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Biomedical subjects

R Walter

Publications and source records attributed to R Walter.

At least 145 records · Page 8Linked to original sources
PubMed1980

Carboxamidopeptidase: purification and characterization of a neurohypophyseal hormone inactivating peptidase from toad skin.

Carboxamidopeptidase, an enzyme which inactivates neurohypophyseal hormones, has been purified 3800-fold in an overall yield of 22% from toad skin, a neurohypophyseal hormone target organ, by (NH4)2SO4 fractionation, DEAE-Sephadex chromatography, and affinity chromatography on immobilized p-aminobenzamidine and concanavalin A-agrose. The purified enzyme is capable of inactivating both [8-arginine]vasopressin (AVP) and oxytocin by hydrolyzing the Arg8-Gly9-NH2 and the Leu8-Gly9-NH2 bonds, respectively, and can hydrolyze the ester substrates, benzoyl-L-arginine ethyl ester (BzArgOEt) and acetyl-L-trypsine ethyl ester, suggesting that the enzyme has both trypsin-like and chymotrypsin-like activities. Carboxamidopeptidase is maximally active at pH 7.5-8.5 for AVP and BzArgOEt and pH 7.0 for oxytocin. Carboxamidopeptidase is inhibited by ovoinhibitor, ovomucoid, Trasylol. lima bean trypsin inhibitor, concanavalin A, antipain, leupeptin, chymostatin, elastatinal, p-nitrophenyl p-guanidinobenzoate, and 4-methylumbelliferyl p-guanidinobenzoate but not by soybean trypsin inhibitor, alpha 1-antitrypsin, hirudin, pepstatin, bestatin, phosphoramidon, or cysteine. The enzyme is also inhibited by the serine protease inhibitor, diisopropyl phosphofluoridate (i-Pr2PF), and by the chloromethyl ketone derivatives of tosyllysine, tosylphenylalanine, and (benzyloxycarbonyl)phenylalanine, as well as by the sulfhydryl group reagent, p-(chloromercuri)benzoate (PCMB). Inhibition by PCMB is reversed by cysteine. The molecular weight determined by gel filtration in the presence of 1 MNaCl is approximately 100 000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is composed of two identical subunits of 48 000 daltons. Each subunit consists of a heavy chain (28 000 daltons) and a light chain (19 000 daltons) joined by a disulfide bond(s). Labeling experiments using [3H]-i-Pr2PF showed that the enzyme active site is located in the heavy chain.

Animals↗
PubMed1980

Development of narcotic tolerance and physical dependence: effects of Pro-Leu-Gly-NH2 and cyclo (Leu-Gly).

Administration of Pro-Leu-Gly-NH2 (MIF) and cyclo (Leu-Gly) blocked the development of tolerance to and physical dependence on morphine, induced by the pellet implanation procedure in mice. Inhibition of tolerance development by peptides was evidenced by the presence of an analgesic response (increase in jump threshold) as determined by measuring the jump threshold to an increasing electric current, after a challenge dose of morphine (40 mg/kg). The same dose of morphine did not alter the jump threshold in morphine tolerant mice which were injected with saline prior to pellet implantation. The inhibition of the development of physical dependence on morphine by these peptides was evidenced by the antagonism of the hypothermic response which occurs during abrupt or naloxone-induced withdrawal. The naloxone-induced withdrawal jumping response was unaffected by these peptides. Dose-response experiments indicated that cyclo (leu-Gly) was much more potent than MIF in these tests. These peptides, when given after the development of tolerance and dependence, did not modify either the analgesic response to morphine or the symptoms of abrupt and naloxone-precipitated withdrawal. The inhibition of development of analgesic tolerance and physical dependence was not associated with changes in brain morphine concentration. The data indicate that these peptides do not interfere withe the morphine-morphine receptor complex formation but alter a subsequent step in the genesis of some aspects of tolerance and dependence processes.

Analgesia↗
PubMed1980

Oxytocin and lysine-vasopressin with N5,N5-dialkylglutamine in the 4 position: effect of introducing sterically hindered groups into the hydrophilic cluster of neurohypophyseal hormones.

