Insulin and C-peptide secretion from B cells in human subjects stimulated with glucose

Two groups of immunoreactive insulin in human sera were reported by Kakita et al. (4), using gel chromatography after acid-alcohol extraction. These analogs were noted not only in circulating human sera but also in incubation medium and incubated human pancreas. The release of these insulin analogs was discussed in a previous report (5). The circulating C-peptide immunoreactivity was separated into two groups on a Bio-Gel column, and the early peak should not be proinsulin but an associated C peptide (6). These analogs of insulin were separated by the methods of ion-exchange chromatography, isoelectric focusing, gel electrophoresis, and gel chromatography. Immunoreactive insulin was also separated into two major bands by standard polyacrylamide gel electrophoresis. The fast migrating band corresponds to the rat insulin II position, and the slower corresponds to rat insulin I, which has one more basic amino acid residue in comparison with rat insulin II. Further studies have been performed in five healthy adults in order to elucidate the physiological relationship between analogs of insulin and C-peptide peak substances in human serum; the results are reported in this paper with a consideration of the mechanism of insulin secretion.     

Hydrolysis of substance P and its analogs by angiotensin-converting enzyme from rat lung. Characterization of endopeptidase activity of the enzyme

Hydrolysis of substance P and nine kinds of substance P analogs by angiotensin-converting enzyme highly purified from rat lung was examined by using amino-group fluorometry and high-performance liquid chromatography. The enzyme hydrolyzed substance P and several analogs, notwithstanding that they did not contain free C-terminal residues. The analyses of cleavage products separated by high-performance liquid chromatography indicated that the enzyme hydrolyzed substance P and its analogs mainly at the bond between Phe8-Gly9 and also at another bond, possibly between Gly9-Leu10, to a lesser extent by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action. The analogs that had D-amino acid residues substituted at the presumed cleavage sites were scarcely hydrolyzed. It was further found that (Pyr6)-fragment (6-11) was hydrolyzed by the enzyme more efficiently than the other fragment-type analogs and was cleaved at a single bond by the endopeptidase activity of the enzyme. Therefore, this fragment was used as a substrate in order to characterized the endopeptidase activity of the enzyme by employing fluorometry. The activity was dependent on chloride ion, and was inhibited by captopril, MK-421, and EDTA. Thus, the endopeptidase activity of the enzyme showed properties similar to those of the dipeptidyl carboxypeptidase activity of the enzyme.     

Synthesis of peptides by the solid-phase method. IV. Des-Arg(9)-bradykinin and analogs

We have synthesized a series of 19 analogs of the octapeptide fragment of bradykinin (BK), des-Arg 9-bradykinin, in order to perform a structure-activity study of this peptide on the newly discovered B1 receptor of bradykinin. The first time, each residue of the octapeptide was replaced by L-alanine to pinpoint biologically important residues. Thereafter, both phenylalanine residues in positions 5 and 8 were substituted by L-tyrosine methyl ether, L-cyclohexylalanine, D-phenylalanine, and L-leucine. This paper describes the synthesis of the analogs by the solid phase method. A Beckman peptide synthesizer was used to assemble the peptides on the resin support. Couplings were performed by the symmetrical anhydribe procedure. After cleavage with liquid HF, the peptides were purified by ion-exchange chromatography on carboxymethyl-cellulose and by gel filtration on Bio-Gel P2 resin. The purity of the octapeptides was then checked by tic, paper electrophoresis, amino acid analysis, and elemental analysis. The new peptides were tested on the rabbit aorta in order to evaluate their kinin-like activities and to see if they act as antagonist. The results of the biological assays are discussed in terms of structure-activity relationships.     

