Isolation and characterization of a specific endogenous Na+,K+-ATPase inhibitor from bovine adrenal

In order to identify a specific endogenous Na+,K+-ATPase inhibitor which could possibly be related to salt-dependent hypertension, we looked for substances in the methanol extract of bovine whole adrenal which show all of the following properties: (i) inhibitory activity for Na+,K+-ATPase; (ii) competitive displacing activity against [3H]ouabain binding to the enzyme; (iii) inhibitory activity for 86Rb uptake into intact human erythrocytes; and (iv) cross-reactivity with sheep anti-digoxin-specific antibody. After stepwise fractionation of the methanol extract of bovine adrenal glands by chromatography on a C18 open column, a 0-15% acetonitrile fraction was fractionated by high-performance liquid chromatography on a Zorbax octadecylsilane column. One of the most active fractions in 0-15% acetonitrile was found to exhibit all of the four types of the activities. It was soluble in water and was distinct from various substances which have been known to inhibit Na+,K+-ATPase such as unsaturated free fatty acids, lysophosphatidylcholines, vanadate, dihydroxyeicosatrienoic acid, dehydroepiandrosterone sulfate, dopamine, lignan, ascorbic acid, etc. This substance was further purified by using an additional five steps of high-performance liquid chromatography with five different types of columns. Molecular mass was estimated as below 350 by fast atom bombardment mass spectroscopy and ultrafiltration. Heat treatment at 250 degrees C for 2 h and acid treatment with 6 N HCl at 115 degrees C for 21 h almost completely destroyed the inhibitory activity of the purified substance for Na+ pump activity. Additionally, alkaline treatment with 0.2 N NaOH at 23 degrees C for 2 h destroyed approximately 70% of the inhibitory activity, whereas boiling for 10 min and various enzyme digestion did not destroy the activity. The dose dependency for the four types of the activities for this substance paralleled those of ouabain, spanning 2 orders of magnitude in concentration range. The inhibitory potencies of the purified substance for Na+,K+-ATPase, Na+ pump, and ouabain binding activities were diminished with increasing K+ concentration, exhibiting a characteristic typical of cardiac glycosides. This substance had no effect on the Ca2+-ATPase activity or the Ca2+ loading rate into the vesicle prepared from skeletal muscle sarcoplasmic reticulum. These results strongly suggest that this water-soluble nonpeptidic Na+,K+-ATPase inhibitor may be a specific endogenous regulator for the ATPase.     

Angiotensin I converting enzyme (ACE) inhibitory peptide derived from the sauce of fermented blue mussel, Mytilus edulis

The angiotensin I converting enzyme (ACE) inhibitory activity of fermented blue mussel sauce (FBMS) was investigated. Blue mussels were fermented with 25% NaCl (w/w) at 20 degrees C for 6 months and the resultant mixture was passed through a 40-mesh sieve, desalted using an electrodialyzer and then lyophilized. The IC(50) value of FBMS for ACE activity was 1.01 mg/ml. An ACE inhibitory peptide was purified from FBMS using Sephadex G-75 gel chromatography, SP-Sephadex C-25 ion exchange chromatography and reversed-phase high-performance liquid chromatography on a C(18) column. The IC(50) value of purified ACE inhibitory peptide was 19.34 microg/ml, and 10 amino acid residues of the N-terminal sequence was EVMAGNLYPG. The purified peptide was evaluated for antihypertensive effect in spontaneously hypertensive rats (SHR) following oral administration. Blood pressure significantly decreased after peptide ingestion. This result suggested that FBMS may have beneficial effects on hypertension.     

Purification and characterization of an antimicrobial peptide produced by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain 4B

An antimicrobial peptide produced by a bacterium isolated from the effluent pond of a bovine abattoir was purified and characterized. The strain was characterized by biochemical profiling and 16S rDNA sequencing as Pseudomonas sp. The antimicrobial peptide was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. A major band on SDS-PAGE suggested that the antimicrobial peptide has a molecular mass of about 30 kDa. The substance was inhibitory to a broad range of indicator strains, including pathogenic and food spoilage bacteria such as Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, among other. The partially purified antimicrobial substance remained active over a wide temperature range and was resistant to all proteases tested. This substance showed different properties than other antimicrobials from Pseudomonas species, suggesting a novel antimicrobial peptide was characterized.     

