Rapid assay for in vitro degradation of luteinizing hormone releasing hormone

A radiochemical method for measuring luteinizing hormone releasing hormone (LHRH) degrading enzymatic activity in vitro was developed using LHRH labeled at the N-terminal 5-pyrrolidone-2-carboxylic acid (<Glu) residue. The intact labeled peptide is separated from the labeled fragments formed by cleavage by a cation-exchange batchwise procedure. The assay reflects the degradation of LHRH specifically in terms of inactivation of hormonal activity, is more rapid than a radioimmunoassay, is independent of LHRH concentration, and is not influenced by high protein concentrations. It can be used for studying the degradation of LHRH by subcellular fractions and enzymes. With this assay a highly active enzymatic degradation system was detected in the rat ovary, a recently discovered target organ for LHRH.     

Studies on the enzymic degradation of luteinizing hormone releasing hormone by rat pituitary plasma membranes

Degradation of luteinizing hormone releasing hormone (LH-RH) by purified plasma membranes from rat pituitaries was investigated. Synthetic LH-RH (0.5 mg/ml) was incubated (20 min, 37°C) with pituitary plasma membranes (750 μg protein/ml). The reaction was stopped by centrifugation at 4°C. The degradation products were isolated by high pressure liquid chromatography using a reversed-phase column. Amino acid analysis of the degradation products indicated that the N-terminal tripeptide (pGlu-His-Trp) and the N-terminal hexapeptide (pGlu-His-Trp-Ser-Tyr-Gly) sequence of LH-RH are the main degradation products. These results suggest that the main cleavage sites of LH-RH by the pituitary plasma membrane-bound enzymes are the Gly⁶-Leu⁷ and the Trp³-Ser⁴ bonds of the neurohormone.     

On the existence and separation of the follicle stimulating hormone releasing hormone from the luteinizing hormone releasing hormone.

Porcine hypothalamic fragments were extracted by 2M AcOH at 4°C, and the extractives were subsequently processed in the presence of one protease inhibitor and one anti-oxidant. Gel filtration was performed on Bio-Gel P-2, and supplementary [³H]-LHRH and [¹⁴C]- 3H]-LHRH, and was differentiated from [¹⁴C]- 相似文献   

Isocratic high-performance liquid chromatographic method for the separation of testosterone metabolites

An isocratic reversed-phase high-performance liquid chromatographic (HPLC) method using an Ultrasphere IP column has been developed for the determination of testosterone and its metabolites after incubation of 4-¹⁴C-labelled or unlabelled testosterone with rat liver microsomes. Compounds were eluted with methanol-water-tetrahydrofuran (35:55:10, v/v, pH 4.0) and detected by ultraviolet (UV) absorption at 245 nm. UV or on-line radioactivity detection can be used although, due to differences in detector cell volumes, peak resolution is slightly better with UV detection. Selectivity was validated by collecting HPLC peaks and verifying their identity by gas chromatography-mass spectrometry after derivatization by N,O-bis(trimethylsily)trifluoroacetamide-trimethylchlorosilane. A three-day validation was performed to determine the linearity, repeatability, reproducibility and accuracy of the method, using corticosterone as internal standard. The method is applicable to the measurement of cytochrome P-450 isoenzyme activities in rat liver.     

The use of bacitracin as an inhibitor of the degradation of thyrotropin releasing factor and luteinizing hormone releasing factor

Bacitracin was found to be an effective inhibitor of the $\text{in}\text{vitro}$ degradation of both thyrotropin releasing factor¹ (TRF) and luteinizing hormone releasing factor (LRF) by guinea pig hypothalamic and whole brain homegenates and rat hypothalamic homogenates and subcellular fractions. Bacitracin was effective in inhibiting the degradation of TRF and LRF, as determined by radioimmunoassay, where it exhibited no interference with the assays. Kinetic studies of the degradation of exogenous synthetic [³H]-TRF demonstrated non-competitive inhibition by bacitracin with K_i = 1.9 × 10^?5 M, while studies on the degradation of [³H] LRF indicated competitive inhibition with K_i = 1.7 × 10^?5 M. Electrophoretic and amino acid analysis revealed that bacitracin itself was not degraded during the course of the $\text{in}\text{vitro}$ incubation.     

