperglycemic hormone forms, "J" and "L," and a peptide, "K," similar to peptides with molt inhibiting activity. Here I report amino acid sequences of these peptides. The amino terminal sequence of the parent peptide, H, (and the homologous fragments) proved refractory to Edman degradation. Data from amino acid analysis and carboxypeptidase digestion of the naturally occurring fragments and of fragments produced by endopeptidase digestion were used together with Edman degradation to obtain the sequences. Amino acid analysis of fragments of the naturally occurring "overlap" peptides (those produced by internal cleavage at one site on H) was used to obtain the sequences across the cleavage sites. The amino acid sequence of the land crab peptide H is Arg-Ser-Ala-Asp-Gly-Phe-Gly-Arg-Met-Glu-Ser-Leu-Leu-Thr-Ser-Leu-Arg-Gly- Ser-Ala-Glu- Ser-Pro-Ala-Ala-Leu-Gly-Glu-Ala-Ser-Ala-Ala-His-Pro-Leu-Glu. In vivo cleavage at one site involves excision of arginine from the sequence Leu-Arg-Gly, whereas cleavage at the other site involves excision of serine from the sequence Glu-Ser-Leu. Proteolysis at the latter sequence has not been previously reported in intact secretory granules. The aspartate at position 4 is possibly covalently modified.     

Direct and Continuous Detection of ATP Secretion from Primary Monolayer Cultures of Bovine Adrenal Chromaffin Cells

A method was developed for direct and continuous detection of secretion of ATP from primary monolayer cultures of bovine adrenal chromaffin cells. ATP, which is costored with catecholamines within adrenal chromaffin cells, was released into the incubation medium, where it reacted with firefly luciferin-luciferase producing light detected by a photomultiplier located directly below the culture well. Acetylcholine, nicotine, the Ca2+ ionophore A23187, BaCl2, and KCl induced release of ATP. Induction of release of ATP by acetylcholine was dose dependent, with a threshold at 10(-7) M and a maximum at 10(-4) M. The dose-response curve for nicotine was bell shaped, with a threshold at 10(-7) M, a maximum at 10(-5) M, and diminished release at higher concentrations, an observation indicative of desensitization. Investigation of the initial rates of ATP secretion revealed that 10(-4) M nicotine actually induced release of ATP at a faster rate than 10(-5) M nicotine. However, the rate of ATP release evoked by 10(-4) M nicotine began to decline by 6 s, a result indicating the onset of receptor desensitization, whereas release induced by 10(-5) M nicotine continued unabated. Induction of release of ATP by acetylcholine or nicotine was biphasic, with a rapid, initial phase of release followed by a plateau at 0.5-1.5 min and a second phase of release beginning at 1.5-2 min, reaching a maximum by 2-3 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphatidylinositol Phosphodiesterase (Phospholipase C) Activity in the Pineal Gland: Characterization and Photoneural Regulation

Phosphatidylinositol phosphodiesterase (PL-C) appears to be a key element in the adrenergic regulation of pineal cyclic AMP levels. In the present study, the rat pineal enzyme was characterized using exogenous [3H]phosphatidylinositol (0.5 mM) as substrate. Half the enzyme activity was found in the cytosolic fraction, but the highest specific concentration was associated with the membrane fraction. Two pH optima (5.5 and 7.5) of enzyme activity were observed for the membrane fraction but only one in the cytosol fraction (pH 5.5). Enzyme activity in both fractions was Ca2+ dependent. In the case of the membrane protein in pH 7.5, the enzyme activity was sensitive to changes in Ca2+ in the 10-100 nM range. Addition of an equimolar concentration of phosphatidylinositol 4-phosphate nearly completely inhibited the hydrolysis of [3H]phosphatidylinositol; other phospholipids (1.0 mM) were less potent. This may reflect our present finding that [3H]phosphatidylinositol 4-phosphate is a better substrate than [3H]phosphatidylinositol for the enzyme. Stimulus deprivation (2 weeks of constant light or superior cervical ganglionectomy) reduced the cytosolic activity by 30% and had no effect on the membrane-associated enzyme.     

Effects of starvation,chilling, and injury on endocrine gland function in Galleria mellonella

Starvation, chilling, and injury of last instar Galleria mellonella larvae typically elicit extra larval molts or a delay in pupation. The primary sites of action and the nature of the signals by which these treatments affect development are not known. However, since the connections of the brain to the nerve cord are crucial for the effects of starvation and chilling, these signals apparently affect the brain-centered program of developmental regulation via the nerve cord. Chilling, and occasionally starvation, cause extra larval molts in last instar larvae treated prior to the nervous inhibition of their corpora allata; release of a cerebral allatotropin, which stimulates the production of juvenile hormone, appears to be involved in this effect. After this time, a delay in pupation is the principal effect of starvation and chilling, and is apparently due to a temporal inhibition of the release of the prothoracicotropic hormone. Chilling also appears to inhibit unstimulated ecdysteroid production by the prothoracic glands. The effect of injury is not mediated by the nerve cord, but appears to involve an inhibitory humoral factor that affects either the brain or the prothoracic glands themselves. Injury also stimulates juvenile hormone production, an effect which is enhanced when the brain is separated from the nerve cord and which is evidenced by a delay of ecdysis and the occasional retention of some larval features in the ecdysed insects. None of the effects of these various treatments on the brain and the endocrine glands persist when the brains or glands are implanted into untreated hosts.     