An unknown endogenous inhibitor of Na/Ca exchange can enhance the cardiac muscle contractility

The cardiac sarcolemma Na/Ca exchanger is a key system for controlling the intracellular calcium levels during the excitation-contraction coupling. Here, we test the hypothesis that the heart tissue contains a putative endogenous factor having a capacity to modulate the Na/Ca exchanger and muscle contractility. The concentrated cardiac extracts inhibit the Na(i)- or Ca(i)-dependent (45)Ca uptakes in isolated cardiac sarcolemma vesicles as well as the Na(o)-dependent Ca efflux, monitored by extravesicular Ca probe fluo-3. The inhibitory activity has been purified approximately 2000-fold by normal and reversed-phase HPLC procedures. The inhibitory activity is eluted from the Sephadex G-10 in the range of 350-550 Da, suggesting that the inhibitory factor is a low-molecular-weight substance. The mass spectra analysis shows a number of signals within m/z 380-560; however, it is not clear at this moment whether these recordings represent the mass of putative inhibitory factor or irrelevant impurities. The endogenous inhibitory factor of Na/Ca exchange does not resemble the properties (HPLC retention time, mass spectra, amino acid analysis, etc.) of autoinhibitory XIP peptide. The addition of inhibitory factor to muscle strip of guinea pig ventricles induces 2- to 5-fold enhancement of isometric contractions, thereby exhibiting a strong positive inotropic effect. This effect is a dose-dependent phenomenon, which can be reversed by washing the inhibitory factor from the organ bath. Assuming a molecular weight of 350-550 Da, the effective concentrations of putative inhibitor must be <10(-6) M. Therefore, the present findings demonstrate that the mammalian heart contains a low-molecular-weight factor that can inhibit Na/Ca exchange and enhance the cardiac contractility.     

Isolation and characterization of a novel angiotensin I-converting enzyme inhibitory peptide derived from the edible mushroom Tricholoma giganteum

The fruiting body of Tricholoma giganteum has many pharmaceutical uses and has long been utilized as a home remedy in Asia. This study describes the extraction and characterization of the first angiotensin I-converting enzyme (ACE) inhibitory peptide from T. giganteum. The maximum ACE inhibitory activity (IC50: 0.31 mg) was obtained when the fruiting body of T. giganteum was extracted with distilled water at 30 degrees C for 3 h. After the purification of ACE inhibitory peptides with ultrafiltration, Sephadex G-25 column chromatography, and reverse-phase HPLC, an active fraction with an IC50 of 0.04 mg and a yield of 0.3% was obtained. The ACE inhibitory peptide was a novel tripeptide, showing very low similarity to other ACE inhibitory peptide sequences, and was sequenced as Gly-Glu-Pro. The purified ACE inhibitor from T. giganteum competitively inhibited ACE, and it maintained inhibitory activity even after incubation with proteases. ACE inhibitor from T. giganteum showed a clear antihypertensive effect in spontaneously hypertensive rats (SHR), at a dosage of 1 mg/kg.     