Preparation of Recombinant Carbohydrate Deficient Active Analogs of Human Chorionic Gonadotropin from Insect Cells

Abstract

Human chorionic gonadotropin (hCG) has four N-glycosyl chains, two in each subunit. Several analogs lacking one or more specific N-linked carbohydrate chains have been purified from insect cells by immunoaffinity chromatography on a monoclonal antibody, B17, column Traces of the hCGβ mutant present, if any, were removed by a second immunoaffinity chromatography on a column of hCGβ specific monoclonal antibody, B158. N-glycosylation was inhibited by the replacement of either Asn or Thr to Gln in the consensus sequence, -Asn × Ser/Thr-, for N-glycosylation. All analogs were overexpressed in High-Five insect cells with the expression levels ranging between 1.5 to 15 μg/ml and were found homogeneous by SDS-PAGE under nonreducing and reducing conditions. Their molecular sizes ranged between 34k to 44k. The receptor binding affinity of all the analogs was unaltered as determined by radio receptor assay using rat ovarian membranes. The availability of these analogs should facilitate studies on the effect of a specific carbohydrate chain on the conformation and in vivo properties of hCG.     

Degradation of cholecystokinin octapeptide,related fragments and analogs by human and rat plasma in vitro

The rate of degradation of cholecystokinin octapeptide, related fragments and analogs by human and rat plasma was investigated, using high pressure liquid chromatography for the separation and identification of the degradation products.CCK tetrapeptide showed a half-life of 13 min in human plasma while its cleavage in rat plasma occurred at a very high rate (half-life of less than 1 min).The kinetics of disappearance of both sulphated and unsulphated CCK-8 indicated more than a single rate of degradation; the faster degrading system showed a half-life of 18 min for unsulphated CCK-8 and of 50 min for the sulphated peptide in human plasma as compared respectively with 5 and 17 min in rat plasma. The cleavage of CCK-8 was inhibited by bestatin and puromycin, suggesting that aminopeptidases play a major role in the breakdown of this peptide.CCK-9 analogs were rapidly converted into their corresponding octapeptides (half-life of 2.7 min in human plasma). This conversion was inhibited by puromycin and bestatin, suggesting the participation of aminopeptidase(s) probably specific for basic amino acids.CCK decapeptide exhibited a greater stability than the nonapeptides (half-life of 30 and 45 min in human and rat plasma respectively) and also gave rise to CCK-8 as degradation product. This cleavage was not affected by aminopeptidase inhibitors but was decreased by aprotinin (Trasylol®), suggesting that trypsin-like and/or kallikrein-like enzyme(s) were involved in the plasma metabolism of this peptide.     

Synthesis and biological activity of GnRH antagonists modified at position 3 with 3-(2-methoxy-5-pyridyl)-alanine.

Degarelix is a potent very long-acting GnRH antagonist after subcutaneous administration. In this paper, we describe the synthesis of two analogs of degarelix incorporating racemic 3-(2-methoxy-5-pyridyl)-alanine (2-OMe-5Pal, 5) at position 3. The two diastereomers were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) and the absolute stereochemistry at position 3 in the peptides was determined by enzymatic digestion with proteinase K. These analogs were tested in vitro for their ability to antagonize the GnRH receptor and in vivo for duration of action in a castrated male rat assay. Analog 7 with D2-OMe-5Pal was potent in vitro (IC50 = 5.22 nM); however, analog 8 with L2-OMe-5Pal at position 3 in degarelix lost potency as an antagonist of the human GnRH receptor (IC50 = 36.95 nM). Both the analogs were found to be short-acting in vivo.     

Diastereoselective synthesis, binding affinity for vitamin D receptor, and chiral stationary phase chromatography of hydroxy analogs of 1,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol.

A series of analogs of 1,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol were obtained with an additional hydroxyl in the aliphatic side chain at carbon atom C-24. These analogs were synthesized by direct and diastereo-selective alpha-hydroxylation of enolates derived from respective vitamin D esters using Davies chiral oxaziridines. The use of (+)-(2R,8aS)-(8, 8-dichlorocamphoryl)sulfonyl oxaziridine resulted in (R) stereochemistry of the new asymmetric center for both series of analogs. Similarly, (-)-(2S,8aR) oxaziridine gave (S) analogs. The diastereomeric purity of hydroxy analogs was determined by high-performance liquid chromatography on a chiral stationary phase. High diastereopurity of hydroxylation of vitamin D esters was obtained without the use of any chiral auxiliary. The binding affinity of (24R)-1,24,25-trihydroxycholecalciferol for the calf thymus intracellular vitamin D receptor was one order of magnitude higher than that of the respective (24S)-diastereomer.     