The synthesis and pharmacological potencies of oxytocin and lysine-vasopressin analogues are reported in which the N5-amide of their glutaminyl residues are dialkylated. These analogues have been studied as an ongoing exploration of the biological effects on the natural hormones of substituting individually one of the amino acid residues, which has a hydrophilic side chain and which are thought to be part of the hydrophilic surface of the hormones. [4-(N5,N5-Dimethylglutamine)]oxytocin (17), [4-(N5,N5-di-n-propylglutamine)]oxytocin (18), and [4-(N5,N5-dimethylglutamine)] lysine-vasopressin (19) were synthesized by clasical solution techniques. Potencies in the in vitro rat uterotonic, avian vasodepressor, rat pressor, and rat antidiuretic assays were determined and are as follows, respectively: for compound 17 3.01 +/- 0.14 units/mg, 4.55 +/- 0.03 units/mg, tachyphylaxis and tachyphylaxis; for compound 18 less than 0.1, less than 0.1, less than 0.05, and less thad 1.88 +/- 0.04 units/mg. The potencies in all cases are significantly less than those of the parent hormone. The results are discussed in terms of the proposed biologically active conformations of the hormones at the uterotonic receptor and at the antidiuretic receptor.

Animals↗
PubMed1980

[5-(N4,N4-Dimethylasparagine),8-lysine]vasopressin: the first 5-position neurohypophyseal hormone analogue to retain to retain significant antidiuretic potency.

In the proposed biologically active conformation of vasopressin at the antidiuretic receptor, the side-chain carboxamide group of the 5-position asparaginyl residue has been previously suggested to be the key active element in the hormone for its initiation of the antidiuretic response. [5-(N4,N4-Dimethylasparagine),8-lysine]vasopressin, the analogue in which the hydrogen atoms of the -NH2 portion of the primary carboxamide have been replaced by methyl groups, has been synthesized and found to retain about 3% of the antidiuretic potency of lysine-vasopressin (i.e., 5.5 +/- 0.3 units/mg). This result suggests that the hydrogen atoms of the carboxamide moiety are not essential for antidiuretic activity. In addition, the analogue possesses rat pressor, avian vasodepressor, and rat uterotonic potencies of 2.55 +/- 0.05, 0.39 +/- 0.03, and less than 0.05 units/mg, respectively.

Animals↗
PubMed1980

Effect of changing the COOH-terminal amide group present in the hydrophilic cluster of oxytocin to dimethylamide.

Oxytocinoic acid dimethylamide was synthesized by stepwise solution techniques as part of an ongoing evaluation of the effects on the biological activity of oxytocin caused by individually changing the groups that comprises the hydrophilic surface of the hormone to more hydrophobic and more bulky groups. The analogue exhibited approximately 3% of the potency of oxytocin in the in vitro uterotonic assay. In addition, it possesses potencies of less than 0.07, less than 0.01, and 0.096 unit/mg in the avian vasodepressor, rat pressor, and rat antidiuretic assays, respectively. In the in vitro uterotonic assay, oxytocinoic acid dimethylamide showed a reduced affinity for the oxytocin receptor, a nonparallel dose-response relationship, and most importantly a reduced intrinsic activity as compared to oxytocin. The results suggest that the replacement of the protons of the primary carboxyamide of Gly9-NH2 of oxytocin by methyl groups displaces the active elements from the orientation for obtaining maximal intrinsic activity in the isolated rat uterus.

Animals↗
PubMed1980

Bidirectional synthesis of [5-aspartic acid] arginine vasopressin on poly-N-acrylylpyrrolidine resin.