Leydig cell receptors for luteinizing hormone releasing hormone and its agonists and their modulation by administration or deprivation of the releasing hormone

Leydig cells isolated from adult rat testes bound ¹²⁵I-labelled luteinizing hormone releasing hormone (LHRH) agonist with high affinity (K_A=1.2 × 10⁹M) and specificity. LHRH and the 3–9 and 4–9 fragments of LHRH agonist competed for binding sites with ¹²⁵I-LHRH agonist but with reduced affinities, whereas fragments of LHRH, and oxytocin and TRH were largely inactive. Somatostatin inhibited binding at high (10^?4M) concentrations but was inactive at 10^?6M and less. Pretreatment of rats for 7 days with 5 μg/day of LHRH agonist reduced binding of ¹²⁵I-LHRH agonist to Leydig cells $\text{in vitro}$ by 25%, whilst inhibition of endogenous LHRH by antibodies for 7 days caused a 40% decrease.     

Enzymatic degradation of luteinizing hormone releasing hormone (LHRH) by mucosal homogenates from the intestine of the common brushtail possum (Trichosurus vulpecula)

The peptidolytic activity of fresh and frozen mucosal homogenates from five regions (duodenum, jejunum, ileum, caecum and colon) of possum intestine from Trichosurus vulpecula towards human Luteinizing Hormone Releasing Hormone (LHRH) was investigated. The rank of order of specific peptidolytic activity of the mucosal homogenates was jejunum > ileum > caecum> duodenum = colon, with a 3 to 4 fold difference between the least and the most active segment in both frozen and fresh samples. The formation of peptides LHRH (1-3), LHRH (1-4) and LHRH (1-5) suggest endopepetidase-24.18, endopeptidase-24.15 and angiotensin converting enzyme (ACE) might be responsible for the peptide degradation in mucosal homogenates. The inhibition of LHRH degradation by mucosal homogenates was evaluated in four regions (jejunum, ileum, caecum and colon) of possum intestine. Ethylenediaminetetraacetic acid (EDTA, 5 mM), sodium deoxycholate (SDA, 10 mM) and bacitracin (3.5 or 9 mM) inhibited the degradation of LHRH in mucosal homogenates from small intestine and hindgut. However, the serine protease inhibitor, soybean trypsin-chymotrypsin inhibitor (SBTI), did not prevent degradation of LHRH. It is concluded that combining peptides with inhibitors may enhance oral delivery of bioactive peptides or proteins to possums.     

Novel ligands for the affinity labelling of luteinizing hormone releasing hormone receptors.

A number of novel luteinizing hormone releasing hormone (LHRH) analogues incorporating biotin together with potential covalent attachment sites have been synthesized. Those based on the des-Gly10-[D-Lys6]-LHRH ethylamide peptide backbone resulted in the most useful characteristics of binding to the LHRH receptor in rat anterior pituitary gland membranes. Of these, des-Gly10-[biotinyl-aminoethylglycyl-D-Lys6]-LHRH ethylamide (XBAL) gave the best specific: non-specific binding ratio, with 44 +/- 6% (+/- S.E.M.) of total binding being specific with a Kd of 131 +/- 16 pM (+/- S.E.M., n = 4) as determined by Scatchard analysis. Two methods have been used to covalently crosslink these analogues with the LHRH receptor; photoaffinity labelling and the use of homobifunctional N-hydroxysuccinimide ester crosslinkers. The photoaffinity analogues gave poor specific: non-specific binding ratios. Of the chemical crosslinkers tested, ethylene glycolbis(succinimidylsuccinate) (EGS) was found to be the most efficient at covalently linking the 125I-XBAL bound to the LHRH receptor site. At an EGS concentration of 5 mM, 23 +/- 3% (+/- S.E.M.) of the specific binding of 125I-XBAL was covalently crosslinked.     

