Purification and analysis of lung and plasma angiotensin I-converting enzyme by high-performance liquid chromatography

We purified angiotensin I-converting enzyme (ACE) from pig and human lung and plasma for comparison of some physicochemical properties between the endothelial membrane-bound form and the soluble form of the enzyme. After affinity chromatography on Sepharose CL-4B/lisinopril, gel-filtration HPLC on Superose 12 achieved homogeneity for both forms as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Whatever the source of ACE, the molecular weight was 300 +/- 40 kDa after calibration of Superose 12 with standard globular proteins and 172 +/- 4 kDa by SDS-PAGE, with or without reduction, a result suggesting interactions between the glycopolypeptide chain and the chromatographic gel possibly related to the overall shape and sugar content of the enzyme. Ion-exchange HPLC analysis on TSK-DEAE showed that the membrane-bound and soluble forms of ACE are not isoenzymes, although isoelectrofocusing did show that the isoelectric point of soluble ACE was lower than those of tissue ACE, suggesting a different glycosylation. No significant difference between porcine and human ACE appeared. HPLC methods seem to be of particular interest for the purification of ACE with a high yield and for the analysis of its putative differently glycosylated isoforms.     

Immunoaffinity purification and partial amino acid sequence analysis of catechol-O-methyltransferase from pig liver

Monoclonal antibodies (mAbs) against the soluble form (S-COMT) of catechol-O-methyltransferase (COMT, EC 2.1.1.6) were produced using a purified preparation of the enzyme from pig liver as antigen. The selected monoclonal antibodies recognized the enzyme with different capacities. One of them (Co60-1B/7) showed a significant cross reaction with S-COMT from rat and human liver. A protein band of 23 kDa was recognized by the mAbs on Western blots of the soluble fraction of pig liver. The mAbs were also able to recognize the membrane-bound form of the enzyme, which was found to be mainly localized in the microsomal fraction of pig and rat liver as well as of the human hepatoma cell line Hep G2. The protein bands detected in microsomes had a molecular mass of 26 kDa in pig and rat liver and displayed a slightly higher molecular mass (29 kDa) in the Hep G2 cell line. A single step method for the immunoaffinity purification of pig liver S-COMT was developed by using a Sepharose 4B column to which the mAb Co54-5F/8 was covalently coupled. Acid elution conditions were optimized to obtain the enzyme in active form with a good yield. SDS-PAGE analysis of the purified preparation revealed a single protein band with a molecular mass of 23 kDa with 154-fold enrichment in enzyme activity over the starting material. Since the N-terminus was blocked, purified enzyme preparations were cleaved with trypsin. Two fragments of 22 and 33 amino acids in length could be sequenced by Edman degradation.     

Species differences in the angiotensin converting enzyme activity of mammalian serum

Angiotensin converting enzyme (ACE; EC 3. 4. 15. 1) activities were compared in the serum of various mammals. Cattle, human, monkey, and swine serum showed enzyme levels from 3.7 to 67 mU/ml. Relatively high enzyme activity was observed in rodents, the rat (Wistar) and mouse (BALB/c) showing levels of 93 +/- 7 and 1052 +/- 165 mU/ml, respectively. Among the mammalian sera examined, that of the guinea pig contained the highest ACE level, 2262 +/- 574 mU/ml. No age-related difference in enzyme activity was observed in 10-day-old to 1-year-old guinea pigs.     

Comparative regional distribution of angiotensin-I-converting enzyme in the rat, rabbit, dog, monkey and human kidneys

Kidney is the main source of the production of renin and angiotensin, while also being one of their main target organs. This study was designed to determine the regional distribution of angiotensin-I-converting enzyme (ACE) in the kidney using a biochemical approach. Interspecies variations were analyzed in human, monkey, rabbit, dog and rat kidneys. Kidney ACE content differed among species with decreasing contents as follows: rabbit greater than human greater than monkey greater than dog greater than rat. In rabbit, human, monkey and dog kidneys, we observed predominant cortical distribution of ACE compared with the medulla or papilla; median cortex/papilla ACE activity ratio was 19, 14, 9 and 7 for the rabbit, human, dog and monkey, respectively. In rat kidney, ACE predominantly distributes in the outer medulla, while cortex ACE content appears to be low. The difference in ACE distribution in the rat kidney and to a lesser extent in the dog kidney when compared to rabbit, monkey or man should be taken into account when extrapolating to the human renal hemodynamic studies, which are frequently performed in rats or dogs.     

