Role of enzymes and inhibitors in leu-enkephalin metabolism in rabbit and human plasma

The hydrolysis of leucine enkephalin by the proteolytic enzymes present in human and rabbit plasma has been studied by kinetic and chromatographic techniques. Data obtained indicate the existence of noticeable intraspecific differences in the kinetics of leu-enkephalin degradation, and of formation of its hydrolysis by-products. The separation of the enzymes active on the substrate and of the inhibitors active on these enzymes evidences the existence of a species specific distribution of both groups of substances. Yet, the dissimilar kinetics of the substrate hydrolysis and of formation of its hydrolysis by-products appear to arise more from diversities in the competition between the enzymes present in plasma and in the role of inhibitors than from the differences in the enkephalin-degrading enzymes. It is suggested that differences observed may be related to the existence of species specific populations of the information-carrying plasma peptides.     

Enkephalin binding systems in human plasma III: Comparative protection of different peptides

We have investigated the hydrolysis and protection from hydrolysis of several peptides by plasma enzymes and by the plasma components previously described as inhibitors of enkephalins' hydrolysis. The results shown indicate that all the peptides actually hydrolyzed are also partially protected from hydrolysis by the enkephalin-protecting substances. Protection is fairly uniform for all the peptides tested, but considerably higher in the case of leu- and met-enkephalin, suggesting a partial specificity of the protecting substances towards opioid peptides.     

Mass Spectrometric Identification and Quantification of Hemorphins Extracted from Human Adrenal and Pheochromocytoma Tissue

Abstract: The hemorphins are a family of recently identified opioid receptor binding peptides derived from the proteolytic processing of the β, γ, δ, and ε chains of hemoglobin. They have previously been identified at high concentration in human pituitary glands and in the CSF of patients with cerebral bleeding. Hemorphins are potent inhibitors of angiotensin converting enzyme and therefore possibly have a role to play in blood pressure regulation. We report the presence of four hemorphin peptides in extracts of normal adrenal tissue and in pheochromocytoma tumors. The hemorphins were quantified and structurally characterized using mass spectrometry. High concentrations of hemorphins were found in all samples, comparable with the levels reported in the literature for pituitary and brain tissue.     

Effects of postnatal treatment with naloxone on plasma gonadotropin,prolactin, testosterone and testicular functions in male rats

Opioid peptides are implicated in the control of gonadotropin and prolactin secretion. The role of opioid antagonist naloxone and its effects on plasma gonadotropin, prolactin, testosterone levels and testicular hyaluronidase, acid phosphatase, [³H]uridine and thymidine incorporation, RNA, DNA and protein concentrations were evaluated in rats after administration of naloxone beginning day 1 through 21 and autopsied on 45, 60 and 90 days of age. Plasma gonadotropin and testosterone levels were significantly elevated after naloxone treatment. Testicular hyaluronidase and acid phosphatase activity increased till 60 days post treatment and declined thereafter. Concentrations of RNA and protein did not change significantly but the concentration of DNA declined at 45 and 60 days of age. These results suggest that endogenous opioid peptides exert regulatory influence on gonadotropin secretion which in turn control the testicular function in the male rat.     

Mechanisms of leu-enkephalin hydrolysis in human plasma

The present work describes the kinetics of enkephalin hydrolysis by plasma enzymes and the fragmentation pattern of both the parent peptide and of the first hydrolysis by-products. The degradation kinetics were followed by positive identification of the hydrolysis fragments by chromatographic methods, by amino acid analysis and by scintillation counting of tritium-labeled enkephalin. In addition, the results presented confirm the role of the low molecular weight plasma components in the control of the hydrolysis of the peripherally-released enkephalins.     