Metabolism of prostaglndin A. II. Isolation, characterization, and synthesis of PGA1 renal.

Since the renal cortex has recently been shown to be a major site of prostaglandin A₁ (PGA₁) metabolism, studies were undertaken to isolate and characterize the major metabolites. Homogenates of rabbit cortex (500g) were incubated with ³H-PGA₁ (50mg) in the presence of NAD⁺ (50mg). Acidic lipid extracts were subjected to linear gradient silicic acid chromatography. Six radioactive peaks were recovered, of which peak 4 was unconverted PGA₁. The major metabolites (1,3) were further subjected to reversed phase partition chromatography and TLC with and without silver nitrate. Three PGA₁ analogs were then synthesized via oxidation of the secondary alcohol group at C-15 by manganese dioxide (15-keto-PGA₁). The second compound was synthesized by hydrogenation of 15-keto-PGA₁ (15-keto 13, 14-dihydro PGA₁). The third compound (13, 14-dihydro PGA₁) was obtained by direct catalytic hydrogenation of PGA₁. Purification of these substances were achieved by a combination of silicic acid and thin layer chromatography. It was found that metabolite 1 cochromatographed on TLC (AgNO3) with synthesized 15-keto 13, 14-dihydro PGA₁. Both compounds were 100 times less potent than PGA₁ in lowering rat blood pressure. Metabolite 3 cochromatographed on TLC (AgNO3) with synthesized 13, 14-dihydro PGA₁. Both were as potent as PGA₁ in lowering rat blood pressure. Metabolites 1 and 3 absorbed UV at 221 nm but not at 280 nm following alkali treatment. These studies suggest that rabbit renal cortex metabolizes PGA₁ to what appears to be biologically active 13, 14-dihydro PGA₁ and biologically inactive 15-keto 13, 14-dihydro PGA₁. It remains possible that the hypotensive effect of PGA₁ is the result of its conversion to its biologically active 13, 14-dihydro derivative.     

Antagonists of angiotensin II containing N-methyl-L-alanine and DL-nipecotic acid in position 7.

[1-sarcosine, 7-N-methyl-L-alanine, 8-isoleucine]-Angiotensin II and [1-sarcosine, 7-DL-nipecotic acid, 8-isoleucine]-angiotensin II were synthesized by the solid-phase method and purified by cation-exchange chromatography and high-pressure liquid chromatography. In the isolated rat uterus these analogs and less than 0.1% of the myotropic activity of angiotensin II and inhibited angiotensin II with pA2 values of 8.2 and 7.8, respectively. In the rat pressor assay (vagotomized ganglion blocked rat) these analogs had 0.9 and 2.8%, respectively, of the pressor activity of angiotensin II. The results show that the proline residue in position 7 of [Sar1,Ile8]-angiotensin II may be replaced by other secondary amino acids without disrupting interactions at angiotensin II receptors.     

Purification and chemical studies on rat urinary kallikrein.

A highly purified kallikrein was obtained from rat urine by chromatography on DE-32 cellulose, affinity chromatography on Bio-gel P-200-Aprotinin and gel filtration over Sephadex G-100 coarse and superfine. A molecular weight of 32,000 by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis was estimated. The aminoacid composition and the esterase activity of the purified material were determined. Biological characterization of the purified kallikrein was tested by liberation of a kinin from rat plasma kininogen, by direct action on the isolated rat uterus and by the lowering of rat arterial pressure after intravenous injection of the enzyme. The preparation of insoluble derivative of Aprotinin is described herein. The polymer used as insoluble support (Bio-gel P-200) was before changed to its corresponding azide, which reacts with Aprotinin; the product maintained the binding property of the Aprotinin with urinary kallikrein.     