In the proposed biologically active conformation of vasopressin at its antidiuretic receptor, the side-chain carboxamide moiety of the 5-position asparaginyl residue has been suggested to be an active element for the initiation of the antidiuretic response. [5-Aspartic acid] arginine vasopressin, the analog in which the -NH2 portion of the primary amide has been replaced by an -OH group, has been synthesized and tested for some of the pharmacological activities of vasopressin. The partially protected nonapeptide intermediate was assembled bidirectionally on a poly-N-acrylylpyrrolidine resin. The 6-position cysteinyl residue was attached to the resin via its side-chain through an S-carbamoyl linkage. First the COOH-terminus was extended by coupling with Pro-Arg(Tos)-Gly-NH2, then the NH2-terminus was extended in a stepwise manner. [5-Aspartic acid] arginine vasopressin was found to possess 86.5 +/- 4.8 units/mg of antidiuretic potency, 17% of the parent hormone. In addition, the analog possesses rat pressor and rat uterotonic potencies of 6.93 +/- 0.15 and 0.38 +/- 0.03 units/mg, respectively. This result suggests that a carboxylic acid moiety on the 5-position aspartyl residue retains sufficient steric features and hydrophilicity in common with the carboxamide moiety present in the hormone to substitute for it as an active element at the antidiuretic receptor.

Acrylic Resins↗
PubMed1980

N.M.R. and equilibrium dialysis studies of the interaction of bovine neurophysin-1 with vasopressin and small peptides.

The binding to bovine neurophysin of lysine-vasopressin and of lysine-vasopressin selectively deuterated at the protons ortho to the tyrosine hydroxyl was studied by proton n.m.r. and equilibrium dialysis. The principal object of these studies was to investigate reports that, at standard salt concentrations, neurophysin contained a second site specific for vasopressin. At pH 6, the effects of neurophysin-I on the line-width, longitudinal relaxation rate and nuclear Overhauser properties of the lysine-vasopressin tyrosine ring protons were interpretable in terms of a slow-exchange 1:1 interaction between lysine-vasopressin and neurophysin. Additionally, n.m.r. competition studies between lysine-vasopressin and L-phenylalanyl-L tyrosinamide suggested 1:1 competition for a single binding site on neurophysin. No evidence pointing to a significant second lysine-vasopressin-binding site was obtained from the n.m.r. studies. The lack of a moderately strong second binding site for lysine-vasopressin at neutral pH was also indicated by equilibrium dialysis studies at relatively high free hormone concentrations. These studies demonstrated only a single thermodynamically significant site for either oxytocin or vasopressin and failed to confirm a reported effect of LiCl on the number of sites available to oxytocin. It is suggested that secondary sites for the hormones are probably markedly weaker and less specific than reported elsewhere.

Amino Acids↗
PubMed1980

Time-dependency of neurohypophyseal peptide attenuation of puromycin amnesia in mice.

[Ile3, Arg8]vasopressin (arginine-vasotocin), as well as the C-terminal tripeptides of the neurohypophyseal hormones arginine and lysine vasopressin, Pro-Arg-Gly-NH2 and Pro-Lys-Gly-NH2, were protective against puromycin-induced amnesia in mice when administered 24h before training. The N-protected tripeptide derivative, Z-Pro-Lys-Gly-NH2, was effective when given 5 days before training. The effectiveness of all peptides to attenuate puromycin-induced amnesia decreased as the interval between training and peptide treatment increased, indicating that the peptides influence memory processes, rather than general arousal. Z-Pro-Lys-Gly-NH2 was active at 24h after training, when the other peptides were no longer effective. Although it seems clear that neurohypophyseal hormones per se can attenuate puromycin-induced amnesia, these results are in line with the possibility that some portion of hormone action may be mediated via formation of longer-lived hormone fragments in the CNS.

Amnesia↗
PubMed1980

3-nitro-2-pyridinesulfenyl (Npys) group. A novel selective protecting group which can be activated for peptide bond formation.

