Substance P Hydrolysis by Human Serum Cholinesterase

Highly purified human serum cholinesterase (EC 3.1.1.8, also known as pseudocholinesterase and butyrylcholinesterase) had peptidase activity toward substance P. Digestion of substance P was monitored by high performance liquid chromatography, which separated three product peptides. The cleavages occurred sequentially. The first peptide to appear as Arg1-Pro2. The Km for this hydrolysis was 0.3 mM; maximum activity was 7.9 nmol min-1 mg-1 of protein, which corresponded to a turnover number of 0.6 min-1. A second cleavage yielded Lys3-Pro4. A third cleavage occurred at the C-terminal, where the amide was removed from Met11 to yield a peptide containing residues 5-11. Both the peptidase and esterase activities of the enzyme were completely inhibited by the anticholinesterase agent, diisopropylfluorophosphate. Substance P inhibited the hydrolysis of benzoylcholine (a good ester substrate) with a KI of 0.17 mM, indicating that substance P interacted with cholinesterase rather than with a trace contaminant. Peptidase and amidase activities for serum cholinesterase are novel activities for this enzyme. It was demonstrated previously that the related enzyme acetylcholinesterase (EC 3.1.1.7) catalyzed the hydrolysis of substance P, but at entirely different cleavage sites from those reported in the present work. Since butyrylcholinesterase is present in brain and muscle, as well as in serum, it may be involved in the physiological regulation of substance P.     

Degradation of bradykinin and its metabolites by rat brain synaptic membranes

Bradykinin (BK) (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) was degraded by rat brain synaptic membranes at a rate comparable to that found for Met-enkephalin, but approximately 40 times the rate for vasopressin and oxytocin. The catabolic pathway for BK and its metabolites was elucidated through the use of high performance liquid chromatography for metabolite identification and peptidase inhibitors for blocking specific cleavage sites. BK was hydrolyzed at three sites: at the -Phe5-Ser6- bond by metalloendopeptidase 24.15, at the -Pro7-Phe8- bond by an apparently novel peptidyl dipeptidase, and at the -Phe8-Arg9 bond by a carboxypeptidase B-like enzyme. Each enzyme contributed about equally to BK degradation under the assay conditions used. Some of the resulting metabolites were further hydrolyzed: BK(1-8) to BK(1-7) + Phe by a DFP inhibitable prolyl carboxypeptidase-like enzyme, BK(1-8) to BK(1-5) + BK(6-8) by metalloendopeptidase 24.15, BK(1-7) slowly to BK(1-5) by a second peptidyl dipeptidase which was captopril inhibited, and Phe-Arg to Phe + Arg by a bestatin-inhibited dipeptidase. A number of properties of the individual enzymes were determined including sensitivity to a variety of peptidase inhibitors. These results provide a starting point for investigating the potential physiological role of each enzyme in BK function in the brain.     

Inhibition of substance P degradation in rat brain preparations by peptide hydroxamic acids

A peptidase activity of rat diencephalon membranes, which acts on the C-terminal hexapeptide sequence of substance P, was characterized using the radiolabeled substrate N alpha-[( 125I]iododesaminotyrosyl)-substance P (6-11)-hexapeptide. This activity presents certain characteristics similar to those of the substance-P-degrading enzyme purified from human brain by Lee et al. [Eur. J. Biochem. 114, 315-327 (1981)]. It is inhibited by metal chelators and some thiol reagents, but is insensitive to inhibitors of serine proteases and aminopeptidases. The activity is different from angiotensin-converting enzyme and enkephalinase, since it is not affected by specific inhibitors of these enzymes. Substance P and substance P C-terminal fragments longer than the pentapeptide inhibited the degradation of the radiolabeled substrate with inhibition constants around 200 microM. Short fragments of the substance P sequence, such as Boc-Phe-Phe-OMe and Boc-Phe-Phe-Gly-OEt, were also found to inhibit the degradation of the substrate. When the metal-chelating hydroxamic acid moiety was attached to the carboxyl terminus of these short peptides, potent inhibitors of the substance-P-degrading activity were obtained, with inhibition constants in the micromolar range. The most potent of these compounds, iododesaminotyrosyl-Phe-Phe-Gly-NHOH (IBH-Phe-Phe-Gly-NHOH), is a competitive inhibitor, with a Ki value of 1.9 microM. The degradation of substance P by rat diencephalon slices was inhibited to the same extent (40-50%) by IBH-Phe-Phe-Gly-NHOH (20 microM) and by phosphoramidon (1 microM). A combination of both reagents reduced the degradation rate by 75-80%, suggesting that both enkephalinase and the substance-P-degrading activity are involved in the metabolism of substance P in this preparation. IBH-Phe-Phe-Gly-NHOH seems to be quite specific for the latter enzyme, since at a high concentration (0.1 mM) it did not affect the degradation of the radiolabeled substrate by alpha-chymotrypsin, papain, or thermolysin.     