Purification and partial immunochemical characterization of proteins of fimbriæ F107 fromEscherichia coli isolated from edema disease of pigs

The paper describes the isolation, purification and characterization of F107-fimbrial proteins, obtained by thermoelution fromEscherichia coli 107/86. Isolation of the pure F107 protein was done by FPLC chromatography, employing Superose 12, Mono Q, and Phenyl-Superose columns. The highest purity of the F107 protein was achieved with Superose 12 HR 10/30. Purity checking by a HPLC system Waters 625 LC (Millipore) proved the absence of protein admixtures in a fraction from Superose 12. Analysis of the molar mass of F107 proteins by SDS PAGE revealed that F107 fimbriæ consist of two proteins, one ofM=43 kDa (minor), and other ofM=18.9 kDa (major). Western blot analysis with rabbit polyclonal antiserum confirmed that the 18.9 kDa protein was the major characteristic unit of F107 fimbriæ.     

Identification of three pertussis toxin substrates (41, 40 and 39 kDa proteins) in mammalian brain. Comparison of predicted amino acid sequences from G-protein alpha-subunit genes and cDNAs with partial amino acid sequences from purified proteins

We have determined the partial amino acid sequences of the 40 kDa protein, one of the three pertussis toxin substrates in porcine brain. Purified 40 kDa protein from porcine brain was completely digested with TPCK-trypsin. Digested peptides were separated by reverse-phase HPLC and subjected to analysis by gas-phase protein sequencing. Several sequences of porcine brain 40 kDa protein completely matched with those which were deduced from the nucleotide sequences of the human Gi2 alpha gene and rat Gi2 alpha cDNA. On the other hand, the previously determined sequences of the rat brain 41 and 39 kDa proteins were in complete agreement with the predicted amino acid sequences of rat Gi1 alpha and Go alpha cDNAs, respectively.     

Comparative analysis of galactoside-binding lectins isolated from mammalian spleens

Galactoside-inhibitable lectins have been isolated from rabbit, rat, mouse, pig, lamb, calf, and human spleens. Native molecular mass, subunit structure, pI, and hemagglutinating activity have been compared for these lectins. The yields of lectin varied from 1.8 mg/kg for rabbit spleen to 79 mg/kg for lamb spleen. Pig, lamb, calf, and human spleen lectins yielded single protein peaks when subjected to Superose 12 fast-protein liquid chromatography. The apparent molecular mass for these lectins was 33-34 kDa. In contrast, rat and mouse spleen lectin preparations were separated into three components ranging from 8.4 to 34 kDa. Superose 12 chromatography of rabbit spleen lectin revealed the presence of at least six components. Gradient slab gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of single polypeptides for pig, calf, lamb, and human lectins corresponding to a molecular mass of 14-14.5 kDa. Multiple polypeptides were detected for the mouse, rat, and rabbit lectins. The molecular mass of the major polypeptides were 15, 15, and 17 kDa for rat, mouse, and rabbit, respectively. The presence of isolectins in all preparations was shown by isoelectric focusing. The major isolectins were acidic proteins with pI 4.38-4.80. Hemagglutination and hemagglutination inhibition assays demonstrated similarities as well as differences among the lectin preparations. Hemagglutinating activity could not be demonstrated in rabbit spleen extracts nor for isolated putative lectin. Human buffy coat cells were reversibly agglutinated by calf and human spleen lectins, demonstrating the presence of leucocyte cell surface lectin receptors.     

