An assay for arginase in chicken kidney.

1. The enzyme arginase in chicken kidney is associated with mitochondria and the mitochondrial membranes must be disrupted to obtain maximum activity. 2. When the membranes were disrupted by sonication, approximately 30% higher 2. When the membranes were disrupted by sonication, approximately 30% higher arginase activity was observed than with the nonsonicated samples. 3. The optimum pH for assay of chick kidney arginase was 9.7-9.8. Prior heat treatment with Mn2+ decreased arginase activity. 4. Highest enzyme activity was obtained by using sonicated preparations and measuring initial reaction velocity during the first 1-2 min of incubation.     

Proteolysis-associated deglycosylation of beta 1-adrenergic receptor in turkey erythrocytes and membranes

A protease that can be inhibited by glutathione, dithiothreitol, and o-phenanthroline but not by ethylenediaminetetraacetic acid converts the 50-kilodalton beta-adrenergic receptor in turkey erythrocyte membranes to a 40-kDa polypeptide which retains the specific ligand binding site. This conversion is attenuated in intact erythrocytes. The large 50-kDa peptide contains N-linked, complex carbohydrates and is retained on wheat germ agglutinin-Sepharose. The 40-kDa product of proteolysis does not bind to the wheat germ agglutinin and can thus be separated from the 50-kDa polypeptide by lectin chromatography. However, the large difference in molecular weights of the two receptor peptides cannot be accounted for solely by the different extent of glycosylation.     

The beta-adrenergic receptor of turkey erythrocyte membranes: conformational modification by beta-adrenergic agonists.

The β-adrenergic receptors of turkey erythrocyte membranes have been identified by the specific binding of the radiolabeled antagonist (?)-|³H|-dihydroalprenolol. Pretreatment of these membranes with either the alkylating agent N-ethylmaleimide or with β-adrenergic agonists does not affect (?)-|³H|-dihydroalprenolol binding to the receptor sites. However, the simultaneous presence of both types of products causes a 50% decline in the number of binding sites. A less pronounced decline occurs when the membranes are pretreated with N-ethylmaleimide in presence of the partial agonist (?)-phenylephrine, and no decline in the presence of the antagonist (?)-|³H|-dihydroalprenolol. β-adrenergic agonists thus appear to induce a conformational change of their receptor, with results in an increased susceptibility to inactivation by N-ethylmaleimide.     

Plate assay for fungal enzymes using cellophane membranes

Fungal mycelia mass and pigments are major obstacles to investigating the secretion of bioactive substances such as enzyme activities using a plate assay. In this study, we applied a cellophane membrane and demonstrated that it can block mycelia mass and conidia (especially pigmented spores that would likely interfere with any subsequent color development-based activity detection) while allowing secreting enzymes to pass through. Visual observation after lifting the cellophane membrane and the collected mycelia and conidia indicated that the bioactivities on specific plates were improved significantly, although some fungal growth hurdle was noted. This proved to be true whether the assays were color development based or not.     

Double antenna structure of chicken prolactin receptor deduced from the cDNA sequence.

Chicken prolactin receptor (cPRLR) deciphered from the cDNA sequence showed a unique double antenna structure in its extracellular domain. The predicted cPRLR preprotein was composed of 831 amino acids and contained a signal peptide and a transmembrane region. The extracellular domain comprised 438 residues, and was divided into two tandemly repeated, highly homologous units, each of which corresponded to the extracellular domains of mammalian prolactin receptors. Both extracellular units of cPRLR possessed two structural features characteristic of the ligand binding units of cytokine/prolactin receptor family, namely two pairs of cysteine residues and a WSXWS motif. These findings strongly suggest that cPRLR contains two repeated ligand binding units, that is a double antenna structure. The cPRLR gene is expressed in a wide range of tissues of laying hen.     

Comparative analysis of microsatellite loci in chicken and turkey.