Enzymatic inactivation of bradykinin by rat brain neuronal perikarya

1. Bradykinin (Bk; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg8) inactivation by bulk isolated neurons from rat brain is described. 2. Bk is rapidly inactivated by neuronal perikarya (4.2 +/- 0.6 fmol/min/cell body). 3. Sites of inactivating cleavages, determined by a kininase bioassay combined with a time-course Bk-product analysis, were the Phe5-Ser6, Pro7-Phe8, Gly4-Phe5, and Pro3-Gly4 peptide bonds. The cleavage of the Phe5-Ser6 bond inactivated Bk at least five fold faster than the other observed cleavages. 4. Inactivating peptidases were identified by the effect of inhibitors on Bk-product formation. The Phe5-Ser6 bond cleavage is attributed mainly to a calcium-activated thiol-endopeptidase, a predominantly soluble enzyme which did not behave as a metalloenzyme upon dialysis and was strongly inhibited by N-[1(R,S)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate and endo-oligopeptidase A antiserum. Thus, neuronal perikarya thiol-endopeptidase seems to differ from endo-oligopeptidase A and endopeptidase 24.15. 5. Endopeptidase 24.11 cleaves Bk at the Gly4-Phe5 and, to a larger extent, at the Pro7-Phe8 bond. The latter bond is also cleaved by angiotensin-converting enzyme (ACE) and prolyl endopeptidase (PE). PE also hydrolyzes Bk at the Pro3-Gly4 bond. 6. Secondary processing of Bk inactivation products occurs by (1) a rapid cleavage of Ser6-Pro7-Phe8-Arg8 at the Pro7-Phe8 bond by endopeptidase 24.11, 3820ACE, and PE; (2) a bestatin-sensitive breakdown of Phe8-Arg9; and (3) conversion of Arg1-Pro7 to Arg1-Phe5, of Gly4-Arg9 to both Gly4-Pro7 and Ser6-Arg9, and of Phe5-Arg9 to Ser6-Arg9, Phe8-Arg9, and Ser6-Pro7, by unidentified peptidases. 7. A model for the enzymatic inactivation of bradykinin by rat brain neuronal perikarya is proposed.     

Properties of a Leu-Phe-Cleaving Endopeptidase Activity Putatively Involved in β-Endorphin Metabolism in Rat Brain

Incubation of beta-endorphin with cytosolic and particulate fractions of rat brain resulted in the formation of several peptides, including gamma-endorphin [beta-endorphin-(1-17)] and beta-endorphin-(18-31), indicating the presence of enzyme activity cleaving the Leu17-Phe18 bond of beta-endorphin. An assay for this Leu-Phe cleaving activity, based on the cleavage of the 14C-labeled substrate acetyl-Val-Thr-Leu-Phe-[epsilon-([14C]CH3)2]Lys-NHCH3, was used to examine the properties of this enzyme activity. beta-Endorphin-(1-31) competitively inhibited the Leu-Phe-cleaving enzyme activity on the pentapeptide substrate. Over 90% of activity was recovered in the cytosolic fraction. Leu-Phe-cleaving activity behaved like a thiol endopeptidase because it was inhibited by low concentrations of N-ethylmaleimide, p-chloromercuribenzoate, p-chloromercuribenzoyl sulfate, and low concentrations of Hg2+. Low concentrations of sulfhydryl compounds stimulated Leu-Phe-cleaving activity. The activity was optimal between pH 8.5 and 9.0. The Km of Leu-Phe-cleaving activity in the cytosolic fraction was 35 microM and in the particulate fraction 88 microM with Vmax values of 193 and 15 nmol mg protein-1 h-1, respectively. The apparent molecular mass of the Leu-Phe-cleaving enzyme was estimated by gel filtration to be approximately 200 kilodaltons. These properties of Leu-Phe-cleaving activity indicate that the Leu-Phe-cleaving enzyme is distinct from any known brain endopeptidase.     