Production and characterization of monoclonal antibodies to porcine brain pyridoxal kinase.

Six monoclonal antibodies that recognize porcine brain pyridoxal kinase have been selected and designated as PK67, PK86, PK91, PK144, PK252 and PK275. A total of six monoclonal antibodies recognizing different epitopes of the enzyme were obtained, of which four inhibited the enzyme activity. When total proteins of porcine brain homogenate separated by SDS-PAGE were subjected to monoclonal antibodies, a single reactive protein band of molecular weight 39 kDa which comigrated with purified porcine pyridoxal kinase was detected. Using the anti-pyridoxal kinase antibodies as probes, the cross reactivities of brain pyridoxal kinase from human and other mammalian tissues and from avian sources were also investigated. Among human and all animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 39 kDa. These results indicate that mammalian brains contain only one major type of immunologically similar pyridoxal kinase, although some properties of the enzymes reported previously differed from one another.     

Identification of three pertussis toxin substrates (41, 40 and 39 kDa proteins) in mammalian brain Comparison of predicted amino acid sequences from G-protein α-subunit genes and cDNAs with partial amino acid sequences from purified proteins

We have determined the partial amino acid sequences of the 40 kDa protein, one of the three pertussis toxin substrates in porcine brain. Purified 40 kDa protein from porcine brain was completely digested with TPCK-trypsin. Digested peptides were separated by reverse-phase HPLC and subjected to analysis by gas-phase protein sequencing. Several sequences of porcine brain 40 kDa protein completely matched with those which were deduced from the nucleotide sequences of the human G_i2α gene and rat G_i2α cDNA. On the other hand, the previously determined sequences of the rat brain 41 and 39 kDa proteins were in complete agreement with the predicted amino acid sequences of rat G_i1α and G_oα cDNAs, respectively.     

Isolation, characterization and immunological determination of basement membrane-associated heparan sulfate proteoglycan

Basement membrane-associated heparan sulfate proteoglycan (HSPG) was extracted from isolated porcine glomerular basement membranes and purified by ion-exchange chromatography. The proteogycan was characterized by specific enzymatic digestions, by amino-acid analysis, by SDS-polyacrylamide gel electrophoresis and by density gradient centrifugation. Polyclonal antibodies were raised against the purified HSPG in rabbits. Antibodies were characterized by enzyme immunoassays, immunoprecipitation and immunohistological methods. They were shown to recognize specifically the core protein of HSPG from porcine, human and rat glomerular basement membrane but did not recognize HSPG from guinea pig or rabbit kidney. The affinity-purified antibodies did not cross-react with other basement membrane proteins like laminin, fibronectin or collagen type IV nor with chondroitin sulfate-rich or keratan sulfate-rich proteoglycans from human or bovine tissue. Using these antibodies an enzyme immunoassay was developed for determination of HSPG in the range of 1-100 ng/ml. Studies with cultured porcine endothelial cells showed that subendothelial basement membrane-associated HSPG may be determined with the enzyme immunoassay.     

Production of monoclonal antibodies and immunohistochemical studies of brain myo-inositol monophosphate phosphatase

Five monoclonal antibodies that recognize porcine brain myo-inositol monophosphate phosphatase (IMPase) have been selected and designated as mAb IMPP 9, IMPP 10, IMPP 11, IMPP 15, and IMPP 17. These antibodies recognize different epitopes of the enzyme and one of these inhibited the enzyme activity. When the total proteins of the porcine brain homogenate separated by SDS-PAGE were probed with monoclonal antibodies, a single reactive protein band of 29 kDa, co-migrating with the purified porcine brain IMPase, was detected. Using the anti-IMPase antibodies as probes, the cross reactivities of the brain IMPase from human and other mammalian tissues, as well as from avian sources, were investigated. Among the human and animal tissues tested, the immunoreactive bands on Western blots appeared to have the same molecular mass of 29 kDa. In addition, there was IMPase immunoreactivity in the various neuronal populations in the rat brain. These results indicate that mammalian brains contain only one major type of immunologically similar IMPase, although some properties of the enzymes that were previously reported differ from each another. The first demonstration of the IMPase localization in the brain may also provide useful data for future investigations on the function of this enzyme in relation to various neurological diseases.     

