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Biomedical subjects

R E Galardy

Publications and source records attributed to R E Galardy.

At least 55 records · Page 3Linked to original sources
PubMed1983

Inhibition of collagenase from Clostridium histolyticum by phosphoric and phosphonic amides.

Di- and tripeptides with sequences present in collagen that are known to occupy the S1' through S3' subsites at the active site of the collagenase from Clostridium histolyticum do not themselves inhibit this zinc protease. Thus glycylproline, glycylprolylalanine, and their C-terminal amides are not inhibitors. N alpha-Phosphorylglycylproline, N alpha-phosphorylglycyl-L-prolyl-L-alanine, and their C-terminal amides are weak inhibitors with IC50's (concentration causing half-maximal inhibition) of 4.6, 0.8, 3, and 1.5 mM, respectively. Extension of glycyl-L-prolyl-L-alanine to L-leucyl-glycyl-L-prolyl-L-alanine gives a tetrapeptide known to occupy the S1, S1', S2', and S3' subsites of collagenase when present in collagen but that still does not itself inhibit the enzyme. (Isoamylphosphonyl)glycyl-L-prolyl-L-alanine, a peptide containing a tetrahedral phosphorus atom at the position of the amide carbonyl carbon of the L-leucylglycyl amide bond of the parent tetrapeptide, inhibits collagenase with an IC50 of 16 microM, at least 1000-fold more potent than the parent peptide. Substitution of the two-carbon ethyl chain of alanine for the five-carbon isoamyl chain of leucine increases the IC50 to 46 microM. Substitution of the n-decyl chain for the isoamyl chain does not change the IC50. (Isoamylphosphonyl)glycyl-glycyl-L-proline contains a tripeptide that does not occupy the S1' through S3' subsites of collagenase when this peptide is present in collagen and thus has an IC50 of 4.4 mM. (Isoamylphosphonyl)glycyl-L-prolyl-L-alanine may be an analogue of the tetrahedral transition state for the hydrolysis of the natural collagen substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗
PubMed1982

Inhibition of angiotensin converting enzyme by phosphoramidates and polyphosphates.

N alpha-Phosphoryl-L-alanyl-L-proline is a reversible competitive inhibitor of angiotensin converting enzyme with a Ki of 1.4 nM. Alkylation of one phosphate oxygen with methyl, ethyl, or benzyl does not change the Ki. The high activity of the O-alkylated inhibitors demonstrates that the two phosphate oxygen anions do not constitute a bidentate ligand of the active site zinc ion. Substitution of valyltryptophan, glycylglycine, or delta-aminovaleric acid for alanylproline in the phosphoramidate raises the Ki to 12 nM, 25 microM, and 178 microM, respectively. Methylation of the alanine nitrogen in phosphorylalanylproline raises the Ki to 29 microM. Polyphosphates inhibit converting enzyme with the following Ki's: phosphate, approximately 300 mM; pyrophosphate, 2 mM; tripolyphosphate, 18 microM; tetrapolyphosphate, 150 microM. The inhibition by tripolyphosphate appears to be competitive and is unaffected by the addition of excess zinc ion. Since the Ki of tripolyphosphate is nearly 10-fold lower than that of N-phosphoryl-delta-aminovaleric acid and is near that of N alpha-phosphorylglycylglycine, its terminal phosphates may bind the zinc site and the cationic site on the enzyme, thus spanning the S1' and S2' sites.

Alkylation↗
PubMed1982

s-Cis...and s-trans isomerism in acylproline analogs. Models for conformationally locked proline peptides.