Proteinase inhibitors in aggregated forms of boar seminal plasma proteins

Boar seminal plasma proteins were separated by gel chromatography on Sephadex G-75 into five fractions (I–V). Serine proteinase inhibitors were found mainly in the protein fraction with relative molecular weight 5–25 kDa. Small amounts of these inhibitors were also found in the high molecular weight protein fraction (M_r>100 kDa). The protein fraction containing most of the proteinase inhibitory activity was further separated by RP HPLC. Isolated proteins were characterized by SDS electrophoresis and immunoblotting, N-terminal amino acid sequencing and by determination of the proteinase inhibitory activity. In the fraction containing proteinase inhibitors, also β-microseminoprotein (β-MSP), AQN 1 and lactoferrin were identified. The possible existence of complexes of protein components in the fraction with relative molecular weight 5–25 kDa was studied in detail using gel chromatographic separation on Sephadex G-50. A part of proteinase inhibitors with M_r 8 kDa was eluted together with AQN 1 spermadhesin. An interaction of isolated spermadhesin AQN 1 and proteinase inhibitor was shown.     

Relationship between plasma concentrations of immunoreactive beta-endorphin and food intake in rats

Periods of increased food intake in male rats were characterized by significant elevations in the plasma concentrations of immunoreactive beta-endorphin (beta-ep). Administration of 2-deoxy-D-glucose (400 mg/kg) produced rapid and concurrent increases in both food intake and plasma beta-ep. Administration of insulin (10 units/kg) produced large delayed increases in food intake but only modest delayed increases in plasma beta-ep. Spontaneous nocturnal feeding was associated with increased plasma beta-ep. Increases in daytime food intake in rats subjected to 24 hr of food deprivation were also characterized by elevated plasma beta-ep. In all cases examined, those feeding behaviors in male rats which were subject to inhibition by naloxone were characterized by elevated concentration of plasma beta-ep.     

Studies of Specificity and Inhibition of Human Cerebrospinal Fluid Dynorphin Converting Enzyme

Abstract

Dynorphin-converting activity was recently discovered in human cerebrospinal fluid.¹ This enzyme (hCSF-DCE) cleaves dynorphin A, dynorphin B and alpha-neoendorphin to release Leu-enkephalin-Arg⁶. To characterize the enzyme further we used several protease inhibitors, including N-peptidyl-O-acyl hydroxylamines which are known to act as potent irreversible inhibitors of serine and cysteine proteinases.^2-4

No irreversible inactivation occurred but strong, reversible effects on the dynorphin-converting activity by some of the inhibitors tested could be observed. Although, hCSF-DCE binds its substrates (dynorphin A and B) in the μM-mM concentration range, it exhibits high specificity in recognizing and cleaving the linkage between the two basic amino acids in the substrate sequence.     

The effects of different exercise training modalities on plasma proenkephalin Peptide F in women

Due to the important interactions of proenkephalin fragments (e.g., proenkephalin [107–140] Peptide F) to enhance activation of immune cells and potentially combat pain associated with exercise-induced muscle tissue damage, we examined the differential plasma responses of Peptide F to different exercise training programs. Participants were tested pre-training (T1), and after 8 weeks (T2) of training. Fifty-nine healthy women were matched and then randomly assigned to one of four groups: heavy resistance strength training (STR, n = 18), high intensity endurance training (END, n = 14), combined strength and endurance training (CMB, n = 17), or control (CON, n = 10). Blood was collected using a cannula inserted into a superficial vein in the antecubital fossa with samples collected at rest and immediately after an acute bout of 6 X 10 RM in a squat resistance exercise before training and after training. Prior to any training, no significant differences were observed for any of the groups before or after acute exercise. With training, significant (P ≤ 0.95) elevations were observed with acute exercise in each of the exercise training groups and this effect was significantly greater in the CMB group. These data indicate that in untrained women exercise training will not change resting of plasma Peptide F concentrations unless both forms of exercise are performed but will result in significant increases in the immediate post-exercise responses. Such findings appear to indicate adrenal medullary adaptations opioid production significantly altered with exercise training.     

