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.     

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.     

Catabolism of substance P in the stomach wall of the rat

The aim of this study was to examine the catabolism of substance P (SP) in the stomach wall of the rat. Catabolism in vitro was investigated by incubation of unlabelled and tritiated SP (prolyl 2,4-3,4(n)-3H SP) with membrane bound-peptidases prepared from the rat gastric corpus. Catabolism was studied in vivo by use of a catheter chronically implanted in the stomach wall to deliver tritiated SP to the gastric tissues and implanted dialysis fibers to collect the catabolic products. The products from both experiments were separated by high pressure liquid chromatography and identified by their retention times or amino acid analysis. Membrane-bound peptidases in vitro hydrolyzed both unlabelled and tritiated SP and the products of hydrolysis were consistent with the cleavage of three bonds: Gln6-Phe7, Phe7-Phe8 and Gly9-Leu10. None of the peptide fragments would be expected to be biologically active. Only those fragments with tritiated Pro residues could be detected in vivo. The major identified products were SP(1-2) and SP(3-4), with smaller amounts of SP(1-4), SP(1-6), SP(1-7), SP(1-8) and SP(1-9). The enzymes that may be responsible for these cleavage patterns are discussed.     

Degradation of substance P by the neuroblastoma cells and their membrane

Cells of the N-18 line of mouse neuroblastoma and their membrane degrade substance P added exogenously. The degradation by the cells and their membrane, examined by high-performance liquid chromatography, is strongly inhibited by EDTA but scarcely inhibited by captopril and phosphoramidon. Gly-Leu-Met-NH2 is the major cleavage product among C-terminal fragments of substance P in both cases. Thus, the degradation of substance P by the neuroblastoma cells and their membrane seems to take place mainly through the hydrolysis between Phe8-Gly9 by EDTA-sensitive protease(s).     

Hydrolysis of substance p and neurotensin by converting enzyme and neutral endopeptidase

Angiotensin I converting enzyme (ACE) and neutral endopeptidase ("enkephalinase"; NEP), were purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln6-Phe7,-Phe8, and Gly9-Leu10 and neurotensin (NT) at Pro10-Tyr11 and Tyr11-Ile12. NEP hydrolyzed 0.1 mM SP, NT and their C-terminal fragments at the following rates (mumol/min/mg): SP1-11 = 7.8, SP4-11 = 11.7, SP5-11 = 15.4, SP6-11 = 15.6, SP8-11 = 6.7, NT1-13 = 2.9, and NT8-13 = 4.0. Purified ACE rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe8-Gly9 and Gly9-Leu10 to release C-terminal tri- and dipeptide (ratio = 4:1). The hydrolysis was Cl- dependent and inhibited by captopril. ACE released mainly C-terminal tripeptide from SP methyl ester, but only dipeptide from SP free acid. Modification of arginine residues in ACE with cyclohexanedione or butanedione similarly inhibited hydrolysis of SP, bradykinin and Bz-Gly-Phe-Arg (80-93%) indicating an active site arginine is required for hydrolysis of SP. ACE hydrolyzed NT at Tyr11-Ile12 to release Ile12-Leu13. SP, NT and their derivatives (0.1 mM) were cleaved by ACE at the following rates (mumol/min/mg): SP1-11 = 1.2, SP methyl ester = 0.7, SP free acid = 8.5, SP4-11 = 2.4, SP5-11 = 0.9, SP6-11 = 1.4, SP8-11 = 0, NT1-13 = 0.2, and NT8-13 = 1.3. Peptide substrates were used as inhibitors of ACE (substrate = FA-Phe-Gly-Gly) and NEP (substrate = Leu5-enkephalin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and biological activity of substance P C-terminal hexapeptide analogues: structure-activity studies

The synthesis of six hexapeptide analogues of C-terminal Substance P fragment containing alpha, beta-dehydrophenylalanine (delta Phe) in the position 7 or 8 is described. The effect of the structural changes on the hypotensive activity and antigenic properties of analogues was compared. It was found that substitution of delta Phe in various analogues of C-terminal hexapeptide of Substance P resulted in different effects on the hypotensive activity. The analogues [Glp6, delta Phe7]SP6-11 and [Glp6, delta Phe8]SP6-11 retained 70% and 45% of hypotensive activity of the C-terminal hexapeptide of Substance P, respectively but they showed a completely destroyed antigenic determinant. The analogues containing additionally Sar or His in the position 9 showed a complete lack of both: hypotensive activity and expression of the antigenic determinant of Substance P.     