Angiotensin converting enzymes from human urine of mild hypertensive untreated patients resemble the N-terminal fragment of human angiotensin I-converting enzyme

Angiotensin I-converting enzyme (ACE) activity was analyzed in human urine collected from mild hypertensive untreated patients. DEAE-cellulose chromatography using linear gradient elution revealed two forms of angiotensin I-converting enzyme, eluted in the conductivity of 0.75 and 1.25 mS. The fractions of each conductivity were pooled and submitted to direct gel filtration in an AcA-34 column, and the apparent molecular weights of urinary ACEs were estimated as 90 kDa (for ACE eluted in 0.75 mS) and 65 kDa (for ACE eluted in 1.25 mS). Both enzymes have a K(i) of the order of 10(-7) M for the specific inhibitors studied, and are able to hydrolyze luteinizing hormone-releasing hormone and N-acetyl-Ser-Asp-Lys-Pro as described for N-domain ACE. By Western blot analysis, both peaks were recognized by ACE-specific antibody Y4, confirming the molecular weight already described. A plate precipitation assay using monoclonal antibodies to the N-domain of ACE showed that both forms of ACE binds with all monoclonal antibodies to the active N-domain ACE, suggesting that these forms of human urine ACEs resemble the N-fragment of ACE. The HP2 ACE (65 kDa) is similar to low molecular weight (LMW) ACE from normal subjects, and the HP2 ACE (90 kDa) is different from high molecular weight (190 kDa) and LMW (65 kDa) normal ACEs. The 90 kDa ACE could have an important role in development of hypertension. It will be fundamental to elucidate the molecular mechanism responsible for the genesis of this isoform.     

Mast cell tryptase from pig lungs triggers infection by pneumotropic Sendai and influenza A viruses. Purification and characterization.

A novel trypsin-type serine proteinase, which processes the precursors of the envelope fusion glycoproteins of pneumotropic Sendai and human influenza A viruses, was purified to homogeneity from pig lungs. On SDS/PAGE, the purified enzyme gave a protein band corresponding to about 32 kDa, and has an apparent molecular mass of 120 kDa, as determined by gel permeation chromatography. Immunohistochemical staining with antibodies against this enzyme revealed that the enzyme is located in pig lung mast cells. The N-terminal 44-amino-acid sequence of the enzyme exhibits about 80% identity with those of mast cell tryptases from other species. Of the inhibitors tested, di-isopropyl fluorophosphate, antipain, leupeptin, benzamidine and a few proteinaceous inhibitors, such as mucus protease inhibitor and aprotinin, inhibited this enzyme activity. Heparin stabilized the enzyme, but high-ionic-strength conditions did not, unlike for human mast cell tryptase. The purified enzyme efficiently processed the fusion glycoprotein precursor of Sendai virus and slowly processed hemagglutinin of human influenza A virus, and triggered the infectivity of Sendai virus in a dose-dependent manner, although human mast cell tryptase beta and rat mast cell tryptase (rat MCP-7) from lungs did not process these fusion glycoproteins at all. These results suggest that mast cell tryptase in pig lungs is the possible trigger of the pneumotropic virus infections.     