Further biochemical characterization of an Na+ pump inhibitor purified from human urine

An increase in endogenous Na+,K+-ATPase inhibitor(s) with digitalis-like properties has been reported in chronic renal insufficiency, in Na+-dependent experimental hypertension and in some essential hypertensive patients. The present study specifies some properties and some biochemical characteristics of a semipurified compound from human urine having digitalis-like properties. The urine-derived inhibitor (endalin) inhibits Na+,K+-ATPase activity and [3H]-ouabain binding, and cross-reacts with anti-digoxin antibodies. The inhibitory effect on ATPases of endalin is higher on Na+,K+-ATPase than on Mg2+-ATPase and Ca2+-ATPase. The mechanism of endalin action on highly purified Na+,K+-ATPase was compared to that of ouabain and was similar in that it reversibly inhibited Na+,K+-ATPase activity; it inhibited Na+,K+-ATPase non-competitively with ATP; its inhibitory effect was facilitated by Na+; K+ decreased its inhibitory effect on Na+,K+-ATPase; it competitively inhibited ouabain binding to the enzyme; its binding was maximal in the presence of Mg2+ and Pi; it decreased the Na+ pump activity in human erythrocytes; it reduced serotonin uptake by human platelets; and it was diuretic and natriuretic in rat bioassay. The endalin differed from ouabain in only three aspects: its inhibitory effect was not really specific for Na+,K+-ATPase; its binding to the enzyme was undetectable in the presence of Mg2+ and ATP; it was not kaliuretic in rat bioassay. Endalin is a reversible and partial specific inhibitor of Na+,K+-ATPase, its Na+,K+-ATPase inhibition closely resembles that of ouabain and it could be considered as one of the natriuretic hormones.     

The antihypertensive activity of angiotensin-converting enzyme inhibitory peptide containing in bovine lactoferrin

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from the bovine lactoferrin hydrolysate using peptic hydrolysis by 2-step of reverse-phase high-performance liquid chromatography. This peptide was identified as Leu-Arg-Pro-Val-Ala-Ala and it produced a concentration-dependent inhibition of ACE activity in vitro with an IC50 value of about 4.14 microM. Also, this inhibition was identified as noncompetitive from the Lineweaver-Burk plot. Moreover, the antihypertensive activity of Leu-Arg-Pro-Val-Ala-Ala was investigated by the intravenous injection into spontaneously hypertensive rats (SHRs). A dose-dependent reduction of systolic blood pressure by this peptide was observed at 60 min after injection and it maximally decreased the blood pressure at a rate of 1 nmol/ml/kg. The blood pressure lowering activity of this peptide was calculated as 210% of captopril (10 pmol/ml/kg) that was used as positive control. Otherwise, identification of this peptide in the blood of SHRs was carried out chromatographically. Reduction of blood pressure coincides with the peak peptide concentration in the serum. Thus, we conclude that this peptide inhibits ACE activity in vitro and lowers systolic blood pressure in spontaneously hypertensive rat.     

Characteristics of an inhibitor of the Na+/K+ pump in human cerebrospinal fluid

Human cerebrospinal fluid (CSF) inhibits the Na+/K+ pump in human red cells and the activity of purified Na+/K+-ATPase (Halperin, J. A., Shaeffer, R., Galvez, L., and Malavé, S. (1985) Proc. Natl. Acad. Sci. U.S. A. 80, 6102-6104, 1983; Halperin, J. A., Martin, A. M., and Malavé, S. (1985) Life Sci. 37, 561-566. We describe here some properties of the CSF inhibitor of the Na+/K+ pump. Active material was extracted from human CSF with 50% methanol and then concentrated and desalted by ultrafiltration. This extract inhibited, in a dose-dependent manner, the ouabain-sensitive influx of K+ into human red cells and the activity of purified Na+/K+-ATPase. Partial separation of the inhibitory activity was achieved by gel filtration and reverse-phase high performance liquid chromatography. Inhibition of both pump and enzyme was specific in that other red cell membrane transport systems or enzymes examined were not influenced by CSF extracts. Dialysis and ultrafiltration experiments indicate that the molecular weight of the inhibitor is approximately equal to 600. The inhibitory activity is sensitive to proteolytic enzymes indicating that the inhibitor might be a small peptide. In the presence of CSF extract the K0.5 for external K+ to stimulate the Na+/K+ pump increased from 1.4 to 3.1 mM, suggesting that the CSF inhibitor competes with external K+ for stimulation of the pump. We estimate that the concentration of the inhibitor in CSF might be approximately equal to 50 pg/ml, a value close to the concentration of other active peptides found in human CSF.     