Animal and cellular models of Gaucher's disease. I. Refractoriness of thio analogs of glucocerebroside to enzymatic hydrolysis

In order to develop a nonmetabolizable analog of glucocerebroside to investigate the distribution and accumulation of this lipid in model systems, thiohemiacetal derivatives were synthesized and their susceptibility to enzymatic hydrolysis by purified human placental glucocerebrosidase was examined. Sulfur analogs were found to be completely refractory to the activity of this enzyme, indicating their potential use in animal and isolated cell models and possibly for the preparation of affinity chromatography columns for the isolation of glucocerebrosidase.     

Glucagon antagonists. Synthesis and inhibitory properties of Asp3-containing glucagon analogs

In an effort to find analogs of glucagon that would bind to the glucagon receptor of the rat liver membrane but would not activate membrane-bound adenyl cyclase, several hybrid molecules were synthesized which contained sequences from both glucagon and secretin. [Asp3, Glu9]Glucagon and [Asp3, Glu9, Arg12]glucagon were inactive in the adenyl cyclase assay even at high concentrations but retained some binding affinity for the receptor. They were able to displace 125I-glucagon completely from its receptor and could completely inhibit the activation of adenyl cyclase by natural or synthetic glucagon. The inhibition index [I/A]50 was approximately 110 for both analogs. [Asp3]Glucagon, [Glu3]glucagon and [Asp3, Lys17, 18, Glu21]glucagon were weak partial agonists, while [Asp3, Glu21]glucagon was inactive and a poor inhibitor. The peptides were synthesized by solid-phase methods and purified to homogeneity by reverse-phase high-performance liquid chromatography on C18 silica columns. These are the first fully synthetic competitive glucagon antagonists to be reported.     

N-α-biotinylated-neuropeptide Y analogs: Syntheses, cardiovascular properties, and application to cardiac NPY receptor visualization

Two monobiotinylated analogs of neuropeptide Y (NPY) were synthesized by coupling the N-hydroxysuccinimidyl esters of biotin and (6-biotinylamido)-hexanoic acid, respectively, to the free alpha-NH2 group of the side chain protected NPY peptide resin. Crude peptides obtained by HF cleavage were purified by RPLC and their integrities were confirmed by amino acid and mass spectral analysis. As with NPY, both biotinylated analogs inhibited 125I-NPY binding and adenylate cyclase activity of rat cardiac ventricular membranes in a dose-dependent manner. N-alpha-[(6-biotinylamido)-hexanoyl]-NPY exhibited potencies comparable to that of NPY whereas N-alpha-biotinyl-NPY was slightly less potent. In the in vivo experiments, however, both the biotinylated analogs exhibited responses comparable to NPY in increasing arterial blood pressure and decreasing heart rate in anesthetized rats. The responses of the biotinyl analogs were longer lasting than those of NPY. Histochemical studies revealed that N-alpha-[(6-biotinylamido)-hexanoyl]-NPY could label the NPY receptors in rat cardiac ventricular tissues. This labeling was specific since intact NPY inhibited the staining. These studies show that biotinyl-NPY analogs exhibit biological potencies comparable to intact NPY and can therefore be used to further probe the NPY-receptor interaction.     

Characterization of immunoreactive motilin from the rat small intestine.

Immunocytochemistry, radioimmunoassay, chromatography, and biological assay using a rabbit isolated duodenal muscle strip preparation were used in attempting to characterize motilin from the rat small intestine. Several different antisera and monoclonal antibodies directed against natural porcine motilin were used. A variety of fixation techniques using Bouin's, paraformaldehyde, and benzoquinone with different staining methods including, fluorescein-conjugated second antibody, peroxidase-antiperoxidase or peroxidase-conjugated second antibody techniques were used. All methods failed to detect immunoreactive motilin cells in the rat small intestine. The same antisera were used in radioimmunoassays for motilin to evaluate extracts of rat intestinal tissue. Two of these detected immunoreactive motilin in gut extracts, and these antisera showed a different distribution for the peptide. Samples containing immunoreactive motilin obtained from cation exchange chromatography on SP-Sephadex-G25 were concentrated and assayed for biological activity in a rabbit duodenal muscle strip preparation. Desensitization of duodenal tissue to porcine motilin could be demonstrated by pretreatment with this peptide. The biological activity of partially purified rat intestinal immunoreactive motilin was not prevented by pretreatment of the tissue with motilin. Further purification of this preparation on Bio-Gel P-10 yielded an immunoreactive motilin peak that co-eluted with natural porcine motilin. Rat intestinal immunoreactive motilin did not co-elute with natural porcine motilin following high pressure liquid chromatography on a Waters microBondapak C18 reversed-phase column using a linear gradient of water-acetonitrile (10-45%) over 30 min. Although of similar molecular size, rat motilin is probably structurally dissimilar to other mammalian motilins.     