Purification and characterization of a substance P-degrading endopeptidase from rat brain

A novel substance P-degrading endopeptidase has been solubilized with Brij 35 from a membrane fraction of rat brain and purified by a procedure involving DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G-100 gel filtration, and Mono-Q HPLC. The activity of the degrading enzyme was monitored by measuring the disappearance of substance P by means of a bioassay and HPLC. SDS-polyacrylamide gel electrophoresis under reducing conditions of the enzyme gave a single band corresponding to a molecular weight of 58,000. The molecular weight of the enzyme was estimated to be 55,000 by gel filtration and the optimum pH for its activity was 7.5.. The purified enzyme cleaved substance P at three bonds, Pro4-Gln5, Gln5-Gln6, and Gln6-Phe7, in the ratio of 2:2:3. EDTA, o-phenanthroline, and p-chloromercuribenzenesulfonic acid strongly inhibited the enzyme, while diisopropyl fluorophosphate, E-64, Z-Gly-ProCH2Cl, phosphoramidon, and captopril had little or no inhibitory effect on it. The cleavage of substance P by the rat brain synaptic membrane was also analyzed under the conditions with or without these inhibitors. The inhibitor-susceptibility of the cleavage sites suggests that the present enzyme, together with endopeptidase-24.11, is involved in the degradation of substance P in the synaptic region.     

Metalloendopeptidase (EC 3.4.24.11) but not angiotensin converting enzyme is involved in the inactivation of substance P by synaptic membranes of the rat substantia nigra

Substance P is a neuropeptide released in vivo from the substantia nigra, the principal substance P nerve terminal region in the rat brain. Its inactivation was investigated in a purified nigral synaptic membrane preparation. The membrane-bound enzyme shares many features with the endopeptidase 24-11 (EC 3.4.24.11): 1) hydrolysis of peptide bonds Gln6-Phe7, Phe7-Phe8 and Gly9-Leu10, 2) sensitivity to the inhibition by phosphoramidon and 3) relative affinity for substance P. Bestatine and captopril inhibit only the hydrolysis of the metabolites. These results suggest that substance P is inactivated in substantia nigra by endopeptidase 24-11 and that a bestatin-sensitive aminopeptidase and angiotensin converting enzyme may play a role in subsequent degradation of the substance P metabolites.     

Hydrolysis of the synthetic chromophoric hexapeptide Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe catalyzed by bovine pepsin A. Dependence on pH and effect of enzyme phosphorylation level

Inactivation of substance P and its C-terminal hexapeptide analog [p-Glu6]substance P6-11 was studied in rat parotid and hypothalamic slices. It was found that in the parotid slice system the decay of substance P induced K+ release occurs concurrently with a decrease in the biologically active concentration of the peptide in the medium. The inactivation was further studied using [p-Glu6]substance P6-11 as substrate in the parotid and in the hypothalamic slice systems. In both tissue preparations the hexapeptide is degraded to small peptide fragments by metalloendopeptidase. Separation of the peptide fragments by high performance liquid chromatography and determination of their amino acid composition showed that in the hypothalamic slice system the major cleavage of the hexapeptide analog occurs between Phe8-Gly9 with minor cleavage sites between Phe7-Phe8 and Gly9-Leu10. In the rat parotid slice system the major cleavage occurs between Gly9-Leu10 with a minor cleavage site between Phe7-Phe8. The degradation of the hexapeptide analog in the hypothalamic system was inhibited 77% and 67% by treatment with 1 mM p-chloromercuriphenylsulfonate and p-chloromercuribenzoate, respectively, whereas in the parotid system these reagents inhibited the degradation of the hexapeptide only by 15% and 8%. These results may indicate that different proteases in the parotid and hypothalamus are involved in degradation of substance P. Kinetic studies, including the use of various inhibitors as well as competition by the peptide hormones somatostatin, LHRH, TRH and Leu-enkephalin-NH2, revealed that in both tissues the hexapeptide analog is a preferred substrate for degradation by protease of considerable specificity towards the C-terminal sequence of substance P. It is suggested that this metalloendopeptidase may be important in the termination of the substance P response.     