Cross-species amplification of 520 chicken microsatellite markers was tested by polymerase chain reaction with genomic DNA of the turkey (Meleagris gallopavo). Each primer pair was tested at six different combinations of annealing temperature and MgCl2 concentration. A total of 280 (54%) of the primer pairs produced amplification products. The majority of these products were similar, if not identical in size to those expected based on the fragment sizes of the corresponding chicken loci. Structure of the dinucleotide repeat and flanking sequences was examined for 13 turkey fragments (amplified with chicken primers) and 5 chicken fragments (amplified with turkey primers). Sequence analysis found a wide array of mutations between species in addition to differences in repeat length. To estimate the usefulness of the amplified loci for genetic mapping in the turkey, allelic polymorphism was determined for 57 of the 280 amplified loci. A total of 20 of 57 markers (35%) were polymorphic with an average of 1.4 alleles per locus. The results of this study suggest that approximately 20% of the chicken microsatellite markers will be useful for mapping the turkey genome.     

An immunoradiometric assay for squirrel monkey prolactin.

Determination of immunoreactive prolactin in squirrel monkeys has been hampered by the lack of specific antibodies. We investigated the adaptability of a commercially available immunoradiometric assay for human prolactin, which employs two separate monoclonal antibodies (MAb I and II) to human prolactin, to determine the presence of squirrel monkey prolactin. We found that immunoreactivity curves for prolactin in squirrel monkey pituitary homogenates and serum were parallel to human prolactin standards, suggesting that the epitopes recognized by these antibodies were common to both human and squirrel monkey prolactin. Both nonglycosylated (23 kD) and glycosylated (26 kD) forms of squirrel monkey prolactin were detected in squirrel monkey pituitary homogenates by Western blot analysis using [125I]-MAb II. Neither sheep nor rat prolactin was recognized by Western blot analysis, indicating that the assay may be specific for primate prolactins. We examined the effect of ketamine HCl, an anesthetic that has been shown to elevate serum prolactin levels in other primates, on prolactin secretion in squirrel monkeys. Serum prolactin levels increased greater than fourfold after the administration of ketamine HCl (30 mg/kg b.w., i.m.) compared with control levels. Serum prolactin levels were unaffected by anesthesia with pentobarbital sodium (15 mg/kg b.w., i.v.). This assay provides a reliable and sensitive method for determining immunoreactive squirrel monkey prolactin.     

A reconstitution assay for the guanine nucleotide regulatory protein of the adenylate cyclase system using turkey erythrocyte membranes as the acceptor preparation

The turkey erythrocyte beta-adrenergic receptor-adenylate cyclase system has the unusual property that neither GTP nor Gpp(NH)p are effective in activating adenylate cyclase unless a beta-agonist is present simultaneously. This property results in essentially no basal activity and the inability of GTP or Gpp(NH)p alone to activate the catalytic moiety. In this study, we have exploited these characteristics to utilize turkey erythrocyte membranes as the acceptor preparation in a reconstitution assay. Rat reticulocyte or turkey erythrocyte membranes that have been activated with isoproterenol and Gpp(NH)p followed by solubilization with sodium cholate serve as the donor source of the guanine nucleotide regulatory protein (N). By reconstituting this Gpp(NH)p-activated N protein, it has been found that: (1) exogenous Gpp(NH)p-associated N could activate the catalytic unit of adenylate cyclase in turkey erythrocyte membranes; (2) this system can be used to assay N protein activity; (3) the endogenous pathway for activation of turkey erythrocyte membrane adenylate cyclase by hormones and fluoride remains qualitatively functional; and (4) the effects of combined activation via the endogenous and exogenous pathways are additive and saturable.     

Catecholamine-stimulated GTPase activity in turkey erythrocyte membranes.

