In vitro stability of growth hormone releasing factor (GRF) analogs in porcine plasma.

The stability of growth-hormone releasing factor (growth regulating factor; GRF) analogs in porcine plasma was examined. GRF analogs were incubated in porcine plasma at 37 degrees C, extracted and subsequently analyzed using high performance liquid chromatography (HPLC). GRF(1-29)-NH2 was rapidly broken down in the plasma with a degradation rate of t1/2 = 13 min. The primary degradation product was identified as GRF(3-29)-NH2. Substitution of Gly15 by Ala15 slightly prolonged the plasma half-life (t1/2 = 17 min) and the major degradative fragment was found to be [Ala15]GRF(3-29)-NH2. The cleavage between the 2 and 3 position of the peptide was not inhibited by trasylol at a concentration of 1,000 KIU/ml but was dramatically reduced by the combined use of diprotin A and trasylol. Absence of the free amino group at the N-terminus and/or substitution of a D-amino acid residue at the penultimate position completely prevented cleavage between the 2 and 3 position in the structural linear GRF analogs. Side-chain to side-chain cyclization between Asp8 and Lys12 amino acid residues significantly improved the stability of GRF in plasma with t1/2 greater than 2 hr. An additional stability was provided by substitution of D-Ala2 for Ala2 in the structural cyclic analog. Cyclization between Lys21 and Asp25 also improved the stability of the GRF peptide in the plasma. Stability was further enhanced by the presence of D-Ala2 or by forming a dicyclic analog through an additional linkage between Asp8 and Lys12.     

Degradation of growth hormone releasing factor analogs in neutral aqueous solution is related to deamidation of asparagine residues. Replacement of asparagine residues by serine stabilizes

The incubation of a solution of the human growth hormone releasing factor analog, [Leu27] hGRF(1-32)NH2 at pH 7.4 and 37 degrees, resulted in extensive degradation of the sample. The major degradation products were identified as the peptides [beta-Asp8, Leu27] hGRF(1-32)NH2 and [alpha-Asp8, Leu27] hGRF(1-32)NH2, produced by deamidation of the Asn8 residue. When tested as growth hormone (GH) secretagogues in cultured bovine anterior pituitary cells, [beta-Asp8, Leu27] hGRF(1-32)NH2 was estimated to be 400-500 times less potent than the parent Asn8 peptide, while [alpha-Asp8, Leu27] hGRF(1-32)NH2 was calculated to be 25 times less potent than the parent Asn8 peptide. Three additional analogs of [Leu27] hGRF(1-32)NH2 containing either Ser or Asn at positions 8 and 28 were prepared and evaluated for their GH releasing activity and stability in aqueous phosphate buffer (pH 7.4, 37 degrees). Based on disappearance kinetics, [Leu27] hGRF(1-32)NH2 had a half-life of 202 h while the other analogs had the following half-lives: [Leu27, Asn28] hGRF(1-32)NH2 (150 h); [Ser8, Leu27, Asn28] hGRF(1-32)NH2 (746 h); and [Ser8, Leu27] hGRF(1-32)NH2 (1550 h). After 14 days, incubated samples of the Asn8 analogs lost GH releasing potency, while the Ser8 analogs retained full potency. The potential for loss of biological activity brought about by deamidation of other engineered peptides and proteins should be considered in their design.     

Kinetic and thermodynamic control of the relative yield of the deamidation of asparagine and isomerization of aspartic acid residues.

Selective deamidation of Asn67 of RNase A to beta-Asp67 and Asp67 residues at neutral pH initially produces greater amounts of the beta-Asp derivative. As the reaction proceeds the relative concentration of [Asp67]-RNase A increases and, at equilibrium, becomes predominant. Such a discrepancy between the kinetic and thermodynamic control on reaction products is discussed in light of information from X-ray three-dimensional analysis and the lower thermodynamic stability of the beta-Asp derivative relative to the parent enzyme.     

Synthesis, biological activity and conformational analysis of cyclic GRF analogs

A novel cyclic GRF analog, cyclo(Asp8-Lys12)-[Asp8,Ala15]-GRF(1-29)-NH2, i.e. cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2, was synthesized by the solid phase procedure and found to retain significant biological activity. Solid phase cyclization of Asp8 to Lys12 proceeded rapidly (approximately 2 h) using the BOP reagent. Substitution of Ala2 with D-Ala2 and/or NH2-terminal replacement (desNH2-Tyr1 or N-MeTyr1) in the cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 system resulted in highly potent analogs that were also active in vivo. Conformational analysis (circular dichroism and molecular dynamics calculations based on NOE-derived distance constraints) demonstrated that cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 contains a long alpha-helical segment even in aqueous solution. A series of cyclo8,12 stereoisomers containing D-Asp8 and/or D-Lys12 were prepared and also found to be highly potent and to retain significant alpha-helical conformation. The high biological activity of cyclo8,12[N-MeTyr1,D-Ala2,Asp8,Ala15]-GRF(1-29)- NH2 may be explained on the basis of retention of a preferred bioactive conformation.     