Kininase from the Latrodectus tredecimguttatus venom. Characteristics of the enzyme as a thiol endopeptidase hydrolyzing the -Pro7-Phe8- bond of bradykinin

The nature of the bradykinin (BK)-hydrolyzing (kininase) activity of peptidhydrolase isolated from spider (Latr. tredecimguttatus) venom has been studied. It was found that the BKase activity of the enzyme is fully inhibited by organic mercurials (10(-5)-10(-6) M) as well as by 5,5'-dithiobis(2-nitrobenzoic acid) (10(-7) M); the latter blocks three SH-groups within the enzyme molecule. Serine and metalloproteinase inhibitors have no effect on the kininase activity. Thin-layer chromatography on silicagel revealed that the highly purified enzyme hydrolyzes the -Pro7-Phe8- bond of BK liberating the C-terminal dipeptide, HPhe-ArgOH. Besides, the kininase splits off the C-terminal tripeptide from angiotensin I by hydrolyzing its -Pro7-Phe8-bond. The enzyme does not exhibit any exopeptidase activity with free and N-substituted tri- and pentapeptides. The data obtained suggest that the Latr. tredecimguttatus kininase can be related to thiol endopeptidases hydrolyzing the peptide bonds formed by proline carboxyl.     

A new feature of angiotensin-converting enzyme in the brain: hydrolysis of substance P

Highly purified rat brain angiotensin-converting enzyme hydrolyzes substance P which contains a C-terminal amino acid with an amidated carboxyl group. The hydrolysis of substance P verified by amino-group fluorometry and by high-performance liquid chromatography is inhibited by captopril, but not by phosphoramidon. The presence of sodium chloride is essential for the hydrolysis. The analyses of cleavage products indicate that the enzyme hydrolyzes substance P between Phe7-Phe8 and Phe8-Gly9 by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action.     

Human lung post-proline endopeptidase: purification and action on vasoactive peptides

Post-proline endopeptidase (PPE, EC 3.4.21.26) was purified 3,450 times from human lung. PPE was routinely assayed with the artificial substrate, carbobenzoxy-glycyl-L-prolyl-p-nitroanilide (Z-Gly-Pro-pNA). The pH optimum was 7.4, and the Mr was 77,000. Thiol blocking agents were strongly inhibitory but serine blocking agents were not inhibitory. No metal ions were required for activity, but heavy metal ions such as Hg2+, Cu2+, Cd2+, and Zn2+ completely inactivated the enzyme. Both dithiothreitol (DTT) and ethylenediaminetetraacetic acid (EDTA) were required to stabilize PPE activity. Michaelis constant values for Z-Gly-Pro-pNA and carbobenzoxy-glycyl-L-prolyl-2-naphthylamide were 0.36 and 0.10 mmol/l, respectively. PPE cleaved vasoactive peptides including bradykinin (BK) and des-(Arg9)-BK (Pro3-Gly4 and Pro7-Phe8 bonds), angiotensins I and II (Pro7-Phe8 bond), substance P (Pro4-Gln5 bond), and oxytocin (Pro7-Leu8 bond). Each of these peptides inhibited PPE-catalyzed hydrolysis of Z-Gly-Pro-pNA competitively. BK had the lowest Ki value (2.35 mumol/l) and oxytocin had the highest Ki value (84.0 mumol/l). PPE was not inhibited by captopril, a potent inhibitor of angiotensin converting enzyme, which also cleaves the Pro7-Phe8 bond of BK.     

Proteolytic Inactivation of Substance P and Neurokinin A in the Longitudinal Muscle Layer of Guinea Pig Small Intestine

Membrane vesicles, showing a 21 +/- 2-fold enrichment in the activity of 5'-nucleotidase and a 11 +/- 4-fold enrichment in the activity of angiotensin-converting enzyme relative to homogenate, were prepared from the myenteric plexus-containing longitudinal muscle layer of guinea pig ileum. Incubation of the vesicles with substance P and neurokinin A led to degradation of the peptides, and metabolites were isolated by reverse-phase HPLC and identified by amino acid composition. Cleavages of substance P between Glu6-Phe7, Phe7-Phe8, and Gly9-Leu10 and of neurokinin A between Gly8-Leu9 were observed and could be inhibited in a dose-dependent manner by phosphoramidon, an inhibitor of neutral endopeptidase 24.11. Formation of these metabolites was not completely inhibited by this agent, indicating that a phosphoramidon-insensitive form of endopeptidase 24.11 was present in the gut. Substance P was resistant to degradation by aminopeptidases, but neurokinin A was a substrate for bestatin-sensitive aminopeptidase(s), so that the neurokinin A (3-10) fragment represented the predominant metabolite in the chromatograms. The rate of formation of all the metabolites was not inhibited by enalapril and not enhanced by an increased Cl- concentration, indicating that angiotensin-converting enzyme was unimportant in the degradation process. Degradation of neurokinin A by the vesicles (Km 30 microM; Vmax 7.2 +/- 0.8 nmol min-1 mg of protein-1) was more rapid than degradation of substance P (Km 25 microM; Vmax 4.4 +/- 0.4 nmol min-1 mg of protein-1).     