Detection of secreted antimicrobial peptides isolated from cell-free culture supernatant of Paenibacillus alvei AN5

An antimicrobial substance produced by the Paenibacillus alvei strain AN5 was detected in fermentation broth. Subsequently, cell-free culture supernatant (CFCS) was obtained by medium centrifugation and filtration, and its antimicrobial activity was tested. This showed a broad inhibitory spectrum against both Gram-positive and -negative bacterial strains. The CFCS was then purified and subjected to SDS-PAGE and infrared spectroscopy, which indicated the proteinaceous nature of the antimicrobial compound. Some de novo sequencing using an automatic Q-TOF premier system determined the amino acid sequence of the purified antimicrobial peptide as Y-S-K-S-L-P-L-S-V-L-N-P (1,316 Da). The novel peptide was designated as peptide AN5-1. Its mode of action was bactericidal, inducing cell lysis in E. coli ATCC 29522 and S. aureus, and non-cell lysis in both S. marcescens and B. cereus ATCC 14579. Peptide AN5-1 displayed stability at a wide range of pH values (2–12) and remained active after exposure to high temperatures (100 °C). It also maintained its antimicrobial activity after incubation with chemicals such as SDS, urea and EDTA.     

Isolation and characterization of a novel platelet aggregation inhibitory peptide from the medicinal mushroom, Inonotus obliquus

This study describes the extraction and characterization of a platelet aggregation inhibitory peptide from Inonotus obliquus. Ethanol extract from I. obliquus ASI 74006 mycelia showed the highest platelet aggregation inhibitory activity (81.2%). The maximum platelet aggregation inhibitory activity was found when the mycelia of I. obliquus ASI 74006 was extracted with ethanol at 80 degrees C for 12 h. The platelet aggregation inhibitor was purified by systematic solvent fractionation, ultrafiltration, Sephadex G-10 column chromatography, and reverse-phase HPLC. The purified platelet aggregation inhibitor is a novel tripeptide with a molecular mass of 365 Da, having a sequence of Trp-Gly-Cys. The purified platelet aggregation inhibitor also showed high platelet aggregation inhibitory activity in Institute of Cancer Research (ICR) mice.     

Purification and partial chemical characterization of the antimicrobial peptide cerein 8A

AIMS: To purify and to characterize the antimicrobial compound cerein 8A. METHODS AND RESULTS: Cerein 8A was isolated by ammonium sulfate precipitation, 1-butanol extraction and ion-exchange chromatography. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. The purified substance corresponded to a 26 kDa peptide band. The native protein eluted at the void volume of Sephadex G-100, but within the included volume when a 1.5 mol l(-1) NaCl buffer was used, indicating that cerein 8A aggregates extracellularly. The antimicrobial activity was lost by treatment with proteases and heat. The ultraviolet spectrum was typical of a polypeptide and the infrared spectrum indicates that the peptide contains acyl group(s) in its structure. Intact Bacillus cereus spores were sensitive to cerein 8A at 1600 AU ml(-1). CONCLUSIONS: Cerein 8A show distinct properties from other antimicrobial peptides of B. cereus, and has a significant inhibitory effect on spores. Significance and Impact of the Study: The characterization of a substance active against important pathogens addresses an important aspect of food safety.     

Inhibition of triosephosphate isomerase from Trypanosoma brucei with cyclic hexapeptides.