Determination of specific GTPase (EC 3.6.1.--) activity in turkey erythrocyte membranes was achieved using low concentration of GTP (0.25 muM), inhibition of nonspecific nucleoside triphosphatases by adenosine 5'(beta,gamma-imino-triphosphate (App(NH)p) and suppression of the transfer of gamma-32P from GTP to ADP with an ATP regeneration system. Under these conditions catacholamines caused a 30--70% increase in GTP hydrolysis. The stimulation of GTPase activity by catecholamines required the presence of Mg2+ or Mn2+. DIfferent batches of membranes revealed the following specific activities (pmol 32Pi/mg protein min): basal GTPase (determined in the absence of catecholamine), 6-- 11; catecholamine-stimulated TTPase, 3--7; and residual non-specific NTPase 3--5. The stimulation of GTPase activity by catecholamines fulfilled the stereospecific requirements of the beta-adrenergic receptor, and was inhibited by propranolol. The concentrations of DL-isoproterenol which half-maximally activated the GTPase and adenylate cyclase were 1 and 1.2 muM, respectively. The following findings indicate that the catecholamine-stimulated GTPase is independent of the catalytic production of cyclic AMP by the adenylate cyclase. Addition of cyclic AMP to the GTPase assay did not change the rate of GTP hydrolysis. Furthermore, treatment of the membrane with N-ethylmaleimide (MalNEt) at 0 degrees C which caused 98% inhibition of the adenylate cyclase, had no effect on the catecholamine-stimulated GTPase. The affinity and specificity for GTP in the GTPase reactions are similar to those previously reported for the stimulation of the adenylate cyclase. The apparent Km for GTP in the basal and the catecholamine-stimulated GTPase reaction was 0.1 muM. These GTPase activities were inhibited by ITP but not by CTP and UTP. It is proposed that a catecholamine-stimulated GTPase is a component of the turkey erythrocyte adenylate cyclase system.     

A filtration assay for solubilized prolactin receptors using polyethyleneimine-treated membrane filters

Free 125I-labeled ovine prolactin can be separated from detergent-solubilized prolactin-receptor complex by filtration on triacetate membrane filters pretreated with polyethyleneimine. Up to 98% of the total 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilized prolactin-receptor complexes from rat liver bound to polyethyleneimine-treated membranes. This simple and rapid technique can be used to quantitate solubilized prolactin-receptor complexes.     

A liquid-phase two-site immunoradiometric assay for human prolactin.

The development of a 'two-site' immunoradiometric assay for human prolactin (hPrl) is described. The assay is based on the addition of radio-iodinated sheep anti-hPrl immunoglobulin G (IgG) and rabbit anti-hPrl serum to standards and unknowns followed by 3 h incubation. The use of solid phase reagents was avoided in order to minimize non-specific effects and the time required for reactants to reach equilibrium. Instead, the separation of hPrl-bound and free labelled antibody is achieved by the addition of sheep anti-(rabbit IgG) serum which precipitates bound labelled antibody by complex formation with rabbit anti-hPrl antibodies which are also hPrl-bound. Varying the order of addition of specific antibodies had a pronounced effect on the 'operating range' and sensitivity of resultant assays. This was attributed to competition between labelled and unlabelled antibodies for binding sites on the hPrl molecule. The immunoradiometric assay employing 'simultaneous addition' of specific antibodies was compared to a 'simultaneous addition' hPrl radioimmunoassay developed using the same sheep antiserum as that used to prepare the radioiodinated sheep anti-hPrl IgG. This immunoradiometric assay is characterized by rapid equilibration of reactants, a wide 'operating range' (the precision of dose estimates was less than 10% over the range 8-10000 mU/l), and high sensitivity (2.6 mU/l, 13 pg). In contrast, the hPrl radioimmunoassay required an incubation of 18 h, demonstrated a much reduced 'operating range' (the precision of dose estimates was less than 10% only over the range 25-1500 mU/l) and reduced sensitivity (9.8 mU/l, 49 pg).     

Sphingomyelinase of chicken erythrocyte membranes.