Product inhibition of carboxypeptidase H

Carboxypeptidase H is one of several enzymes required for the processing of peptide hormone precursors. In this study, inhibition of carboxypeptidase H by its peptide products was investigated. Carboxypeptidase H activity in bovine adrenal medulla chromaffin granules and rat adrenal medulla homogenate was inhibited by the peptides Met- and Leu-enkephalin, vasopressin, oxytocin, luteinizing hormone-releasing hormone, substance P, and thyrotropin-releasing hormone, with oxytocin and ACTH 1-14 having the least effect, at concentrations of 2-20 mM. Inhibition by amidated peptide products (vasopressin, oxytocin, luteinizing hormone-releasing hormone, substance P, and thyrotropin-releasing hormone) show that the final products of the precursor processing pathway can regulate carboxypeptidase H. These levels of peptides are similar to known intragranular peptide concentrations indicating that product and feedback inhibition of carboxypeptidase H may play a role in the control of neuropeptide synthesis. The proenkephalin-derived peptides Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg6-Gly7-Leu8, and Met-enkephalin-Arg6-Phe7 competitively inhibited bovine and rat carboxypeptidase H with Ki values of 12.0, 6.5, 7.0, and 5.5 mM, respectively. The significantly greater Ki for Met-enkephalin may reflect the effects of higher intragranular concentration of Met-enkephalin, since one proenkephalin molecule contains four copies of Met-enkephalin and only one copy of each of the other enkephalin peptides. Thus, the products from one multivalent precursor molecule may equivalently inhibit carboxypeptidase H activity. Product inhibition of carboxypeptidase H and perhaps other processing enzymes may serve to limit the maximum peptide concentration within the secretory vesicle.     

Effect of 6-Phosphogluconate on Phosphoglucose Isomerase in Rat Brain In Vitro and In Vivo

The activity of phosphoglucose isomerase, its kinetic properties, and the effect of 6-phosphogluconate on its activity in the forward (glucose 6-phosphate----fructose 6-phosphate) and the reverse (fructose 6-phosphate----glucose 6-phosphate) reactions were determined in adult rat brain in vitro. The activity of phosphoglucose isomerase (in nmol/min/mg of whole brain protein) was 1,865 +/- 20 in the forward reaction and 1,756 +/- 32 in the reverse reaction at pH 7.5. It was 1,992 +/- 28 and 2,620 +/- 46, respectively, at pH 8.5. The apparent Km and Vmax of phosphoglucose isomerase were 0.593 +/- 0.031 mM and 2,291 +/- 61 nmol/min/mg of protein, respectively, for glucose 6-phosphate and 0.095 +/- 0.013 mM and 2,035 +/- 98 nmol/min/mg of protein, respectively, for fructose 6-phosphate. The activity of phosphoglucose isomerase was inhibited intensely and competitively by 6-phosphogluconate, with an apparent Ki of 0.048 +/- 0.005 mM for glucose 6-phosphate and 0.042 +/- 0.004 mM for fructose 6-phosphate as the substrate. With glucose 6-phosphate as the substrate, at concentrations from 0.05 to 0.5 mM, the activity of the enzyme was inhibited completely in the presence of 0.5-2.0 mM 6-phosphogluconate. With 0.05-0.2 mM fructose 6-phosphate as the substrate, it was inhibited greater than or equal to 85% at the same concentrations of the inhibitor. No significant changes were observed in the values of Km, Vmax, and Ki for phosphoglucose isomerase in the brain of 6-aminonicotinamide-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of brain endopeptidases: influence of size and sequence of substrates structurally related to bradykinin.