Investigation of bradykinin metabolism in human and rat plasma in the presence of the dual ACE/NEP inhibitors GW660511X and omapatrilat.

Several studies have suggested that the accumulation of bradykinin, or that of one its metabolites BK1-8, is involved in the occurrence of side effects such as AE associated with the use of various ACEi. In this work a novel approach combining HPLC-UV on-line with oaTOF-MS and ICPMS was applied to investigate in human and rat plasma the metabolism of labelled BK (79/81 Br-Phe5) BrBK in the presence of two new dual ACE/NEP inhibitors (GW660511X and omapatrilat) currently under clinical trial. In human plasma the BrBK half-life values in the absence or in the presence of GW660511X (3.8 microM) or omapatrilat (32 nM) were 38.7 +/- 2.4, 51.2 +/- 4.7 and 114.7 +/- 9.3 min, respectively and BrBK was degraded into BrBK1-8, BrBK1-7, BrBK1-5 and Br-Phe. In the presence of inhibitors, however, the levels of these resultant metabolites were different. Unlike GW660511X, omapatrilat abolished the production of BrBK1-5 and BrBK1-7, suggesting a better ACE inhibition effect over GW660511X as no NEP activity was found. In addition the production of BrBK1-8 was enhanced in the presence of these inhibitors with a greater accumulation being observed with omapatrilat. The production of Br-Phe5 was reduced with GW660511X while no significant change was observed with omapatrilat after 4 h of incubation. In rat plasma the BrBK half-life values in the absence or in the presence of GW660511X (530 nM) or omapatrilat (50 nM) were 9.31 +/- 1.7, 22.06 +/- 3.1 and 25.3 +/- 1.7 min, respectively and BrBK was degraded into BrBK1-8, BrBK1-7, BrBK1-5 and Br-Phe5 plus BrBK2-9, BrBK4-8 and BrBK2-8 metabolites not found in human plasma. GW660511X and omapatrilat reduced the production of BrBK1-5 and BrBK1-7 with more effect being observed with omapatrilat. GW660511X and omapatrilat increased the production of both BrBK1-8 and Br-Phe5 but not that of BrBK4-8 and BrBK2-8. This study shows that the potency of GW660511X in comparison with omapatrilat is more than 100-fold lower in human, but less than 10-fold lower in rat plasma, suggesting that rat may not be a suitable in vivo model for the evaluation of ACE/NEP inhibition in relation to effects in humans.     

8-Hydroxyquinoline-based inhibitors of the Rce1 protease disrupt Ras membrane localization in human cells

Ras converting enzyme 1 (Rce1) is an endoprotease that catalyzes processing of the C-terminus of Ras protein by removing -aaX from the CaaX motif. The activity of Rce1 is crucial for proper localization of Ras to the plasma membrane where it functions. Ras is responsible for transmitting signals related to cell proliferation, cell cycle progression, and apoptosis. The disregulation of these pathways due to constitutively active oncogenic Ras can ultimately lead to cancer. Ras, its effectors and regulators, and the enzymes that are involved in its maturation process are all targets for anti-cancer therapeutics. Key enzymes required for Ras maturation and localization are the farnesyltransferase (FTase), Rce1, and isoprenylcysteine carboxyl methyltransferase (ICMT). Among these proteins, the physiological role of Rce1 in regulating Ras and other CaaX proteins has not been fully explored. Small-molecule inhibitors of Rce1 could be useful as chemical biology tools to understand further the downstream impact of Rce1 on Ras function and serve as potential leads for cancer therapeutics. Structure–activity relationship (SAR) analysis of a previously reported Rce1 inhibitor, NSC1011, has been performed to generate a new library of Rce1 inhibitors. The new inhibitors caused a reduction in Rce1 in vitro activity, exhibited low cell toxicity, and induced mislocalization of EGFP-Ras from the plasma membrane in human colon carcinoma cells giving rise to a phenotype similar to that observed with siRNA knockdowns of Rce1 expression. Several of the new inhibitors were more effective at mislocalizing K-Ras compared to a potent farnesyltransferase inhibitor (FTI), which is significant because of the preponderance of K-Ras mutations in cancer.     