Two series of oligopeptides have been synthesized. Their effects on the activity of purified triosephosphate isomerase from Trypanosoma brucei and various other organisms have been studied. Using detailed three-dimensional structure information, the first series consisted of both cyclic and linear hydrophilic peptides that were designed to mimic the beta turns of the subunit interface loops of the trypanosome triosephosphate isomerase dimer. None of these exerted any inhibitory effect. The second series consisted of more hydrophobic cyclic peptides, originally designed to inhibit a hepatic transport system. Several of these were very effective in inhibiting the trypanosome triosephosphate isomerase, but not the homologous enzymes from rabbit, dog, yeast or Escherichia coli. The most active peptide, cyclo[-Trp-Phe-D-Pro-Phe-Phe-Lys(Z)-], exerted 50% inhibitory activity at a concentration of 3 microM. The nature of the inhibitory action of one of these compounds cyclo[-Trp-Tyr(OSO3Na)-D-Pro-Phe-Thr(OSO3Na)-Lys(Z)-] was studied in more detail. Its inhibition was noncompetitive and reversible and more than one peptide was able to bind/active site.     

Substance P induces alterations on cerebral lipids involved in membrane fluidity

This study was undertaken to examine the variations in rat brain of cholesterol, phospholipid and phospholipid fatty acid composition induced by substance P. The cholesterol content was increased by substance P; concomitantly, an increase of the ratio cholesterol/phospholipid was observed. These changes do not appear to be responsible of the stimulation observed in Na+,K+-ATPase activity by substance P action. Phospholipid fatty acid analysis revealed that the peptide induced a decrease in both linoleic and arachidonic acids content.     

Localization of pancreatic enzyme secretion-stimulating activity and trypsin inhibitory activity in zymogen granule of the rat pancreas

The intracellular localization of pancreatic enzyme secretion-stimulating activity in rat pancreas was investigated. We found and purified a pancreatic enzyme secretion-stimulating peptide from rat bile/pancreatic juice. The peptide is trypsin-sensitive (showing temporary trypsin inhibitory activity), and it is hypothesized that it acts as a trypsin-sensitive mediator in the feedback regulation of diet-induced pancreatic enzyme secretion. The zymogen granule fraction was purified 5-fold by ultracentrifugation by the Percoll density gradient method. The purity of the zymogen granule fraction was determined from the specific amylase activity and electron microscopic morphology. The specific enzyme activities of amylase and trypsin and the trypsin inhibitory activity increased in parallel during the purification, and the pancreatic enzyme secretion-stimulating activity was also localized in the zymogen granule fraction. These results suggest that the pancreatic enzyme secretion-stimulating peptide originates from the acinar cells, and that it is secreted through exocytosis of zymogen granules into the small intestine, its ratio to trypsin thus remaining constant. This idea supports our hypothesis that the stimulating peptide acts as a mediator for the feedback regulation of pancreatic enzyme secretion by trypsin.     

Isolation and identification of hemin as an endogenous Na+/K(+)-ATPase inhibitor from porcine blood cells.

A substance which is a potent inhibitor of Na+/K(+)-ATPase activity and competitively displaces [3H]ouabain binding to this enzyme was isolated from porcine blood cells. From its chemical and physiochemical properties, this activity was identified as hemin (chloroprotohemin IX). Hemin showed a dose dependent curve for Na+/K(+)-ATPase inhibitory activity similar to that of ouabain and displaced [3H]ouabain binding as potent as 1/100 of ouabain itself.     

Novel peptide inhibitor (SPAI) of Na+, K+-ATPase from porcine intestine

Three unique inhibitors (SPAI-1, -+2, and -3) were first purified from porcine duodenal extract based on the Na+, K+-ATPase inhibitory activity. These peptide inhibitors had four disulfide bridges in common. The sequencing results of their S-carboxymethyl derivatives, lysilendopeptidase fragments, and chymotryptic peptides disclosed their entire primary structures. Both SPAI-2 and -3 consisted of 61 amino acids, respectively, and had almost the same sequences except for two amino acid substitutions, while SPAI-1 was found to lack the N-terminal twelve amino acid sequence of SPAI-2. The kinetics study revealed that SPAIs inhibited Na+, K+-ATPase by the competitive mode against Na+ and were uncompetitive with K+.     