Two thiol-activated endopeptidases with pH optima near pH 7.5 were isolated from the supernatant fraction of rabbit brain homogenates by DEAE-cellulose chromatography, gel filtration and isoelectrofocusing. Peptide bond hydrolysis was measured quantitatively by ion-exchange chromatography with an amino acid analyzer. Brain kininase A hydrolyzes the Phe5-Ser6 peptide bond in bradykinin (Bk), Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9. It is isoelectric near pH 5.2 and has a molecular weight of approximately 71 000. The enzyme also hydrolyzes the Phe-Ser peptide bond in Lys-Bk, Met-Lys-Bk, des-Arg1-Bk, Lys9-Bk, Pro-Gly-Phe-Ser-Pro-Phe-Arg, and Gly-Pro-Phe-Ser-Pro-Phe-Arg, but does not hydrolyze (0.1%) this bond in des-Phe8-Arg9-Bk. Brain kininase B hydrolyzes the Pro7-Phe8 peptide bond in Bk. It is isoelectric at pH 4.9 and has a molecular weight of approximately 68 000. Brain kininase B also hydrolyzes the Pro-Phe bond in Lys-Bk, Met-Lys-Bk, Lys9-Bk, Ser-Pro-Phe-Arg, and Phe-Ser-Pro-Arg. Pretreatment of denatured kininogen with brain kininase A or B did not reduce the amount of trypsin-releasable Bk from this precursor protein, indicating that the Bk sequence, when part of a large protein, is not a substrate for either enzyme. However, kininase A and B hydrolyze the octadecapeptide Gly-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gin-Val. The data show that a large part of the C-terminal portion of bradykinin is important for the brain kininase A activity and, for both enzymes, the size of the peptide and presumably the residues adjacent to the scissle bond are important in determining the rate of peptide bond hydrolysis by these endopeptidases.     

Modulation of rat brain cytosolic phosphatidate phosphohydrolase: effect of cationic amphiphilic drugs and divalent cations

The effects of three cationic amphiphilic drugs on rat brain cytosolic phosphatidate phosphohydrolase and their mechanisms of action were studied utilizing membrane-bound, emulsified, and emulsified sonicated phosphatidate as substrates. With the membrane-bound substrate, chlorpromazine, desmethylimipramine, and propranolol inhibited the activity in a dose-dependent fashion with an IC50 of 30-50 microM. In the presence of the emulsified substrate, chlorpromazine was a more potent inhibitor than desmethylimipramine or propranolol but 200 microM was needed for 50% inhibition of activity. Addition of heat-inactivated microsomes to the emulsified substrate, to simulate the conditions with the membrane-bound substrate, did not alter this value. Both Mg2+ and Ca2+ stimulated the enzyme activity but only Ca2+ counteracted the effect of chlorpromazine. Kinetic studies indicate that chlorpromazine acts as a noncompetitive inhibitor of the enzyme. Emulsified sonicated phosphatidate was a good substrate at low (less than 10 microM) concentrations. It was a poor substrate at 1 mM, but at this concentration chlorpromazine stimulated the activity instead of inhibiting. This drug altered the integrity of phosphatidate vesicle membranes as visualized by electron microscopy. The different results obtained with the three types of substrate indicate the importance of the configuration of phosphatidate for the expression of enzyme activity and for its susceptibility to the action of cationic amphiphilic drugs.     

Importance of secondary structure for endothelin binding and functional activity.

ET-1[16-Phe] and ET-1[12-Pro] were prepared in order to investigate the importance of secondary structure of ET-1 for receptor binding and function. ET-1[16-Phe] displayed the greatest binding and contractile potency of the ET-analogs tested in rabbit pulmonary artery, rat aorta, and rat left atria. ET-1[12-Pro] also exhibited low nanomolar binding in these tissues but showed less contractile activity than ET-1[16-Phe] or ET-1. The results indicate that the helical region between residues Lys9 and Cys15 of ET-1 is not critical for receptor binding and functional activity. However, replacement of His16 with Phe altered the charge characteristics of the C-terminal region of ET-1 producing the most potent ET-1 analog yet reported.     