Kinetics of dipeptidyl peptidase IV proteolysis of growth hormone-releasing factor and analogs.

The kinetics and selectivity of proteolysis of synthetic human growth hormone-releasing factor and analogs by purified human placental dipeptidyl peptidase IV (DPP IV) were studied by HPLC. The initial rates of Ala2-Asp3 cleavage (pH 7.8, 37 degrees C, So = 0.15 mM) were all approx. 5 mumol min-1 mg-1 for the parent hormone, GRF(1-44)-NH2, and the fragments, GRF(1-29)-NH2 and GRF(1-20)-NH2. Lower activities observed for GRF(1-11)-OH, GRF(1-3)-OH, and cyclic lactam analogs indicate S1'-Sn' binding. Assays of [Trp6]-GRF(1-29)-NH2 versus [D-Trp6]-GFR(1-29)-NH2 indicate an S4' binding cavity. Peptides with D-configuration at P2, P1 or P1' and desNH2Tyr1 and N-MeTyr1 analogs of GRF were not cleaved. Catalytic parameters for the P1-substituted analogs [X2,Ala15]-GRF(1-29)-NH2 were found to vary with X as follows, Km: Abu less than Ala less than Pro less than Val less than Ser less than Gly much less than Leu; kcat: Pro greater than Ala greater than Abu greater than Ser greater than Gly much greater than Leu greater than Val; kcat/Km: Abu greater than Pro greater than Ala much greater than Ser greater than Gly = Val much greater than Leu. Km is at a minimum and kcat/Km at a maximum, for a hydrophobic P1 side-chain of about 0.25 nm in length, i.e., the ethyl side-chain of alpha-aminobutyric acid (Abu) is very close to optimal. These results further define the S1 selectivity of DPP IV and may be useful in the design of DPP IV resistant GRF analogs that can be produced by recombinant DNA methods and the design of DPP IV inhibitors.     

Semisynthesis of human growth hormone-releasing factor by trypsin catalyzed coupling of leucine amide to a C-terminal acid precursor.

Human growth hormone-releasing factor, GRF(1-44)-NH2, was synthesized by trypsin catalyzed coupling of Leu-NH2 to Arg43 of the precursor, GRF(1-43)-OH, prepared by solid phase peptide synthesis. The semisynthetic GRF(1-44)-NH2 was fully characterized and showed full potency in the rat pituitary in vitro bioassay. Conversion to GRF(1-44)-NH2 was limited to 60-70% in both 75% v:v N,N'-dimethylacetamide and 95% v:v 1,4-butanediol due to competing transpeptidations at Arg41 and Arg38 generating [Leu42]-GRF(1-42)-NH2 and [Leu39]-GRF(1-39)-NH2 side-products, respectively. The rates of formation and yields of GRF(1-44)-NH2 versus pH, Leu-NH2 concentration, and solvent composition were also studied.     

Synthesis and biological activity of novel C-terminal-extended and biotinylated growth hormone-releasing factor (GRF) analogs.

A series of novel hGRF(1-29)-NH2 analogs were synthesized and biotinylated. The immunological and biological activities of these analogs were then characterized. To distance the biotin moiety from the putative bioactive core, a C-terminal spacer arm consisting of -Gly-Gly-Cys-NH2 (-GGC) was added to hGRF(1-29)-NH2 (hGRF29) and analogs, with subsequent biotinylation performed at the cysteine residue. Neither addition of the C-terminal spacer arm nor biotinylation affected affinity of these analogs for GRF antibody. Relative to hGRF(1-44)-NH2 (hGRF44: potency = 1.0), the biotinylated analogs were equipotent in vitro to their nonbiotinylated, parent compounds: [desNH2Tyr1,D-Ala2,Ala15]hGRF29-GGC-(tpBiocyt in)-NH2 (4.7) = [Ala15]hGRF29-GGC-(tpBiocytin)-NH2 (3.9) greater than hGRF29-GGC-(tpBiocytin)-NH2 (0.8). Based upon cumulative GH release data in vivo (0-60 min postinjection), [desNH2Tyr1,D-Ala2,Ala15]hGRF29-GGC-(tpBiocyt in)-NH2, [Ala15]hGRF29-GGC-(tpBiocytin)-NH2, and hGRF29-GGC-(tpBiocytin)-NH2 displayed 8.6, 5.5, and 0.8 times, respectively, the potency of hGRF44. These in vivo potency values were not significantly different from the corresponding parent compounds (i.e., with or without the C-terminal spacer arm). In summary, biotinylated hGRF analogs have been developed that retain full immunoreactivity and potent bioactivity (in vitro and in vivo), thus permitting their use in GRF receptor isolation, ELISA, and histochemical procedures.     