N-Acetyl-2,3-dehydroproline, N-acetyl-5-oxo-L-proline, N-acrylyl-L-proline, N-acetyl-L-azetidine-2-carboxylic acid and N-acetyl-D,L-pipecolic acid have been examined in 2H2O by 1H and 13Cn.m.r. for the purpose of finding s-cis or s-trans locked acylprolines. Conformationally locked acylprolines could be incorporated into proline-containing peptide hormones such as angiotensin and thyroliberin in order to determine the rotational state of the peptide bond to proline in the hormone receptor complex. The populations of trans and cis rotational isomers were determined as a function of p2H in order to assign the trans and cis isomers and to compare the populations in all the acylprolines at neutral p2H, where the cis isomer is normally present. Proton spectra were also recorded at from 7 degrees to 75 degrees in order to qualitatively determine the exchange rate between the isomers. The majority of these analogs exhibit a cis-trans isomerization similar to that of N-acetyl-L-proline in the ratio of trans to cis rotational isomer found at neutral p2H (about 1:1), the temperature dependence of the population ratio (none), and the coalescence temperature for proton resonances (greater than 75 degrees). However, N-acetyl-5-oxo-L-proline was found to be greater than 98% s-trans at neutral pH, compared to 50% s-trans in N-acetyl-L-proline, and therefore a good candidate for synthesis of an s-trans locked peptide hormone. N-Acetyl-2,3-dehydroproline rapidly exchanges between s-cis and s-trans in contrast to all other proline analogs examined and exhibits coalescence of the beta-proton cis and trans resonances at 45 degrees. Titration with the shift reagent Pr+++ was employed to confirm the assignments of the cis and trans methyl resonances of all of the N-acetyl compounds except N-acetyl-5-oxo-L-proline.

Magnetic Resonance Spectroscopy↗
PubMed1982

The rate of s-cis/s-trans isomerization in angiotensin II is at least 70-fold greater than in His-Pro and is not rate limiting in receptor binding.

The dipeptide L-histidyl-L-proline interconverts between the s-cis and s-trans rotational isomers of the amide bond with an average rate constant of 2.0 ks-1 at 22.5 degrees. This rate is independent of pH throughout the pH range 5.5-8.5 as determined by pH jump from low pH with potentiometric recording of the slow approach to equilibrium pH, and by integration of the histidine C-2 and C-4 proton n.m.r. resonances as a function of time following pD jump from low pD. From the temperature dependence of the n.m.r. experiments this interconversion has an Arrhenius activation energy of 20 kcal X mol-1 and a free energy of activation of 21 kcal X mol-1, similar to other simple acyl- and aminoacylprolines. The octapeptide angiotensin II containing L-histidyl-L-proline at position 6-7 interconverts at least 70-fold faster than the free dipeptide. This rate is too fast to be rate limiting in the binding of angiotensin II to its receptor in rat adrenal cortex.

Angiotensin II↗
PubMed1982

Renin-angiotensin and development of collateral circulation after renal ischemia.

Aortic ligation between the origins of the renal arteries in the rat produces a left renal ischemia, renin-dependent hypertension, and a transitory hindlimb paralysis of less than 2 h. Removal of the left ischemic kidney at the time of aortic ligation curtails the rise of blood pressure, plasma renin activity is normal, and paralysis is still present 24 h after surgery. Administration of an angiotensin-converting enzyme inhibitor or saralasin also prevents recuperation from paralysis after aortic ligation. Independent manipulation of the mean arterial pressure or plasma renin activity by pretreatment with reserpine or deoxycorticosterone before surgery shows that the presence or absence of paralysis is dependent on the plasma renin activity and not on the high blood pressure. Blood flow measurements show that paralysis is due to a persistent impairment of blood supply to the hindlimb muscle and not to ischemia of the spinal cord. Infusion of angiotensin II to aortic-ligated, left-renoprival animals tends to restore blood flow to muscle. It is concluded that after renal ischemia the renin-angiotensin system, independent of its hypertensive effect, restores blood flow by stimulating the development of collateral circulation.

Animals↗
PubMed1981

Biological activity of an angiotensin II--ferritin conjugate on rabbit aortic smooth muscle cells.

Specific binding sites for [Asp1,Ile5]angiotensin II (angiotensin) have been demonstrated in homogenates and subcellular fractions of aortic medial smooth muscle cells, but the localization of the angiotensin receptor responsible for contraction has not been determined [Devynck, M. A., & Meyer, P. (1976) Am. J. Med. 61, 758-767]. To establish the location of this receptor, we have prepared a membrane-impermeable analogue of angiotensin by acylating its N-terminal amino group with the N-hydroxysuccinimide ester of succinylated ferritin. This angiotensin-ferritin conjugate possessed the same intrinsic activity as angiotensin but was approximately 200 times less potent in inducing contraction in rabbit aortic strips. The stability of the conjugate was investigated, and approximately 5% of the contractile activity of the angiotensin-ferritin conjugate was attributable to low molecular weight components that were present before or after exposure to aortic strips. The time required for aortic strips to reach a plateau of contraction in response to angiotensin-ferritin was significantly longer than that required by free angiotensin to produce the same level of contraction. With enzymatically dispersed aortic smooth muscle cells, however, the time taken to produce contractions by both angiotensin and angiotensin-ferritin was indistinguishable. [Sar1,-Ala8]angiotensin II, a competitive inhibitor of angiotensin, completely suppressed contractions induced by angiotensin or angiotensin-ferritin in aortic strips or dispersed aortic smooth muscle cells. These results suggest that angiotensin need not directly penetrate the plasma membrane to cause contraction and imply that the angiotensin receptor responsible for initiating contraction of aortic smooth muscle cells is located on the plasma membrane.