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).     

Immunochemical analysis of substance P using its synthetic fragments and analogs

Synthetic fragments and analogs were used to characterize specificity of antisera to substance P. Both, the C-terminal hexapeptide and the pentapeptide completely inhibited binding of 125I-[Tyr8]substance P by these antisera, showing the antigenic identity with substance P. Synthetic fragments shorter than peptide (7-11) did not react with anti-substance P antisera in this system. Substitution of amino acids in different positions in the fragments (6-11) or (7-11) by histidine or glycine revealed that all five amino-acid residues take part in a structure of the antigenic determinant.     

Interaction of substance P antagonists with substance P receptors on dispersed pancreatic acini

In the present study we examined the abilities of three analogs of substance P, [D-Pro2-, D-Phe7-, D-Trp9]-substance P, [D-Pro2-, D- Trp7 ,9]-substance P and [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P to alter substance P-induced changes in pancreatic acinar cell function and to occupy substance P receptors. At 30 microM, each analog of substance P lacked agonist activity and inhibited amylase secretion stimulated by substance P receptor agonists. The inhibition was reversible and specific for peptides that interact with substance P receptors (physalaemin, substance P, eledoisin, kassinin ). The analogs of substance P did not inhibit the actions of cholecystokinin, caerulein, gastrin, carbamylcholine, secretin, vasoactive intestinal peptide, PHI, ionophore A23187 or 8Br -cAMP. At high concentrations, [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P, but not [D-Pro2-, D- Trp7 ,9]-substance P or [D-Pro2-, D-Phe7-, D-Trp9]-substance P, caused a small but significant inhibition of bombesin-stimulated amylase release. For each analog of substance P, the inhibition was competitive in nature in that there was a rightward shift of the dose-response curve for physalaemin-stimulated amylase secretion with no change in efficacy. From Schild plots of the ability of [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P to inhibit either substance p- or physalaemin-stimulated amylase release, the slopes were not different from unity. For each analog of substance P, there was a close correlation between its ability to inhibit substance P- or physalaemin-stimulated amylase release and its ability to inhibit binding of 125I-labeled substance P or 125I-labeled physalaemin. [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P was 2-fold more potent than [D-Pro2-, D- Trp7 ,9]-substance P which was 4-fold more potent than [D-Pro2-, D-Phe7-, D-Trp9]-substance P, (i.e., pA2 6.1, 5.9, and 5.2, respectively). For each analog, the dose-response curve for its ability to inhibit physalaemin-stimulated amylase release was superimpossible on the dose-response curve for its ability to inhibit binding of 125I-labeled physalaemin. These results indicate that each of these analogs of substance P is a specific competitive inhibitor of the action of the substance P on dispersed acini from guinea-pig pancreas, and that their abilities to inhibit substance P-induced changes in acinar cell function can be accounted for by their abilities to occupy the substance P receptor.     

Failure of tachykinins including substance P and its fragments to influence proteoglycan and protein synthesis in bovine chondrocytes in vitro.

Adult bovine articular chondrocytes were exposed to substance P, neurokinins A and B or substance P fragments, SP1-4, SP1-6 and SP7-11 in vitro. Proteoglycan synthesis was assessed by measuring proteoglycans which were released into the culture medium or incorporated into the cell layer. The intact tachykinins or substance P fragments had no direct effect on proteoglycan synthesis. Nor was total protein production affected. Gel chromatography, under dissociative conditions, revealed that sulphated proteoglycans detected in the medium or cell layer following treatment of chondrocytes with substance P, contained proteoglycans of similar molecular weight to those produced by cells exposed only to diluent controls. Therefore, we conclude that the acceleration of arthritis by substance P does not appear to be mediated through an effect on chondrocyte synthetic function.     

Comparison of spantide II and CP-96,345 for blockade of tachykinin-evoked contractions of smooth muscle.