Mechanisms of glucagon degradation at alkaline pH

Glucagon is unstable and undergoes degradation and aggregation in aqueous solution. For this reason, its use in portable pumps for closed loop management of diabetes is limited to very short periods. In this study, we sought to identify the degradation mechanisms and the bioactivity of specific degradation products. We studied degradation in the alkaline range, a range at which aggregation is minimized. Native glucagon and analogs identical to glucagon degradation products were synthesized. To quantify biological activity in glucagon and in the degradation peptides, a protein kinase A-based bioassay was used. Aged, fresh, and modified peptides were analyzed by liquid chromatography with mass spectrometry (LCMS). Oxidation of glucagon at the Met residue was common but did not reduce bioactivity. Deamidation and isomerization were also common and were more prevalent at pH 10 than 9. The biological effects of deamidation and isomerization were unpredictable; deamidation at some sites did not reduce bioactivity. Deamidation of Gln 3, isomerization of Asp 9, and deamidation with isomerization at Asn 28 all caused marked potency loss. Studies with molecular-weight-cutoff membranes and LCMS revealed much greater fibrillation at pH 9 than 10. Further work is necessary to determine formulations of glucagon that minimize degradation and fibrillation.     

Identification of deamidation and isomerization sites on pharmaceutical recombinant antibody using H(2)(18)O

Asparagine (Asn) deamidation and aspartic acid (Asp) isomerization are spontaneous and common alterations occurring in pharmaceutical protein drugs in solution. Because those reactions may cause functional changes, it is important to identify the product-related substances, especially when biopharmaceuticals are under development. In this study, we used H(2)(18)O to identify Asn deamidation and Asp isomerization sites on a recombinant humanized monoclonal antibody (mAb) by using high-performance liquid chromatography-mass spectrometry (HPLC-MS). This strategy takes advantage of reactions whereby (18)O is incorporated into the protein molecule. The mAb was lyophilized and reconstituted in H(2)O or H(2)(18)O, followed by incubation at 50 degrees C for 1 month. Samples were reduced/carboxymethylated and digested by trypsin and then subjected to HPLC-MS and HPLC-tandem mass spectrometry (MS/MS) analysis. Among all of the peptide fragments analyzed, there were two in which deamidation and/or isomerization was observed. In one peptide fragment, an obvious mass shift ( approximately 3Da) at Asn was observed in the newly produced peptide when the mAb was incubated in H(2)(18)O, whereas it was barely feasible to identify this mass shift in H(2)O. In the other peptide fragment, isomerization of Asp was identified after incubation in H(2)(18)O, although it was impossible to distinguish when using H(2)O. By means of this procedure, identification of deamidation and isomerization sites can be accomplished easily even when they are difficult or impossible to detect by the usual peptide mapping.     

The pH dependence of the conformation of angiotensin peptides by nuclear magnetic resonance: cis-trans isomerism of proline 7.

The pH dependence of the proton NMR spectrum of [Asn1, Val5] angiotensin II in aqueous solution shows the existence of one major and one minor conformation above pH 6.5, the minor conformation representing 12 +/- 2% of the total peptide. A similar observation has been made for (Asn1, Val5) angiotensin I and Val-Tyr-Val-His-Pro-Phe. This effect is not due to the presence of angiotensin-like impurities in the peptide samples. We have shown two expected impurities, [beta-Asp1, Val5] angiotensin II and [Asn1, 3-Bzl-Ty4, Val5] - angiotensin II, to be absent, and a third impurity [Asn1, Val5, D-His6] angiostensin II, to be present at less than or equal to 2.1 mol%, too little to account for the observed amount (12 +/- 2%) of minor conformation. The carbon-13 spectrum of the hexapeptide at high pH shows that the major conformation has Pro7 in the trans form and the minor conformation has Pro7 in the cis form.     