Enzymes of serine metabolism in normal and neoplastic rat tissues

Enzymes involved in the pathway of de novo serine biosynthesis (L-phosphoserine aminotransferase) and in alternative pathways of serine utilization (L-serine hydroxymethyltransferase, L-serine dehydratase and L-serine aminotransferase) were assayed in normal adult and fetal rat tissues and in a range of transplantable sat tumors. Serine dehydratase and serine aminotransferase activities were essentially confined to normal adult liver and kidney, whereas phosphoserine aminotransferase and serine hydroxymethyltransferase activities showed a more ubiquitous tissue distribution. In particular, phosphoserine aminotransferase and serine hydroxymethyltransferase activities were appreciable in neoplastic tissues, in the absence of the other enzymes of serine utilization. The pattern of enzyme distribution suggests that the synthesis of serine de novo is metabolically coupled to its utilization for nucleotide biosynthesis in tumors of differing tissue origins.     

Mapping of human plasma kallikrein active site by design of peptides based on modifications of a Kazal-type inhibitor reactive site

Human plasma kallikrein (huPK) is a proteinase that participates in several biological processes. Although various inhibitors control its activity, members of the Kazal family have not been identified as huPK inhibitors. In order to map the enzyme active site, we synthesized peptides based on the reactive site (PRILSPV) of a natural Kazal-type inhibitor found in Cayman plasma, which is not an huPK inhibitor. As expected, the leader peptide (Abz-SAPRILSPVQ-EDDnp) was not cleaved by huPK. Modifications to the leader peptide at P'1, P'3 and P'4 positions were made according to the sequence of a phage display-generated recombinant Kazal inhibitor (PYTLKWV) that presented huPK-binding ability. Novel peptides were identified as substrates for huPK and related enzymes. Both porcine pancreatic and human plasma kallikreins cleaved peptides at Arg or Lys bonds, whereas human pancreatic kallikrein cleaved bonds involving Arg or a pair of hydrophobic amino acid residues. Peptide hydrolysis by pancreatic kallikrein was not significantly altered by amino acid replacements. The peptide Abz-SAPRILSWVQ-EDDnp was the best substrate and a competitive inhibitor for huPK, indicating that Trp residue at the P'4 position is important for enzyme action.     

Different Effects of Steroidal Therapy on Neuropeptide-Active Enzymes in Male and Female Human Saliva

The hydrolysis of a model neuropeptide (leucine enkephalin) was studied in the presence of saliva obtained from normal and allergic male and female volunteers in the absence and in the presence of steroidal treatment. Possible variations in the formation of substrate hydrolysis by-products were studied in whole samples and after steric exclusion chromatography fractionation. The results obtained confirm already-described variations in substrate hydrolysis in allergic as compared to control saliva, as well as the effect of steroidal treatment on the activity of the substrate-active enzymes. In addition, whereas in male saliva, therapy was associated with a net decrease of substrate hydrolysis, in female saliva hydrolysis remained near the levels measured in the absence of treatment. Finally, therapy induced modifications of enzyme apparent molecular weight distribution that appear to be similar for all substrate-active enzyme classes, but different in male and female saliva. In male saliva, therapy decreased the activity of the enzymes eluted at high apparent molecular weight, while it increased the activity of the enzymes of low apparent molecular weight. Because the increase was considerably less than the decrease, the net effect was to decrease the activity of the substrate-active enzymes, nearly to the low levels measured in the controls. In female saliva the therapy-associated decrease in the activity of the enzymes eluted at high apparent molecular weight was offset by the increase in the activity of those eluted at low apparent molecular weight, consequently, substrate hydrolysis remained near the level measured in the absence of treatment, a level that was higher than that measured in the controls.     