CP-96,345, a quinuclidine, is a potent inhibitor of substance P for the NK1 receptor of bovine brain, but has reduced potency for the corresponding receptor of the rat and mouse, and none for NK2 or NK3 receptors. A related quinuclidine showed similar but lower potency than CP-96,345 for NK1. CP-96,345 was more potent than the spantide I of 1984, D-Arg1,Pro2,Lys3,Pro4,Gln5,Gln6,D-Trp7,Phe8,D-Trp9, Leu10,Leu11,NH2. Our continued designs for antagonists of substance P led to spantide II in 1990 which is: D-NicLys1,Pro2,3-Pal3,Pro4,D-Cl2Phe5,Asn6,D-Trp7 ,Phe8,D-Trp9,Leu10,Nle11-NH2. The pA2 values of spantide II and CP-96,345 for guinea pig taenia coli were 7.6 and 6.8, respectively. The pIC50 values for blockade of tachykinin-mediated neurotransmission in the rabbit iris sphincter were 6.1 and 5.4, respectively. Spantide II was nearly 10 times more potent than CP-96,345 in these two assays.     

Modulation of endopeptidase activity by calcitonin gene related peptide: a mechanism affecting substance P action?

Peptides with hormonal or neuronal activity are derived by enzymatic processing from pro-hormones, which by themselves are biologically inert. Processing and other enzymatic conversions may occur step-wise, leading to the formation of a cascade of biologically active (or inactive) peptides. The neurokin in substance P is known to be metabolically transformed both by amino- and endopeptidases. More N-terminal substance (1-7) has been found than C-terminal (2-11 to 5-11) fragments in various CNS areas. The substance P (1-7) fragment also shows biological activity e.g., providing analgesia, lowering blood pressure, inhibiting aggressive behavior and (in contrast to substance P) inhibiting grooming behavior. An endopeptidase generating substance P (1-7) and to a lesser extent, substance (1-8), has been isolated and characterized from human cerebrospinal fluid (CSF) and bovine spinal cord, as a metalloenzyme with essential SH-groups. Substance P co-exists with calcitonin gene related peptide (CGRP) in a large population of non-myelinated primary afferent ('pain') fibers. Intrathecal injection of substance P causes behavioral and physiological responses which are potentiated and prolonged by CGRP. It was found that CGRP competes with substance P for the endopeptidase. It is suggested that the main action of CGRP in the spinal cord is to inhibit substance P degradation.     

Reactive oxygen species induced structural alterations of substance P

Substance P (SP(1-11)) was exposed to a continuous flux of superoxide (O2-) or hydroxyl radicals ((.)OH) in a hypoxanthine (HX)/xanthine oxidase (86 mU) system in the presence of 1 mM deferoxamine and 40 mM D-mannitol or 50 muM FeCI(3). 6H(2)O and 50 muM EDTA, respectively. O2- caused fragmentation between the Phe(7) and Phe(8), whereas (.)OH induced cleavage also between the Phe(8) and Gly(9). Reactive oxygen species H(2)O(2) and HCIO did not cause fragmentation, but modification of the amino acid side chains and/or aggregation with altered hydrophobicity in reverse phase high performance liquid chromatography compared to native SP(1-11). Furthermore, exposure of SP(1-11) to phorbol myristate acetate preactivated neutrophils resuited in products similar to those observed upon exposure to superoxide or hydroxyl radicals in a cell-free HX/xanthine oxidase system. This study suggests that, in contrast to rigid proteins, fragmentation is relatively easily induced in a small peptide like SP(1-11), perhaps due to strain on the peptide and t-carbon bonds caused by the movable, random coil configuration acquired by SP(1-11) in an aqueous solution. Oxidative modification might modulate paracrine actions of SP(1-11) at site of inflammation.     

Angiotensin converting enzyme (ACE) and neprilysin hydrolyze neuropeptides: a brief history, the beginning and follow-ups to early studies