Potent trypsin-resistant hGH-RH analogues.

A series of analogues of hGH-RH-(1-29)-NH2 designed to have metabolic stability has been synthesized. Standard Boc-SPPS was employed, modified to permit the guanidinylation of amino side-chains after chain assembly but before release from the resin. [Dat1, Har(11, 12, 20, 21, 29), Ala15, Nle27, Asp28]-, [Dat1, Har(11, 20, 29), Orn12, Ala15, Nle27, Asp28]-, and [Dat1, Gap(11,12, 21, 29), Ala15, Har20, Nle27, Asp28]-hGH-RH-(1-29)-NH2 were completely resistant to trypsin and about 50 times as potent as hGH-RH-(1-29)-NH2 itself when injected subcutaneously in rats. These peptides are candidates for clinical application in the therapy of GH deficiency.     

Effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides

The nonenzymatic rates of deamidation of Asn residues in a series of pentapeptides with the sequences VSNXV and VXNSV, where X is one of 10 different amino acids, were determined at neutral, alkaline, and acid pH values. The results demonstrate that in neutral and alkaline solutions the amino acid residue on the amino side of the Asn had little or no effect on the rate of deamidation regardless of its charge or size. The group on the carboxyl side of Asn affected the rate of deamidation significantly. Increasing size and branching in the side chain of this residue decreased the rate of deamidation by as much as 70-fold compared to glycine in the N-G sequence, which had the greatest rate of deamidation. In acidic solution, the rate of deamidation of the Asn residue was not affected by the amino acid sequence of the peptide. The products for each deamidation reaction were tested for the formation of isoAsp residues. In neutral and alkaline solutions, all products showed that the isoAsp:Asp peptide products were formed in about a 3:1 ratio. In acidic solution, the Asp peptide was the only deamidation product formed. All peptides in which a Ser residue follows the Asn residue were found to undergo a peptide cleavage reaction in neutral and alkaline solutions, yielding a tripeptide and a dipeptide. The rate of the cleavage reaction was about 10% of the rate of the deamidation pathway at neutral and alkaline pH values. The rates of deamidation of Asn residues in the peptides studied were not affected by ionic strength, and were not specific base catalyzed. General base catalysis was observed for small bases like ammonia. A model for the deamidation reaction is proposed to account for the observed effects.     

Response of growth hormone release to human growth hormone-releasing factor and its analogs in the bovine

Responses of growth hormone (GH) release to synthetic human growth hormone-releasing factor (hGRF)-44-NH2 analogs were determined, and the GH-releasing potency based on dose per kg of body weight (bw) was compared with that of hGRF-44-NH2 in female dairy calves. Four- and 12-month-old calves were injected intravenously with 0.25 microgram of hGRF-44-NH2 or its analogs per kg of bw. Blood samples were collected before, and during 180 min after each injection, and plasma GH concentrations were measured by radioimmunoassay. Areas under the GH response curves for 180 min after injection of hGRF-44-NH2 and its analogs were used as an index of the GH-releasing potency of each peptide. The GH-releasing potency of hGRF(1-26)-NH2 was significantly lower than that of hGRF-44-NH2 (P less than 0.05). On the other hand, hGRF(1-29)-NH2 possessed similar potency to hGRF-44-NH2. [D-Tyr1]-hGRF-44-NH2 showed prolonged GH-releasing activity, though its potency was similar to that of hGRF-44-NH2. Also, [D-Ala2]-hGRF(1-29)-NH2 exhibited prolonged GH-releasing activity, and its potency was 2.5 (P less than 0.05) and twice (P less than 0.05) as great as that of hGRF-44-NH2 and hGRF(1-29)-NH2, respectively. These results demonstrate that the N-terminal 29 amino acid residues of hGRF possess the activity site required for full GH release in vivo, and [D-Ala2]-hGRF(1-29)-NH2 has longer and greater activity, on a dose basis, than hGRF-44-NH2 in the calves.     

The influence of albumin on vitamin D metabolism in fetal chick osteoblast-like cells

Human pancreatic growth hormone releasing factor (1-29)-amide [hpGRF (1-29)-NH2] and the following analogs: [D-Tyr-1]-hpGRF(1-29)-NH2, [D-Ala-2]-hpGRF(1-29)-NH2, [D-Asp-3]-hpGRF(1-29)-NH2, and [N-Ac-Tyr-1]-hpGRF (1-29)-NH2 were synthesized using solid phase methodology and tested for their ability to stimulate growth hormone (GH) secretion in the rat and the pig in vivo. [D-Ala-2]-hpGRF (1-29)-NH2 was approximately 50 times more potent than the parent molecule in eliciting GH secretion in the rat. The other analogs were less active, but all were more potent than the 1-29 amide in the rat. [D-Tyr-1]-hpGRF(1-29)-NH2 was 10 times more potent, [D-Asp-3]-hpGRF(1-29)-NH2 7 times more potent, and the acetylated molecule approximately 12 times more potent than hpGRF(1-29)-NH2.     