High molecular weight transglutaminase inhibitor produced by a microorganism (Streptomyces lavendulae Y-200)

Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena such as blood clotting, wound healing, apoptosis, and cell differentiation. Streptomyces lavendulae Y-200, isolated from soil, produced a substance that inhibited transglutaminases. The inhibitory substance was purified from the cultured medium by procedures of acid precipitation, deoxyribonuclease treatment, and gel filtration chromatography. The partially purified sample was dark brown. The inhibitory activity was stable under acidic, alkaline, and high temperature conditions, and resistant to the treatment with proteinases such as trypsin and Pronase. The molecular weight of the inhibitory substance was estimated to be between 10(4) and 10(5) from its permeability through ultrafilter membranes. The acid hydrolysate of the inhibitory substance contained amino acids and sugars. The inhibitory substance inhibited both calcium-dependent and calcium-independent transglutaminases in a competitive manner with a glutamine substrate. The extent of inhibition caused by the calcium-dependent transglutaminase increased with increasing calcium concentration. The results obtained here may help identify a novel regulatory substance of transglutaminase in biological systems.     

Purification and Characterization of the Antioxidative Substance Produced by Aspergillus sojae K

An antioxidative substance produced by Aspergillus sojae K is absolutely soluble in water and strongly inhibits autoxidation of Na-ascorbate. The substance, produced extracellularly in the culture ftuid by the mold, was purified by ion exchange chromatography, gel filtration, and HPLC. The substance was purified 34-fold with an activity recovery of 38% from culture fluid. Purity of the substance was confirmed with a single peak through HPLC using an analytical column as well as a single spot on TLC. The purified substance consists of equimolar aspartic acid and glycine, indicating that the substance is a peptide. From mass spectral analysis the molecular weight was 710, but the precise sequence of the amino acids is not clear. The substance is stable at 70°C, but about 80% of the activity was lost at 80°C after 60 min. Besides, the substance is completely stable at pH 3–14. This substance efficiently suppressed the oxidation of fish oil.     

A lymphocyte chemotactic peptide released from immunoglobulin G by neutrophil neutral thiol protease.

A thermostable and dialyzable peptide, released from rabbit IgG by rabbit neutrophil neutral thiol protease, exhibited a distinct chemotactic activity for rat lymphocytes; it was assumed to be derived from the Fc fragment (but not from the Fab fragment) by the enzyme. This substance seemed to be effective for adherent cells (B cells) from rat spleen, but not for nonadherent cells (T cells). The chemotactic peptide was purified by gel filtration on Sephadex G-50 and G-15 and then by high-voltage paper electrophoresis. As previously described, the IgG residue after release of dialyzable peptide(s) exhibited chemotactic activity for neutrophils but not for macrophages.     

Involvement of the Na+-independent Cl-/HCO3- exchange (AE) isoform in the compensation of myocardial Na+/H+ isoform 1 hyperactivity in spontaneously hypertensive rats

Enhanced activity of Na+/H+ isoform 1 (NHE-1) and the Na+-independent Cl-/HCO3- exchange (AE) is a feature of the hypertrophied myocardium in spontaneously hypertensive rats (SHR). The present study explored the possibility that sustained intracellular acidosis due to increased myocardial acid loading through AE causes NHE-1 enhancement. To this aim, SHR were treated for 2 weeks with a rabbit polyclonal antibody against an AE3 isoform that was recently developed and proven to have inhibitory effects on myocardial AE activity. We then compared the AE activity in the left ventricle papillary muscles isolated from untreated SHR with antiAE3-treated SHR; AE activity was measured in terms of the rate of intracellular pH recovery after an intracellular alkali load was introduced. AE activity was diminished by approximately 70% in SHR treated with the antiAE3 antibody, suggesting that the AE3 isoform is a major carrier of acid-equivalent influx in the hypertrophied myocardium. However, the antibody treatment failed to normalize NHE-1 activity that remained elevated in the myocardium of normotensive rats. The data therefore rule out the possibility that NHE-1 hyperactivity in hypertensive myocardium was due to sustained intracellular acidosis induced by increased AE activity that characterizes SHR myocardial tissue.