The novel 3-nitro-2-pyridinesulfenyl (Npys) group, which is useful for the protection and the activation of amino and hydroxyl groups for peptide synthesis, is reported. The Npys group is readily introduced by treatment of amino acids with 3-nitro-2-pyridinesulfenyl chloride. The Npys group is easily removed by treatment with very dilute HCl, e.g. 0.1-0.2 N HCl in dioxane, but is is resistant to trifluoroacetic acid and 88% formic acid. Npys is also selectively removed under neutral conditions using triphenylphosphine or 2-pyridinethiol 1-oxide without affecting benzyloxycarbonyl (Z), tert-butyloxycarbonyl (Boc), 2-(4-biphenylyl)propyl(2)oxycarbonyl (Bpoc), 9-fluorenylmethyloxycarbonyl (Fmoc), benzyl (Bzl) or tert-butyl (tBu) protecting groups. The N-Npys and O-Npys groups when activated in the presence of RCOOH by the addition of tertiary phosphine form peptide or ester bonds via oxidation-reduction condensation. The important features of the Npys group are demonstrated through the synthesis of peptides in solution and by solid phase methodology without a formal deprotection procedure. In solid phase synthesis, 4-(Npys-oxymethyl) phenylacetic acid is used as the key intermediate for the introduction of the trifluoroacetic acid resistant 4-(oxymethyl) phenylacetamido linking group to the resin.

Amino Acid Sequence↗
PubMed1979

Post-proline cleaving enzyme. Synthesis of a new fluorogenic substrate and distribution of the endopeptidase in rat tissues and body fluids of man.

Synthesis and application of the first fluorogenic substrate, N-carbobenzoxyglycylprolyl-4-methylcoumarinyl amide (Z-Gly-Pro-MeCouNH) for the determination of the post-proline cleaving enzyme (EC 3.4.21.-) were reported. Maximal activity of the enzyme purified from lamb kidney for the new substrate was observed at pH 7.0. This substrate showed a higher affinity (Km = 0.02 mM) for the enzyme than the proline containing substrates studied previously and allowed the detection of 10-50 ng post-proline cleaving enzyme activity per ml sample after a 1 min incubation period. Distribution of post-proline cleaving enzyme and other proline specific peptidases in rat tissues was studied using Z-Gly-Pro-MeCouNH and other proline-containing substrates. High post-proline cleaving enzyme activity was observed in testis, liver and skeletal muscle. Inhibition experiments indicated that post-proline cleaving enzyme activity was completely inactivated by 0.1 mM diisopropylphosphofluoridate and Z-Gly-Pro-chloromethylketone, as had been found in the case of the enzyme isolated from lamb kidney. Activity in human body fluids was also tested for levels of post-proline cleaving enzyme activity using Z-Gly-Pro-MeCouNH and semen was found to show the highest cleaving activity.

Aminohydrolases↗
PubMed1979

Distribution, survival and biological effects in mice of a behaviorally active, enzymatically stable peptide: pharmacokinetics of cyclo(Leu-Gly) and puromycin-induced amnesia.

Cyclo(Leu-Gly), the enzymatically resistant diketopiperazine formally derived from the C-terminal dipeptide sequence of oxytocin, exhibits activity in several behavioral systems. The distribution of cyclo(Leu-14C(U)Gly) in brain, and the time course of the disappearance of this labeled peptide from brain and plasma after subcutaneous injection into mice have been studied. The intact peptide was distributed equally in the five cerebral areas studied, for up to 96 hours after injection. Two exponential components were determined for peptide disappearance rates in plasma and brain; peptide half-lives in plasma up to 10 hr and from 24--96 hr after injection were, respectively, 0.8 and 33 hr; in brain, 1.0 and 42 hr. The peptide was found to accumulate in brain intracellular space to some degree. The time course of distribution of labeled cyclo(Leu-Gly) in subcellular fractions of mouse brain was also examined, and the concentration of peptide in the synaptosomal fraction was significantly correlated with the degree of protection against puromycin-induced amnesia of a maze-learning test. The results obtained not only confirm that cyclo(Leu-Gly) penetrates brain tissue intact and remains intact after peripheral administration in order to exert its behavioral effects, but, moreover, suggest an intriguing dynamic relationship between peptide concentration in the synaptosomal fraction and behavioral activity.

Amnesia↗