Tryptic hydrolysis of hGH-RH(1-29)-NH2 analogues containing Lys or Orn in positions 12 and 21.

Two analogues of the 29 amino acid sequence of human growth hormone-releasing hormone, namely [Nle27]hGH-RH(1-29)-NH2 and [Orn(12,21),Nle27]hGH-RH(1-29)-NH2, have been synthesized and subjected to digestion by trypsin. The course of degradation was followed using RP-HPLC and ESI-MS. Several intermediates and final products of degradation were identified and conclusions regarding the rate of cleavages at different positions occupied by Lys and Arg residues were drawn. The analogue containing ornithine was found to be less susceptible to hydrolysis by trypsin: the 12-13 and 21-22 peptide bonds were completely resistant to the cleavage. The results show that by replacing Lys with Orn, a possibility exists to design new peptides, which could be more stable in biological fluids.     

Catabolism of rat growth hormone-releasing factor(1-29) amide in rat serum and liver.

Clinical and veterinary uses of growth hormone-releasing factor [GRF(1- 29)NH2] require the design of analogs that are resistant to proteolysis by serum and liver degrading enzymes. This study investigated rat GRF(1-29)NH2 processing in serum and liver homogenate by means of high pressure liquid chromatography (HPLC). Synthetic rGRF(1-29)NH2 (30 microM) was incubated (0-120 min, 37 degrees C) in serum (49 +/- 8 mg prot./ml). The rGRF(1-29)NH2 (10 microM) was also incubated (0-120 min, 37 degrees C) with liver homogenate (200 +/- 6 micrograms prot./ml). Time course studies of rGRF(1-29)NH2 disappearance showed apparent half-lives of 18 +/- 4 min and 13 +/- 3 min in serum and liver homogenate, respectively. This was accompanied by the appearance of degradation products that were all less hydrophobic than the native peptide. In the serum, two major metabolites were detected and isolated by preparative HPLC. Combined results of amino acid analysis, sequencing, and chromatography with synthetic homologs revealed the presence of rGRF(1-20)OH and (3-20)OH. A small amount of rGRF(12-29)NH2, coeluting with rGRF(3-20)OH, was also found by sequencing. In the liver, rGRF(1-18)OH, (3-18)OH, and (1-10)OH were identified. The peptide bond Ala2-Asp3 (DPP IV cleavage site) was hydrolyzed in both serum and liver. Other tissue-specific cleavage sites were Arg11-Arg12 and Arg20-Lys21 (trypsin-like cleavage site) in the serum, and Tyr10-Arg11 and Tyr18-Ala19 (chymotrypsin-like cleavage site) in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deamidation of asparagine residues in a recombinant serine hydroxymethyltransferase

Serine hydroxymethyltransferase purified from rabbit liver cytosol has at least two Asn residues (Asn(5) and Asn(220)) that are 67 and 30% deamidated, respectively. Asn(5) is deamidated equally to Asp and isoAsp, while Asn(220) is deamidated only to isoAsp. To determine the effect of these Asn deamidations on enzyme activity and stability a recombinant rabbit liver cytosolic serine hydroxymethyltransferase was expressed in Escherichia coli over a 5-h period. About 90% of the recombinant enzyme could be isolated with the two Asn residues in a nondeamidated form. Compared with the enzyme isolated from liver the recombinant enzyme had a 35% increase in catalytic activity but exhibited no significant changes in either affinity for substrates or stability. Introduction of Asp residues for either Asn(5) or Asn(220) did not significantly alter activity or stability of the mutant forms. In vitro incubation of the recombinant enzyme at 37 degrees C and pH 7.3 resulted in the rapid deamidation of Asn(5) to both Asp and isoAsp with a t(1/2) of 50-70 h, which is comparable to the rate found with small flexible peptides containing the same sequence. The t(1/2) for deamidation of Asn(220) was at least 200 h. This residue may become deamidated only after some unfolding of the enzyme. The rates for deamidation of Asn(5) and Asn(220) are consistent with the structural environment of the two Asn residues in the native enzyme. There are also at least two additional deamidation events that occur during prolonged incubation of the recombinant enzyme.     