Our investigations started when synthetic bradykinin became available and we could characterize two enzymes that cleaved it: kininase I or plasma carboxypeptidase N and kininase II, a peptidyl dipeptide hydrolase that we later found to be identical with the angiotensin I converting enzyme (ACE). When we noticed that ACE can cleave peptides without a free C-terminal carboxyl group (e.g., with a C-terminal nitrobenzylamine), we investigated inactivation of substance P, which has a C-terminal Met(11)-NH(2). The studies were extended to the hydrolysis of the neuropeptide, neurotensin and to compare hydrolysis of the same peptides by neprilysin (neutral endopeptidase 24.11, CD10, NEP). Our publication in 1984 dealt with ACE and NEP purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln(6)-Phe(7), Phe(7)[see text]-Phe(8), and Gly(9)-Leu(10) and neurotensin (NT) at Pro(10)-Tyr(11) and Tyr(11)-Ile(12). Purified ACE also rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe(8)-Gly(9) and Gly(9)-Leu(10) to release C-terminal tri- and dipeptide (ratio = 4:1). The hydrolysis was Cl(-) dependent and inhibited by captopril. ACE released only dipeptide from SP free acid. ACE hydrolyzed NT at Tyr(11)-Ile(12) to release Ile(12)-Leu(13). Then peptide substrates were used to inhibit ACE hydrolyzing Fa-Phe-Gly-Gly and NEP cleaving Leu(5)-enkephalin. The K(i) values in microM were as follows: for ACE, bradykinin = 0.4, angiotensin I = 4, SP = 25, SP free acid = 2, NT = 14, and Met(5)-enkephalin = 450, and for NEP, bradykinin = 162, angiotensin I = 36, SP = 190, NT = 39, Met(5)-enkephalin = 22. These studies showed that ACE and NEP, two enzymes widely distributed in the body, are involved in the metabolism of SP and NT. Below we briefly survey how NEP and ACE in two decades have gained the reputation as very important factors in health and disease. This is due to the discovery of more endogenous substrates of the enzymes and to the very broad and beneficial therapeutic applications of ACE inhibitors.     

Purification of the main somatostatin-degrading proteases from rat and pig brains, their action on other neuropeptides, and their identification as endopeptidases 24.15 and 24.16.

The main somatostatin-degrading proteases were purified from rat and pig brain homogenates and characterized as thiol- and metal-dependent endoproteases. Two types of proteases with apparent native and subunit molecular masses of 70 kDa and 68 kDa could be differentiated in both species. Beside somatostatin, both hydrolyzed several other neuropeptides with chain lengths between 8 and 30 amino acid residues. Cleavage sites were generally similar or identical, but some clear exceptions were observed for enzymes from both species which could be used to differentiate between the two proteases. The 68-kDa protease cleaved somatostatin at three bonds (Asn5-Phe6, Phe6-Phe7 and Thr10-Phe11) and neurotensin only at the Arg8-Arg9 bond, whereas the 70-kDa protease digested somatostatin at only two bonds (Phe6-Phe7 and Thr10-Phe11) and neurotensin as well as acetylneurotensin-(8-13) additionally (pig protease) or almost exclusively (rat protease) at the Pro10-Tyr11 bond. Relative rates for the digestions of various peptides were, however, more dependent on the species than on the type of protease. Cleavage sites for angiotensin II, bradykinin, dynorphin, gonadoliberin and substance P were, apart from different rates, identical for both proteases. In both species the 68-kDa protease was found to be mainly, but not exclusively, soluble and not membrane-associated, whereas the inverse was detected for the 70-kDa protease. Based on distinct molecular and catalytic properties, the 68-kDa protease is supposed to be congruent with the endopeptidase 24.15 (EC 3.4.24.15), the 70-kDa protease with endopeptidase 24.16 (EC 3.4.24.16, neurotensin-degrading endopeptidase). This investigation demonstrates that both proteases hydrolyze various neuropeptides with similar cleavage sites, but with species-dependent activity. Species-independent distinctions are the exclusive action of endopeptidase 24.16 on acetylneurotensin-(8-13) and liberation of free Phe from somatostatin only by endopeptidase 24.15.     

Chemical replacement of P1' arginine residue at the first reactive site of peanut protease inhibitor B-III

The contribution of the P1' residue at the first reactive site of peanut protease inhibitor B-III to the inhibition was analyzed by replacement of the P1' Arg(11) with other amino acids (Arg, Ser, Ala, Leu, Phe, Asp) after selective modification of the second reactive site. The Arg derivative had the same trypsin inhibitory activity as the native inhibitor (Ki = 2 X 10(-9) M). The Ser derivative inhibited more weakly (Ki = 2 X 10(-8) M). The Ala and Leu derivatives inhibited trypsin very weakly (Ki = 2 X 10(-7) M and 4 X 10(-7) M, respectively), and the Phe and Asp derivatives not at all. These results suggest that the P1' arginine residue is best for inhibitory activity at the first reactive site of B-III, although it has been suggested that a P1' serine residue at the reactive site is best for inhibitory activity of Bowman-Birk type inhibitors.     