Most of the chicken erythrocyte's sphingomyelin is hydrolyzed when the chicken red blood cells are incubated in hypotonie solution at 37 °C.Addition of detergents, such as Triton X-100 or Na-cholate, is essential for hydrolysis of external [³H ]sphingomyelin by the erythrocyte membranes.Pure plasma membranes show relatively high sphingomyelinase activity while no activity could be detected in the soluble fraction of the cells. Mg²⁺ and Mn²⁺ activate the enzyme while Ca²⁺ and EDTA strongly inhibit its activity. The optimal pH of the membrane-bound sphingomyelinase lies between pH 7.0–9.0. The detergents Triton X-100 and Na-cholate, at concentrations of 0.5% ($\text{w}\text{v}$) solubilize the membrane-bound enzyme. Human erythrocytes fail to exhibit sphingomyelinase activity.The correlation between the sphingomyelinase activity and its localization is discussed.     

Protein and energy self-selection of turkey hens. Serum prolactin and luteinizing hormone concentrations.

1. The incidence of broodiness was four times as high among turkey hens fed a complete control diet than among hens allowed to self-select their diet from two different feed sources, one being relatively high in protein and the other relatively high in energy (i.e. split-diet). 2. Among non-broody birds, hens fed the split-diet had a significantly lower serum prolactin concentration in the third month of production as compared to control hens. 3. Hens in their second season of egg production had significantly lower serum luteinizing hormone concentrations during the latter stages of egg production than did first season hens.     

Distinguishing bombesin receptor subtypes using the oocyte assay.

Physiological responses to mammalian bombesin-like peptides were studied in Xenopus oocytes injected with mRNA isolated from Swiss 3T3 cells and rat esophagus in order to identify and characterize bombesin receptor subtypes. Both groups respond similarly to either gastrin releasing peptide or neuromedin B, but only the response to neuromedin B in oocytes expressing the esophagus mRNA is not blocked by a specific gastrin releasing peptide receptor antagonist, des-Met-[D-Phe6]Bn(6-13) ethyl ester. Complete desensitization of gastrin releasing peptide-evoked responses in oocytes expressing esophagus mRNA does not abolish neuromedin B-evoked responses. A single application of neuromedin B abolishes responses to subsequently applied gastrin releasing peptide in oocytes expressing esophagus, but not Swiss 3T3, mRNA. RNA blot hybridization studies using a Swiss 3T3 gastrin releasing peptide receptor cDNA probe show no detectable hybridization in esophagus mRNA samples. These data suggest that a gastrin releasing peptide receptor is expressed in the esophagus and that it is distinct from that expressed in Swiss 3T3 cells and may represent a third subtype of mammalian bombesin receptor.     

Isoforms of turkey prolactin: evidence for differences in glycosylation and in tryptic peptide mapping.

1. Three isoforms of turkey pituitary prolactin have been isolated, including a nonglycosylated isoform of 22,500 mol. wt and two glycosylated isoforms of 24,500 mol. wt. 2. The glycosylated turkey prolactins differed in carbohydrate composition, with one isoform apparently containing only O-linked carbohydrate. 3. Tryptic peptide maps showed a few peptides distinctly different among the three prolactin isoforms. 4. Amino acid sequencing of the first 40 residues of the three prolactin isoforms showed arginine at position 24 and histidine at position 27, for the nonglycosylated form, but no identifiable amino acids were detected at this position for the glycosylated isoforms.     

Radioligand assay for 5 -3 -hydroxysteroids. I. 3 -Hydroxy-5-androstene-17-one and its 3-sulfate

A specific, sensitive and reliable radioligand assay for plasma dehydroepiandrosterone (DHA) and its sulfate has been developed. Antisera were obtained by immunizing rabbits with a DHA-17-albumin conjugate. DHA was separated from cross-reacting Δ⁵-steroids by thin layer chromatography. DHA-sulfate was solvolyzed prior to chromatography. Separation of antibody bound and free steroid was achieved with γ-globulin-dextran-coated charcoal. The standard curve was linear on a logit-log plot from 0.1 to 10 ng.