Deamidation and succinimide formation by gamma-N-methylasparagine: potential pitfalls of amino acid analysis

The biological function of the post-translationally methylated amino acid gamma-N-methylasparagine (gamma-NMA) in proteins is unknown. We are examining the premise that amide methylation protects against deamidation. The free amino acids Asn, gamma-NMA, Gln, and delta-N-methylglutamine (delta-NMG) were incubated at elevated temperature and a variety of pH conditions to assay for deamidation. Gln disappears 12- to 14-fold more rapidly than delta-NMG, and Asn hydrolyzes to Asp and NH3 as expected. However, the gamma-NMA deamidation rate is severely overestimated by simply measuring the disappearance of starting material because gamma-NMA undergoes a cyclization reaction in preference to deamidation. At pH 1 the predominant gamma-NMA reaction is formation of stable 3-amino-N-methylsuccinimide (NMS) and this occurs greater than 10-fold faster than Asn deamidation. At pH 4.0, 7.4, and 9.0 NMS is readily formed but it is unstable and partitions between the parent compound, gamma-NMA, and a second species, alpha-N-methylasparagine. At pH 7.4 and 9.0 gamma-NMA disappears 4-fold slower than Asn but the methyl amide hydrolysis rate is diminished by as much as 13-fold. The Asn incubations over the pH range 1-9 yield scant evidence of a succinimide intermediate. It is concluded that the amide methylation provides a unique reaction pathway and stabilization for the N-methylsuccinimide species. Amino acid analysis by o-phthalaldehyde postcolumn reaction fails to detect isoasparagine, alpha-N-methylasparagine, and NMS. Amino acid analysis by precolumn derivatization with phenyl isothiocyanate destroys NMS and therefore cannot quantitate this compound. The ninhydrin postcolumn derivatization method is able to detect and quantitate all of these amino acid species.     

A new type of functional VIP receptor has an affinity for helodermin in human SUP-T1 lymphoblasts

A new type of VIP receptor was characterized in human SUP-T1 lymphoblasts. The order of potency of unlabeled peptides, in the presence of [125I]helodermin, was: helodermin(1-35)-NH2 = helodermin(1-27)-NH2 greater than helospectin greater than VIP = PHI greater than [D-Ser2]VIP greater than [D-Asp3]VIP greater than [D-His1]VIP greater than or equal to [D-Ala4]VIP greater than or equal to secretin = GRF. This specificity was distinct from that of all VIP receptors described so far in that: (i) the affinity for helodermin (Kd = 3 nM) was higher than that of VIP (Kd = 15 nM) and PHI (Kd = 20 nM); and (ii) position 4 played an important role in ligand binding. The labeled sites were likely to be functional receptors as adenylate cyclase in crude lymphoblastic membranes (200-10,000 x g pellets) was stimulated by peptides, in the presence of GTP, with the following order of potency: helodermin(1-35)-NH2 greater than helodermin(1-27)-NH2 greater than helospectin = VIP = PHI.     

Multiple cleavage sites of cholecystokinin heptapeptide by "enkephalinase"

Degradation of Boc CCK7 (Boc Tyr1 (SO3H)-Met2-Gly3-Trp4-Met5-Asp6-Phe7-NH2), a fully active analog of CCK8, by purified rabbit kidney neutral metalloendopeptidase (enkephalinase) was studied as a basis for the rational design of potent peptidases-resistant analogs of cholecystokinin. Characterization of the metabolites was performed by HPLC using several elution procedures. Three cleavage sites were evidenced: one major at the Asp6-Phe7 bond and two minor at Gly3-Trp4 and Trp4-Met5 bonds. All cleavages were fully inhibited by thiorphan, a potent inhibitor of enkephalinase. The relative importance of the different cleavages was established using several cholecystokinin analogs. At 25 degrees C the half-disappearance time was 18 min for Boc CCK7, Boc[diNle2,5]CCK7 and 70 min for Boc[diNle2,5 D.Asp6]CCK7. Although, half-life of Boc CCK7 and Boc[diNle2,5]CCK7 were identical, the replacement of Met by Nle, a more hydrophobic aminoacid, greatly favoured the cleavage at the Trp4-Nle5 bond which became the major breakdown. This feature was exemplified by the substitution of L.Asp by D.Asp, preventing the Trp4-Nle5 cleavage, which gave rise to the most enkephalinase-resistant analog in this series.