Effects of luteinizing hormone releasing hormone on gonadotrophins in the hamster

The changes in serum gonadotrophins in male hamsters following one injection of 15 μg luteinizing hormone releasing hormone (LHRH) (Group A) were compared with those following the last injection of LHRH in animals receiving an injection approximately every 12 hr for 4 days (Group B) or 12 days (Group C). Peak follicle stimulating hormone (FSH) levels (ng/ml) were 1776±218 (Group A), 2904±346 (Group B), and 4336±449 (Group C). Peak luteinizing hormone (LH) values (ng/ml) were 1352±80 (Group A), 410±12 (Group B), and 498±53 (Group C). Serum FSH:LH ratios, calculated from the concentrations measured 16 hr after the last LHRH injections, were higher in Groups B and C than in Group A. Similar injections of LHRH (100 ng or 15 μg/injection) for 6 days elevated the serum FSH:LH ratio in intact males. Five such LHRH injections (100 ng/injection) blunted the rise in serum LH in orchidectomized hamsters. Direct effects of LHRH on gonadotrophin secretory dynamics or altered brain-pituitary-testicular interactions may alter the ratio of FSH to LH in the hamster.     

Isocratic ion-exchange chromatographic assay for the nucleotide gemcitabine triphosphate in human white blood cells

An isocratic bio-analytical assay for the nucleotide gemcitabine triphosphate (2',2'-difluorodeoxycytidine 5'-triphosphate, dFdCTP) in human white blood cells (leukocytes) has been developed and validated. The method is based on ion-exchange liquid chromatography and ultraviolet detection (275 nm). dFdCTP is isolated from the matrix by extraction with perchloric acid while the sample is chilled on ice. After neutralization with potassium hydroxide and removal of the potassium perchlorate precipitate, with the sample still chilled on ice, the mixture is injected into the chromatograph. The method has been validated in the range 0.4-20 microM, 0.4 microM (approximately 20 pmol/10(6) cells) being the lower limit of quantification, using erythrocytes as a substitute for leukocytes. Precisions and accuracies both meet the current requirements for a bioanalytical assay. The stability of dFdCTP in intact mononuclear blood cells on ice is strongly limited (half-life approximately 100 min) and after freezing the half-life of the analyte in the cellular lysate is approximately 30 min. On the other hand, no degradation was observed for dFdCTP for at least approximately 24 h in perchloric acid extracts on ice or in neutralized extracts at ambient temperature. The applicability of the assay was demonstrated in white blood cells of a patient with advanced non-small cell lung cancer receiving i.v. gemcitabine.     

The effects of thyrotropin releasing hormone (TRH) on the gastrointestinal tract.

Thyrotropin-releasing hormone (TRH) immunoreactivity is distributed throughout the gastrointestinal tract and the pancreas. We have studied the effect of TRH on several gastrointestinal functions in intact, unanesthetized dogs. Intravenous TRH stimulated gastric action potentials (p<0.01) and transiently inhibited tetragastrin-stimulated gastric acid secretion (p<0.05). TRH had no effect on basal or secretin-stimulated pancreatic exocrine secretion. TRH did not alter water absorption in dogs with Thiry-Vella loops constructed from proximal jejunum.     

The structural features of effective antagonists of the luteinizing hormone releasing hormone