Effects of salinomycin and vitamin B(6) on in vitro metabolism of phenylalanine and its related compounds by ruminal bacteria,protozoa and their mixture

An in vitro study was conducted to examine the effects of salinomycin (SL) and vitamin B(6) (B(6)) on the production of phenylalanine (Phe) from phenylpyruvic acid (PPY) and phenylacetic acid (PAA) and of PAA from Phe and PPY by mixed rumen bacteria (B), mixed rumen protozoa (P) and their mixture (BP). Rumen microorganisms were collected from fistulated goats fed lucerne cubes (Medicago sativa) and a concentrate mixture (3 : 1) twice a day. Microbial suspensions were anaerobically incubated at 39 degrees C for 12 h. Phe and some other related compounds in both supernatants and microbial hydrolysates of the incubations were analyzed by HPLC. When PPY was used as a substrate, it completely disappeared without additives and converted mainly to Phe and PAA on the average by 396 and 178, 440 and 189, and 439 and 147 &mgr;M in B, P and BP, respectively, during the 12 h incubation period. The rate of disappearance showed no significant differences between the microbial suspensions with and without SL and B(6) during the incubation period. The production of Phe from PPY with SL was enhanced (p<0.05) by 40, 20 and 19% in B, P and BP, respectively, while PAA production from PPY with SL was inhibited (p<0.05) by 35, 37 and 38% in B, P and BP, respectively, during the 12 h incubation period. On the other hand, with B(6), the production of Phe and PAA from PPY tended to be enhanced by 14 and 17, 9 and 11, and 7 and 22% in B, P and BP, respectively, during the 12 h incubation period. When PAA added as a substrate was incubated in the incubation medium without any additives, it disappeared by 483, 462 and 507 &mgr;M and converted mainly to Phe on the average by 231, 244 and 248 &mgr;M in B, P and BP, respectively. The disappearance of PAA with SL was inhibited (p<0.05) by 16, 15 and 20%, in B, P and BP, respectively, whereas the disappearance of PAA with B6 was almost the same as that without B(6) in B and BP suspensions but tended to be enhanced by more than 9% in P suspensions during the 12 h incubation period. The production of Phe from PAA with SL tended to be inhibited by 12, 11 and 8% in B, P and BP, respectively, during the 6 h incubation period, but the inhibition was weakened during the 12 h incubation period, whereas Phe production from PAA with B(6) tended to be enhanced by 13, 16 and 8% in B, P and BP, respectively. When Phe was added as a substrate, the net Phe disappearance without additives was 549, 365 and 842 &mgr;M and converted mainly to PAA on the average by 254, 205 and 461 &mgr;M in B, P and BP, respectively. The net disappearance of Phe with SL was inhibited (p<0.05) by 38, 28 and 46%, whereas the net disappearance of Phe with B(6) was enhanced (p<0.05) by 9, 8 and 7% in B, P and BP, respectively. The production of PAA from Phe with SL was inhibited (p<0.05) by 73, 54 and 76% in B, P and BP, respectively. On the other hand, with B(6), PAA production from Phe was enhanced (p<0.05) by 19, 18 and 20% in B, P and BP, respectively. Based on these results, it seems that SL inhibited Phe disappearance and enhanced the synthesis of Phe from PPY, though not from PAA, and accumulated free Phe in the medium, whereas B(6) also enhanced Phe synthesis both from PPY and PAA, which could provide additional amino N for animals.     

Comparative study of substance P and its fragments: analgesic properties, effect on behavior and monoaminergic processes

The effect of substance P (SP) and of its fragments 5-11, 8-11, 9-11, 10-11 administered into the brain ventricles in doses of 5, 25 and 50 nM on the behavior and content of biogenic monoamines of the rat brain was studied. The analgetic properties of the substances under consideration and those of fragment SP 10-11 in doses of 5, 25, 50 and 100 nM were also subjected to examination. It was found that SP and fragment 5-11 stimulate and enhance the locomotor activity in rats, while fragments 8-11 and 9-11 provoke hypoactivity. The substances under study increase the serotonin and dopamine turnover, whereas SP and fragment 8-11 lower the serotonin content as well. After administration of SP and fragment 5-11 analgesia was seen to transform to hyperalgesia depending on the dose. Fragments 8-11 and 9-11 produce analgetic effect. It is suggested that both SP fragments and the whole SP molecule can influence the neurochemical process that regulate behavior and pain perception.     