Active site sequence of hepatic fructose-2,6-bisphosphatase. Homology in primary structure with phosphoglycerate mutase

The reaction mechanism of rat hepatic fructose-2,6-bisphosphatase involves the formation of a phosphohistidine intermediate. In order to determine the sequence around the active site histidine, the enzyme was incubated with [2-32P]fructose 2,6-bisphosphate, denatured, and treated with trypsin or endoproteinase Lys-C. The resultant labeled 32P-phosphopeptides were purified by gel filtration, anion exchange chromatography, and reverse phase high pressure liquid chromatography. The sequence of the tryptic peptide was determined to be HGESELNLR, while the partial sequence of the endoproteinase Lys-C peptide was IFDVGTRYMVNRVQDHVQSRTAYYLMNIHVTPRSIYLRHGESEL. The active site sequence was compared with the active site sequence of other enzymes that catalyze phospho group transfer via a phosphohistidine intermediate. Active site sequences of phosphoglycerate mutase and bisphosphoglycerate synthase were highly homologous with the active site of fructose-2,6-bisphosphatase implying a structural similarity and a common evolutionary origin.     

gamma-Glutamylcysteine synthetase. Further purification, "half of the sites" reactivity, subunits, and specificity.

gamma-Glutamylcysteine synthetase was purified from rat liver by an improved method involving chromatography on Sepharose-aminohexyl-ATP to a specific activity of about 1600 units/mg, or approximately twice that previously obtained; it is thus the most active preparation of this enzyme thus far isolated. The earlier preparation, which is homogeneous on polyacrylamide gel electrophoresis, exhibits "half of the sites" reactivity in that it binds a maximum of 0.5 mol of the inhibitor L-methionine-S-sulfoximine phosphate per mol of enzyme. In contrast, the present enzyme preparation binds 1 mol of methionine sulfoximine phosphate per mol of enzyme; it also differs from the enzyme obtained earlier in exhibiting much less ATPase activity and less activity in catalyzing ATP-dependent cyclization of glutamate. gamma-Glutamylcysteine synthetase dissociates in sodium dodecyl sulfate into two nonidentical subunits of apparent molecular weights 74,000 and 24,000; after cross-linking with dimethyl-suberimidate, a species having a molecular weight of about 100,000 was found on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. New information has been obtained about the interaction of the enzyme with glutamate analogs; thus, the enzyme is active with such glutamate analogs as beta-glutamate, N-methyl-L-glutamate, and threo-beta-hydroxy-L-glutanate, and it is effectively inhibited by cis-1-amino-1,3-dicarboxycyclonexane, 2-amino-4-phosphonobutyrate, and gamma-methylglutamate.     

Liquid chromatographic-atmospheric pressure chemical ionization mass spectrometric determination of anandamide and its analogs in rat brain and peripheral tissues

A simple and selective method for the determination of anandamide (arachidonoylethanolamide), an endogenous cannabinoid receptor ligand, and its analogs with liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS) was developed. The calibration curve for standard anandamide was linear over the range 625 fmol-125 pmol per injection (r=0.999) with a precision of 1.0% (C.V.) at 25 pmol. The detection limit attained was 200 fmol per injection at a signal-to-noise ration of 2. Anandamide and its analogs were extracted from rat brain and peripheral tissues according to the method of Folch, and the recovery of anandamide from rat brain homogenates was 67.0–72.6%. The method was applied to their determination in rat brain and peripheral tissues.     