Angiotensin II↗
PubMed1981

Photochemical inactivation of the angiotensin receptor of rabbit aorta by N alpha-(2-nitro-5-azidobenzoyl)-[1-aspartic acid,5-isoleucine]angiotensin II.

The photoaffinity label N alpha-(2-nitro-5-azidobenzoyl)-[Asp1,Ile5]angiotensin II (nitroazidobenzoyl-angiotensin II) has a pD2 of 7.22 in the rabbit aortic strip assay compared to 8.44 for [Asp1,Ile5]angiotensin II and can be completely inhibited by [Sar1, Ala8]angiotensin II. Photolysis of a single dose of nitroazidobenzoyl-angiotensin II (0.1 microM) with aortic strips for 3 min with light of wavelengths of greater than 320 nm, followed by washing, resulted in loss of approximately 25% of the strip's response to a subsequent dose of nitroazidobenzoyl-angiotensin II. Photolysis of three consecutive doses of label resulted in loss of 62% of the strips response to a subsequent dose. Repetitive exposure to label in the dark and repetitive photolyses without label resulted in loss of no more than 20% of the strips ability to respond to angiotensin II. Photolysis with three consecutive doses of 0.1 microM nitroazidobenzoyl-angiotensin II in the presence of 0.5 microM of the competitive inhibitor [Sar1,Ala8]angiotensin II decreased the observed inhibition from 62 to 22%. The long-lasting inhibition observed after photolysis with 0.1 microM nitroazidobenzoyl-angiotensin II therefore occurred via photolysis of some part of the nitroazidobenzoyl chromophore. This inhibition was mediated via the receptor that binds [Sar1,Ala8]angiotensin II and, therefore, angiotensin II.

Affinity Labels↗
PubMed1980

Irreversible photoactivation of a pancreatic secretagogue receptor with cholecystokinin COOH-terminal octapeptides.

The photochemically reactive analog of cholecystokinin octapeptide (CCK-8), 2-nitro-5-azidobenzoyl-Gly-Asp-Tyr-(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2 (NAB-Gly-CCK-8), has been used to stimulate irreversibly the receptor for CCK-8 on preparations of dispersed guinea pig pancreatic acini. When continuously present with acini in the dark, NAB-Gly-CCK-8 is a reversible stimulant of secretory protein discharge with an ED50 of 0.4 nM. After six cycles of photolysis of acini with NAB-Gly-CCK-8 followed by extensive washing and assay in peptide-free medium in the dark, the photoactivated label induces irreversible discharge. Irreversible discharge is proportional to the concentration of NAB-Gly-CCK-8 present during photolysis and is nearly equivalent in intrinsic activity to that induced by CCK-8 in the dark. Inclusion of a 5-fold excess of prephotolyzed NAB-Gly-CCK-8 during photolysis partially protects against irreversible discharge, suggesting that the receptor irreversibly stimulated by NAB-Gly-CCK-8 is likely a receptor for peptides in the CCK family. Irreversible discharge requires metabolic energy supplied by oxidative phosphorylation and is dependent on extracellular Ca2+ and, thus, resembles that induced by reversible secretagogues.

Animals↗
PubMed1979

s-Cis and s-trans isomerism of the His-Pro peptide bond in angiotensin and thyroliberin analogues.