High-resolution NMR studies of fibrinogen-like peptides in solution: structure of a thrombin-bound peptide corresponding to residues 7-16 of the A alpha chain of human fibrinogen

The interaction of the following human fibrinogen-like peptides with bovine thrombin was studied by one- and two-dimensional NMR techniques in aqueous solution: acetyl-Phe(8)-Leu(9)-Ala(10)-Glu-(11)-Gly(12)-Gly(13)-Gly(14)-Val(15)-Ar g(16)- Gly(17)-Pro(18)-NHMe (F6), acetyl-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16) (tF6), acetyl-Asp(7)-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16)-Gly(17)-Pro- Arg(19)-Val(20)-NHMe (F8), and acetyl-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16) (tF8). At pH 5.3 and 25 degrees C, the Arg(16)-Gly(17) peptide bonds in both F6 and F8 were cleaved instantaneously in the presence of 0.5 mM thrombin, producing truncated peptides tF6 and tF8 and other peptide fragments. On the basis of observations of line broadening, thrombin was found to bind to the cleavage products, tF6 and tF8, of peptides F6 and F8. Peptide tF8 may have a higher affinity for thrombin than peptide tF6, as suggested by the more pronounced thrombin-induced line broadening on the proton resonances in peptide tF8. Transferred NOE (TRNOE) measurements were made of the complexes between thrombin and peptides tF6 and tF8. Medium- and long-range NOE interactions were found between the NH proton of Asp(7) and the C beta H protons of Ala(10), between the C alpha H proton of Glu(11) and the NH proton of Gly(13), and between the ring protons of Phe(8) and the C alpha H protons of Gly(14) and the C gamma H protons of Val(15). Sets of structures of the decapeptide tF8 were deduced by use of distance geometry calculations based on sequential and medium- and long-range TRNOEs from the thrombin-bound peptide. A predominant feature of these structures is the nonpolar cluster formed by the side chains of residues Phe(8), Leu(9), and Val(15) that are directly involved in binding to thrombin. This structural feature is brought about by an alpha-helical segment involving residues Phe(8)-Ala(10), followed by a multiple-turn structure involving residues Glu(11)-Val(15). These results provide an explanation for the observations that Asp(7), Phe(8), and Gly(12) are strongly conserved in mammalian fibrinogens and that the mutations of Asp(7) to Asn(7) and of Gly(12) to Val(12), result in delayed release of fibrinopeptide A, producing human bleeding disorders.     

A comparison of the effects of substance P and shorter analogues on the synaptosomal ATPases activities in the rat brain

The influence in vitro of SP and C-terminal fragments of analogues SP(5-11) (pyroGlu5, Tyr8); SP(6-11) (pyroGlu6, Tyr8); SP(6-11) (pyroGlu6, D-Phe7); SP(6-11) (pyroGlu6, D-Phe8) on the (Ca, Mg) and (Na, K) ATPases activities from synaptosomal membranes of cerebral cortex and hippocampus of rat brain were compared. The data obtained in this study indicate the following: 1. Substance P stimulates the activities of (Na, K) and (Ca, Mg) ATPases more effectively in synaptosomal membranes from hippocampus than cerebral cortex. 2. Heptapeptide SP(5-11) (pyroGlu5, Tyr8) causes a more distinct increase of (Ca, Mg) ATPase activity in cortical synaptosomal membranes than SP does. 3. The change of L-Phe conformation to D in position 7 in hexapeptide induces reduction of enzymes activities in hippocampus. 4. Especially important for the maintenance of biological activity of drugs is the replacement of Gln5 with pyroGlu6 and conformation of Phe residues. 5. SP and shorter analogues of fragments SP C-terminal SP regulate the active cation transport in synaptosomal membranes of cerebral cortex and hippocampus.