Immunohistochemical localization of oxytocin and vasopressin in the adrenal glands of rat,cow, hamster and guinea pig

Summary The distribution of oxytocin and vasopressin in the adrenals of rat, cow, hamster and guinea pig has been studied by use of immunohistochemical techniques. In all the species studied the adrenal cortex contained both peptides; the staining in the zona glomerulosa being more intense than that in zona fasciculata or zona reticularis. The medulla, however, showed considerable species variation. In the cow, both peptides appear to be present in the adrenergic and noradrenergic cells, though staining was particularly prominent in cortical islands interspersed within the medullary tissue. In the rat, groups of medullary cells positive for both peptides were found, though it was not possible to associate these groups with particular chromaffin cell types. In the hamster oxytocin was present only in adrenaline-containing cells, whereas vasopressin was present in all medullary cells. The guinea pig medulla, which contains only adrenaline-secreting cells, was positive for both peptides. The possibilities that vasopressin and oxytocin have an autocrine or paracrine role in functioning of the adrenal gland is discussed.     

Effects of infection with a granulosis virus on larval growth, development and ecdysteroid production in the cabbage looper, Trichoplusia ni

ABSTRACT. Granulosis virus-infected Trichoplusia ni (Hûbner) larvae exhibited an increased larval life span with no supernumerary moult and no pupation. Weight gain was not affected. Insects infected shortly after hatching were slower in reaching the fourth and fifth stadia than were control insects. Haemolymph ecdysteroid titres were lower in virus-infected insects than control insects, but these differences were only significant ( P <0.05) in the fifth stadium. Electron microscopic examination of the pro thoracic glands revealed extensive granulosis virus infection, and glands from virus-infected insects produced no RIA-detectable ecdysteroids in vitro. Injection of 20-OII-ecdysone into virus-infected larvae at various concentrations and times did not induce pupation.     

Gastric emptying and serum insulin levels after intake of glucose-polymer solutions

To examine the gastric emptying characteristics of four drinks varying in carbohydrate composition and concentration, five men ingested 600 ml of one of the different drinks on four separate occasions. All drinks contained Na+ 71 mmol.l-1, Cl- 60 mmol.l-1, Mg+2 5 mmol.l-1 and citrate 7 mmol.l-1; the carbohydrate component was either 3% glucose, 3% glucose-polymer (GP), 5% GP or 10% GP. With 99mTc-diethylene-triaminepenta-acetic acid (DTPA) as a marker, a scintillation camera and computer were used to measure the rate of gastric emptying. The half-emptying times (T 1/2) were inversely related to the glucose content of the solutions. The T 1/2 for 3% PG was 22.4 +/- 4.4 min (mean +/- SE) and for 10% GP 50 +/- 3.3 min (p less than 0.005). There was no significant difference in T 1/2 between the 3% glucose and 3% GP solutions. The increments in blood glucose (highest blood levels from 7.4 +/- 0.3 mmol.l-1 to 8.9 +/- 0.8 mmol.l-1), serum insulin (from 28 +/- 6 mU.l-1 to 77 +/- 13 mU.l-1) and C-peptide (from 3.6 +/- 0.4 micrograms.l-1 to 5.8 +/- 0.9 micrograms.l-1) were related to the amount of carbohydrate ingested. In all cases the serum insulin levels were high enough to inhibit the liberation of free fatty acids from the adipose tissue. It is concluded that the amount of carbohydrate in glucosyl units in the solution is a major determinant of gastric emptying.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadectomy induces laminin biosynthesis and basement membrane assembly in anterior pituitary glands of adult rats

Summary Laminin biosynthesis and basement membrane assembly in anterior pituitary glands of gonadectomized rats were studied by immuno-electron microscopy and radioimmunoassay. Three weeks after gonadectomy, rats received intravenous injections of sheep anti-laminin IgG conjugated to horseradish peroxidase, and glands were fixed and processed for microscopy 1 h later. Peroxidase reaction product uniformly labeled all perivascular and glandular epithelial basement membranes. In addition, reaction product was also found in abnormally multi-layered basement membranes seen especially beneath gonadotrophs, and unusual basement membrane-like structures projecting between gonadotrophs were also labeled. Pituitary sections from gonadectomized rats labeled with pre-embedding immunoperoxidase and post-embedding immungold techniques also localized intracellular laminin within biosynthetic organelles and light body vesicles of gonadotrophs. Neither abnormal basement membrane structures nor intracellular laminin were detected in pituitaries of nongonadectomized, control rats. Radioimmunoassays of pituitary homogenates showed nearly twice as much soluble laminin ( 15 ng/gland) in gonadectomized rats than in controls ( 8 ng/gland), which paralleled gland growth, but serum laminin concentrations did not differ ( 10 ng/ml in both groups). When anterior pituitary glands of gonadectomized rats that received injections of anti-laminin IgG-HRP were fixed 5 days after injection, lengths of unlabeled basement membrane were distributed between labeled lengths. This indicated that new basement membrane was spliced into old by a process similar to that seen in normal development. Supplementation of gonadectomized rats with testosterone, however, arrested laminin biosynthesis and basement membrane assembly and reversed glandular hypertrophy. These results indicate that, in an absence of sex hormone feedback, renewed synthesis of basement membrane components occurs in the anterior pituitary and is probably necessary to support the additional growth and differentiation of gonadotrophs and other pituitary cells.