alpha-Neo-endorphin: receptor binding properties of the tritiated ligand

Tritiated porcine alpha-neo-endorphin has been prepared from its corresponding iodinated analog. The iodinated analog (diiodotyrosine at position 1) was synthesized, along with its non-iodinated counterpart, by the solid-phase method. Catalytic exchange of this iodinated analog in the presence of tritium yielded tritiated porcine alpha-neo-endorphin having a specific activity of 45.5 Ci/mmole. Both the native, iodinated and tritiated alpha-neo-endorphin analogs were shown to be homogenous by chromatography on carboxymethylcellulose, paper chromatography, paper electrophoresis, high performance liquid chromatography and amino acid analysis. For the first time binding of alpha-neo-endorphin to rat membrane preparations is described using [3H2-Tyr1]alpha-neo-endorphin as the ligand. The binding is time-dependent and saturable with respect to alpha-neo-endorphin. Scatchard analysis was bi-phasic with KDs of 0.20 and 3.75 nM. Displacement binding studies indicate that the receptor for alpha-neo-endorphin has "kappa" and possibly "epsilon" binding characteristics.     

Putative inositol 1,4,5-trisphosphate binding proteins in rat brain cytosol.

In previous works, we synthesized a series of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) analogs, with a substituent on the second carbon of the inositol ring. Using these analogs, the Ins(1,4,5)P3 affinity media were also synthesized (Hirata, M., Watanabe, Y., Ishimatsu, T., Yanaga, F., Koga, T., and Ozaki, S. (1990) Biochem. Biophys. Res. Commun. 168, 379-386). When the cytosol fraction from the rat brain was applied to an Ins(1,4,5)P3 affinity column, an eluate with a 2 M NaCl solution was found to have remarkable Ins(1,4,5)P3-binding activity. The active fraction was further fractionated with gel filtration chromatography, and two proteins with an apparent molecular mass of 130 or 85 kDa were found to be Ins(1,4,5)P3-binding proteins but with no Ins(1,4,5)P3 metabolizing activities. Partial amino acid sequences determined after proteolysis and reversed-phase chromatography revealed that the protein with an apparent molecular mass of 85 kDa is the delta-isozyme of phospholipase C and that of 130 kDa has no sequence the same as the Ins(1,4,5)P3-recognizing proteins hitherto examined. Ins(1,4,5)P3 at concentrations greater than 1 microM strongly inhibited 85-kDa phospholipase C delta activity, without changing its dependence on the concentrations of free Ca2+ and H+. Among inositol phosphates examined, Ins(3,4,5,6)P4 inhibited the binding of [3H]Ins(1,4,5)P3 to the 130-kDa protein at much the same concentrations as seen with Ins(1,4,5)P3. This report seems to be the first evidence for the presence of soluble Ins(1,4,5)P3-binding proteins in the rat brain, one of which is the delta isozyme of phospholipase C.     

Solubilization and Partial Purification of α-Amino-3- Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites from Rat Brain

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) binding sites were solubilized from rat brain membranes using 1% Triton X-100 in 0.5 M potassium phosphate buffer containing 20% glycerol. The solubilized binding sites were stable, permitting biochemical and pharmacological characterization as well as partial purification. Pharmacological and binding analyses indicated that the solubilized binding sites were similar to the membrane-bound sites. Both the solubilized and the membrane-bound preparations contained high- and low-affinity AMPA binding sites in the presence of potassium thiocyanate. A similar rank order for inhibition of [3H]AMPA binding by several excitatory amino acid analogs was obtained for the soluble and membrane-bound preparations. [3H]AMPA binding to both soluble and membrane-bound preparations was increased in the presence of potassium thiocyanate. The solubilized AMPA binding sites migrated as a single peak with gel filtration chromatography, with an Mr of 425,000. Beginning with the solubilized preparation, AMPA binding sites were purified 54-fold with ion-exchange chromatography and gel filtration. The characterization and purification of these soluble binding sites is potentially useful for the molecular characterization of this putative excitatory amino acid receptor subtype.