Summary The structure-activity data of 6 years on 395 analogs of the luteinizing hormone releasing hormone (LHRH) have been studied to determine effective substituents for the ten positions for maximal antiovulatory activity and minimal histamine release. The numbers of substituents studied in the ten positions are as follows: (41)¹-(12)²-(12)³-(5)⁴-(47)⁵-(52)⁶-(16)⁷-(18)⁸-(4)⁹-(8)¹⁰. In position 1, DNal and DQal were effective with the former being more frequently the better substituent. DpClPhe was uniquely effective in position 2. Positions 3 and 4 are very sensitive to change. D3Pal in position 3 and Ser in position 4 of LHRH were in the best antagonists. PicLys and cPzACAla were the most successful residues in position 5 with cPzACAla being the better substituent. Position 6 was the most flexible and many substituents were effective; particularly DPicLys. Leu⁷ was most often present in the best antagonists. In position 8, Arg was effective for both antiovulatory activity and histamine release; ILys was effective for potency and lesser histamine release. Pro⁹ of LHRH was retained. DAlaNH₂ ¹⁰ was in the best antagonists.Abbreviations AABLys N -(4-acetylaminobenzoyl)lysine - AALys N -anisinoyl-lysine - AAPhe 3-(4-acetylaminophenyl)lysine - Abu 2-aminobutyric acid - ACLys N -(6-aminocaproyl)lysine - ACyh 1-aminocyclohexanecarboxylic acid - ACyp 1-aminocyclopentanecarboxylic acid - Aile alloisoleucine - AnGlu 4-(4-methoxy-phenylcarbamoyl)-2-aminobutyric acid - 2ANic 2-aminonicotinic acid - 6ANic 6-aminonicotinic acid - APic 6-aminopicolinic acid - APh 4-aminobenzoic acid - APhe 4-aminophynylalanine - APz 3-amino-2-pyrazinecarboxylic acid - Aze azetidine-2-carboxylic acid - Bim 5-benzimidazolecarboxylic acid - BzLys N -benzoyllysine - Cit citrulline - Cl₂Phe 3-(3,4-dichlorphenyl)alanine - cPzACAla cis-3-(4-pyrazinylcarbonylaminocyclohexyl)alnine - cPmACAla cis-3-[4-(4-pyrimidylcarbonyl)aminocyclohexyl]alanine - Dbf 3-(2-dibenzofuranyl)alanine - DMGLys N -(N,N-dimethylglycyl)lysine - Dpo N -(4,6-dimethyl-2-pyrimidyl)-ornithine - F₂Ala 3,3-difluoroalanine - hNal 4-(2-naphthyl)-2-aminobutyric acid - HOBLys N -(4-hydroxybenzoyl)lysine - hpClPhe 4-(4-chlorophenyl)-2-amino-butyric acid - Hse homoserine, 2-amino-4-hydroxybutanoic acid - ICapLys N -(6-isopropylaminocaproyl)lysine - ILys N -isopropyllysine - Ind indoline-2-carboxylic acid - INicLys N -isonicotinoyllysine - IOrn N -isopropylornithine - Me₃Arg N^G,N^G,N^G-trimethylarginine - Me₂Lys N ,N -dimethyllysine - MNal 3-[(6-methyl)-2-naphtyl]alanine - MNicLys N -(6-methylpicolinoyl)lysine - MPicLys N -(6-methylpicolinoyl)lysine - MOB 4-methoxybenzoyl - MpClPhe N-methyl-3-(4-chlorphenyl)lysine - MPZGlu glutamic acid,-4-methylpiperazine - Nal 3-(2-naphthyl)alanine - Nap 2-naphthoic acid - NicLys N -nicotinoyllysine - NO₂B 4-nitrobenzoyl - NO₂Phe 3-(4-nitrophenyl)alanine - oClPhe 3-(2-chlorphenyl)alanine - Opt O-phenyl-tyrosine - Pal 3-(3-pyridyl)alanine - 2Pal 3-(2-pyridyl)alanine - 2PALys N -(3-pyridylacetyl)lysine - pCapLys N -(6-picolinoylaminocaproyl)lysine - pClPhe 3-(4-chlorophenyl)alanine - pFPhe 3-(4-fluorophenyl)-alanine - Pic picolinic acid - PicLys N -picolinoyllysine - Pip piperidine-2-car-boxylic acid - PmcLys N -(4-pyrimidylcarbonyl)lysine - Ptf 3-(4-trifluromethyl phenyl)alanine - Pz pyrazinecarboxylic acid - PzAla 3-pyrazinylalanine - PzAPhe 3-(4-pyrazinylcarbonylaminophenyl)alanine - Qal 3-(3-quinolyl)alanine - Qnd-Lys N -quinaldoyllysine - Qui 3-quinolinecarboxylic acid - Qux 2-quinoxalinecarboxylic acid - Tic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid - TinGly 2-thienylglycine - tNACAla trans-3-(4-nicotinoylaminocyclohexyl)-alanine - tPACAla trans-3-(4-picolinoylaminocyclohexyl)alanine     

Biosynthesis of the luteinizing hormone releasing hormone in mitochondrial preparations and by a possible pantetheine-template mechanism

Data show that the luteinizing hormone releasing hormone (LHRH) is biosynthesized by mitochondrial preparations from hypothalami which were obtained by centrifugation of a Ficoll gradient system. Incubation, extraction, use of carrier synthetic LHRH, and chromatography, yielded fractions showing associated hormonal activity and radioactivity. Other investigators reported biosynthesis of the thyrotropin and the growth hormone releasing hormones by soluble enzymes, but these enzymes might have been derived from mitochondria and/or synaptosomes. These particles appear implicated, but the true site of biosynthesis of these hormones is unknown. The mechanism at the site could possibly involve a pantetheine-protein template.