The dipeptide His-Pro isomerizes from all-s-trans to partly s-cis when titrated in D2O from acidic to neutral pD as observed by 13C and 1H nuclear magnetic resonance of the proline side chain. This isomerization is reported by the His C-2 and C-4 protons and carbons which show distinct, well-resolved resonances for each isomer. The influence of the His-Pro peptide bond rotational state on the histidine protons far removed from the bond has not been previously observed in model compounds or peptides. The peptides thyroliberin (TRH), [3-MeHis2]-TRH, and [3-MeHis6]-, [Sar1,Al8]-, and Nalpha-acetylangiotensin II were found to similarly isomerize from all-s-trans to partly s-cis as reported by their His C-2 and C-4 proton resonances. The His C-2 and C-4 protons in the peptides [1,3-diMeHis2]-TRH and [1-MeHis6]-, and [homoHis6]-angiotensin do not report this isomerization. Angiotensin II has previously been found to exhibit the same isomerization. The reporting of the s-trans to s-cis isomerization by the His C-2 proton appears to be correlated with the known potencies of the five angiotensin peptides in rat uterine strips and of the three TRH peptides by radioimmunoassay of released thyrotropin.

Angiotensin II↗
PubMed1978

Preparation of functional smooth muscle cells from the rabbit aorta.

A procedure for dissociating the rabbit aorta into single, functional smooth muscle cells is described. After removal of adventitia and intima, slices of media were incubated with purified collagenase, elastase, and soybean trypsin inhibitor in a Krebs-Ringer buffer modified with Hepes, amino acids, and a [Ca2+] of 0.2 mM. After enzymatic digestion and mechanical shear, the yield of dispersed cells was approximately 25% based on DNA recovered. Greater than 95% of the cells excluded trypan blue and approximately 80-90% adhered to tissue culture dishes. By phase contrast microscopy, most of the cells were elongate and approximately 10 micron X 30 micron in size. The remainder were either spherical or highly crenated and contracted. Electron microscopy of the cells showed that immediately after dissociation greater than 95% could be identified as smooth muscle, though most had undergone some degree of structural change compared to cells in situ. Depending on the preparation, from 5 to 50% of these cells contracted in response to agonists. Cells shortened by 10-15% and developed numerous evaginations when stimulated by angiotensin II norepinephrine, or carbamylcholine. Cells relaxed after washout of agonists and could subsequently be restimulated. Specific inhibitors of each of the agonists blocked the contractile response. Dispersed cells cultured for 1-5 days contracted in even higher numbers than the freshly prepared cells, suggesting restoration of hormone binding and/or contractile function in culture. This preparation provides a system in which the physiology of individual vascular smooth muscle cells may be studied.

Animals↗
PubMed1978

Synthesis and characterization of 2-nitro-5-aziodobenzoylglycyloxytocin, an oxytocin photoaffinity label.

The oxytocin analogue, 2-nitro-5-azidobenzoylglycyloxytocin (NAB-Gly-oxytocin), has been synthesized and purified. The analogue is a full agonist for the stimulation of osmotic water flow in the toad urinary bladder (one-half maximal activity at 3.2 X 10(-6)M). It also enhances [14C]urea permeability in this tissue. Repetitive photolysis in the presence of NAB-Gly-oxytocin (8 X 10(-6)M) results in a progressive permanent inhibition of oxytocin stimulated urea permeability but does not alter hormone induced 3H2O movement. The inhibition is dependent on the photogeneration of the aryl nitrene intermediate and is relieved by protecting the hormone receptor with excess oxytocin (10(-6)M) during the photolysis. These results suggest that the photodependent permanent inhibition of the response to oxytocin in the toad bladder is due to covalent incorporation of the photoaffinity label, NAB-Gly-oxytocin, into the hormone receptor.

Affinity Labels↗
PubMed1978

Correlation of the biological activity and solution conformation of [Asp1,Ile5]- and [Phe4,Tyr8]angiotensin II.

Angiotensin II is known to undergo a reversible conformational transition and a change in potency in rat uterus in vitro with pK approximately 6.5. We have shown by carbon-13 NMR that the conformational transition involves all-trans to partly cis isomerization of the His6-Pro7 peptide bond. Isomerization from all-trans at pH 6.8 to approximately 16% cis at pH 8.0 is therefore correlated with a 10-fold increase in biological activity for [Asp1,Ile5]-angiotensin II in rat uterus in vitro. Isomerization from all-trans at pH 6.8 to approximately 16% cis at pH 8.0 in the competitive inhibitor [Phe4,Tyr8]angiotensin II is correlated with exhibition of virtually no agonist activity at low pH to full agonist activity at high pH. An angiotensin II conformation with Pro7 in the cis form may therefore be the conformation with maximal binding or biological activity at the cellular receptor.

Angiotensin II↗