Demonstration of two distinct tachykinin receptors in rat brain cortex

Eledoisin and substance P are members of a class of peptides termed tachykinins. They share a similar spectrum of biological activities but their relative potencies in various pharmacological assays differ. We have investigated whether there is more than one receptor for these tachykinins in rat brain cortex membranes. 125I-Bolton Hunter-conjugated eledoisin specifically binds to rat brain cortex membranes with high affinity. The binding is inhibited over 95% by unlabeled eledoisin (6.6 microM). Scatchard analysis of the binding of this ligand is curvilinear suggesting that there are two binding sites with KD values of 0.9 +/- 0.7 nM and 20 +/- 10 nM. We tested various analogs and fragments of substance P and eledoisin for their ability to inhibit the binding of 125I-Bolton Hunter-conjugated eledoisin and 125I-Bolton Hunter-conjugated substance P to these membranes. The following peptides are more potent as inhibitors of the 125I-Bolton Hunter-conjugated eledoisin binding site than of the 125I-Bolton Hunter-conjugated substance P binding site: nonradioactive Bolton Hunter-conjugated eledoisin (greater than 100-fold), eledoisin (12-fold), kassinin (22-fold), neuromedin K (greater than 58-fold), and pyroglutamyl substance P(6-11)hexapeptide (4-fold). In contrast, substance P (21-fold), physalaemin (8-fold), and substance P methyl ester (1200-fold) were more potent as inhibitors of 125I-Bolton Hunter-conjugated substance P binding. These results suggest that these two ligands may bind to distinct receptors. 125I-Bolton Hunter-conjugated substance P binds specifically to rat parotid cell receptors, but 125I-Bolton Hunter-conjugated eledoisin does not, indicating that parotid cells contain only one of the receptor subtypes. The cortex membrane binding of both ligands is stimulated by low concentrations of MnCl2 (ED50 = 0.05 mM) and is inhibited by guanylyl-5'-(beta, gamma-imido)diphosphate (IC50 = 0.5 microM).     

Enzymic dephosphorylation of D-myo-inositol 1,4-bisphosphate in rat brain.

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,4-bisphosphate [Ins(1,4)P2] phosphatase activities were measured in both 180,000 g (60 min) particulate and supernatant fractions of rat brain homogenates. Although Ins(1,4,5)P3 was mostly hydrolysed by a particulate phosphatase [Erneux, Delvaux, Moreau & Dumont (1986) Biochem. Biophys. Res. Commun. 134, 351-358], Ins(1,4)P2 phosphatase was predominantly soluble. The latter enzyme was Mg2+-dependent and sensitive to thiol-blocking agents (e.g. p-hydroxymercuribenzoate). In contrast with Ins(1,4,5)P3 phosphatase activity measured in the soluble fraction, Ins(1,4)P2 phosphatase was insensitive to 0.001-1 mM-2,3-bisphosphoglycerate. Lithium salts, widely used in psychiatric treatment, inhibited both Ins(1,4)P2 and Ins(1)P1 phosphatase activities of the crude soluble fraction. In particular, 50% inhibition of phosphatase activity, with 2 microM-Ins(1,4)P2 as substrate, was achieved at 3-5 mM-LiCl. At these concentrations, LiCl did not change Ins(1,4,5)P3 phosphatase activity measured in the same fraction with 1-4 microM-Ins(1,4,5)P3 as substrate. Chromatography of the soluble fraction of a rat brain homogenate on DEAE-cellulose resolved three phosphatase activities. These forms, peaks I, II and III, dephosphorylated Ins(1,4,5)P3, Ins(1)P1 and Ins(1,4)P2 respectively. If LiCl (10 mM) was included in the assay mixture, it inhibited both peak-II Ins(1)P1 phosphatase and peak-III Ins(1,4)P2 phosphatase, suggesting the existence of at least two Li+-sensitive phosphatases.     

Successive cleavage of N-terminal Arg1--Pro2 and Lys3-Pro4 from substance P but no release of Arg1-Pro2 from bradykinin, by X-Pro dipeptidyl-aminopeptidase

X-Pro dipeptidyl-aminopeptidase (EC 3.4.14.1) purified homogeneously from the human submaxillary gland was proved to hydrolyze N-terminal dipeptide Arg1-Pro2 and subsequent dipeptide Lys3-Pro4 from substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-gly-Leu-Met-NH2). Km and V values of hydrolysis of substance P were 2.0 mM and 3.6 mumol/min per mg protein, respectively. In contrast, the N-terminal Arg-Pro of bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) was not cleaved by the enzyme.