Competitive, uncompetitive, and mixed inhibitors of the alkaline phosphatase activity associated with the isolated brush border membrane of the tapeworm Hymenolepis diminuta

Several compounds were tested as inhibitors of the alkaline phosphatase (AlkPase) activity associated with the isolated brush border membrane of the tapeworm, Hymenolepis diminuta. Molybdate, arsenate, arsenite and beta-glycerophosphate (BGP) were competitive inhibitors of the hydrolysis of p-nitrophenyl phosphate, while levamisole and clorsulon were uncompetitive and mixed inhibitors, respectively. Molybdate was also a competitive inhibitor of the hydrolysis of BGP and 5'-adenosine monophosphate, and levamisole was an uncompetitive inhibitor of BGP hydrolysis. The apparent inhibitor constants (Ki') for molybdate and levamisole were virtually identical regardless of the substrate, and these data support the hypothesis that the AlkPase activity is represented by a single membrane-bound enzyme with low substrate specificity. Quinacrine, Hg2+, and ethylenediaminetetraacetate were also potent inhibitors of the AlkPase activity, but the mechanisms by which these latter three inhibitors function were not clear.     

Anionic and cationic components from protein aggregates in bovine seminal plasma and their effects of sperm motility

Bovine seminal plasma proteins are in an aggregated form of high molecular weight in their native state. By immobilisation on a cation exchanger with exposure to disaggregating conditions (i.e., acetonitrile and low pH), the high-molecular-weight aggregates could be dissociated to slowly release the low-molecular-weight components. The anionic component released from the cation exchanger during disaggregation was collected by adsorption on a hydrophobic interaction column. The cationic component remaining on the cation exchanger was eluted with NaOH. Both components were found on gel permeation chromatography to be <5 kDa. SDS—PAGE of the various fractions showed that component of low molecular weight were still in an aggregated form. These components resulting from the disaggregation process have detrimental effects on sperm motility and the effects were more substantial compared with that of whole seminal plasma. All the cationic components were significantly detrimental to sperm motility, especially the fractions of low molecular weight. The anionic fractions reduced sperm motility when in an aggregated state. The isolated anionic peptide was not detrimental in its free form. In all fractions the peptides tended to re-aggregate to a higher molecular weight under neutral conditions, however, the isolated anionic peptide (molecular weight <1,500) failed to do so. © Wiley-Liss, Inc.     

Large and small forms of cholecystokinin in human plasma: measurement using high pressure liquid chromatography and radioimmunoassay

Defining the role of cholecystokinin (CCK) in gut physiology and disease has proved difficult because of problems with development of radioimmunoassays and because CCK exists in several different molecular forms which have different biological actions. In order to measure small (8 amino acid residues, CCK 8) and large (33 and 39 residues) forms of CCK in plasma we have developed high pressure liquid chromatographic (HPLC) fractionation of plasma prior to radioimmunoassay. Fasting plasma CCK 8 and CCK $\text{33}\text{39}$ levels were usually undetectable (< 3 and < 6 pmol/1, respectively). After a liquid fat meal both CCK 8 and CCK $\text{33}\text{39}$ levels were significantly elevated at 5 min (11.3±3.3 and 11.6±2.6 pmol/1, respectively). Peak CCK 8 levels occurred at 30 min (15.0±4.4 pmol/1) while peak CCK $\text{33}\text{39}$ levels occurred at 120 min (16.7±4.9 pmol/1. Total CCK levels showed a biphasic response to the meal. These CCK 8 and CCK $\text{33}\text{39}$ responses to oral fat are consistent with a role for these hormones in the regulation of gallbladder emptying and pancreatic secretion.     

微囊藻属多肽类酶抑制剂研究进展

本文综述了微囊藻属蓝藻多肽类酶抑制剂的产生、结构特征、药理活性以及应用前景。