Interspecies cross-reactivity of monoclonal antibodies to various epitopes of human plasminogen

The immunological cross-reactivities of three conformationally specific monoclonal antibodies to distinct epitopes on human plasminogen toward plasminogens purified from 14 additional species have been examined. Antibody 10-F-1, which is produced against an epitope on the kringle 4 region of human plasminogen, shows a high degree (greater than 80%) of cross-reactivity against baboon, goat, monkey, ovine, and rabbit plasminogens; more limited (20-50%) cross-reactivity against bovine, equine, goose, guinea pig, mouse, rat, and porcine plasminogens; and little comparable cross-reactivity against canine and chicken plasminogens. Antibody 10-H-2, generated to an epitope of the kringles 1-3 region of human plasminogen, shows extensive cross-reactivity (72%) only toward monkey plasminogen, more limited (22-35%) cross-reactivity toward equine and rabbit plasminogens, and much less cross-reactivity toward any other of the above plasminogens. Antibody 10-V-1, also produced against an epitope on the kringle 1-3 region of human plasminogen, which is distinct from the 10-H-2 epitope, shows extensive cross-reactivity (72-100%) with baboon, monkey, and rabbit plasminogens; more limited cross-reactivity with equine (48%) and mouse (28%) plasminogens; and a low level of such reactivity with the remaining plasminogens. These studies show that the extent of interspecies cross-reactivity of various plasminogens greatly depends upon the epitope in question. The K4 region of these molecules appears more extensively conserved than the K1-3 region, at least in regard to the particular epitopes examined in this study.     

Affinity labeling of vertebrate oxidosqualene cyclases with a tritiated suicide substrate.

Pig and rat liver oxidosqualene cyclase (OSC) enzymes were purified to homogeneity and showed single bands on SDS-polyacrylamide gel electrophoresis with molecular masses of 75 kDa (pig) and 78 kDa (rat). Pig liver OSC was purified for the first time (441-fold with a yield of 39%). Chemical affinity labeling of pure or crude preparations of the liver cyclases using the mechanism-based irreversible inhibitor of OSC, [3H]29-methylidene-2,3-oxidosqualene ([3H]29-MOS), showed a single radioactive band at 75 kDa (pig) and 78 kDa (rat). Affinity labeling experiments were also performed with dog and human microsomal preparations and with yeast and plant cyclases. All of the vertebrate OSC enzymes were specifically labeled with [3H]29-MOS and gave a single band with molecular masses ranging from 70 to 80 kDa (rat, 78 kDa; dog, 73 kDa; pig, 75 kDa; and human, 73 kDa). In contrast, yeast lanosterol cyclase and plant cycloartenol cyclase were not labeled, demonstrating subtle differences in the active sites of animal, plant, and fungal enzymes.     

Immunochemical and immunohistochemical studies,using antisera against porcine 25 kDa amelogenin, 89 kDa enamelin and the 13–17 kDa nonamelogenins,on immature enamel of the pig and rat

Summary Enamel proteins were extracted from the newly formed layer of immature porcine enamel, and the 25 kDa amelogenin, 89 kDa enamelin and 13–17 kDa nonamelogenins were purified. Specific antisera were raised against these proteins. Antibodies specific to the C-terminal region (residues 149–173) of the 25 kDa amelogenin were generated by absorption of the anti-25 kDa amelogenin serum with 20 kDa amelogenin, which contains residues 1–148 of the antigen. Immunoelectrotransfer blotting of the extracted porcine enamel proteins showed that the anti-25 kDa amelogenin serum recognized the 25 kDa and other low and high molecular weight amelogenins. The C-terminal specific anti-25 kDa amelogenin serum reacted only with amelogenins having molecular weights over 23 kDa. The anti-89 kDa enamelin serum recognized the 89 kDa enamelin and lower molecular weight proteins, but neither the amelogenins nor the 13–17 kDa nonamelogenins. The antiserum against the 13–17 kDa nonamelogenins showed no cross reactivity to the 89 kDa enamelin, but recognized higher molecular weight nonamelogenins. In immunohistochemical preparations of the porcine tooth germs, the 25 kDa amelogenin-like immunoreactivity over immature enamel decreased in a gradient from the enamel surface to the middle layer. In the inner layer immunoreactivity was concentrated over the prism sheaths. The C-terminal specific 25 kDa amelogenin-like immunoreactivity was intense at the outer layer of immature enamel and decreased sharply toward the middle layer. Prism sheaths were intensely stained by the antiserum to the 13–17 kDa nonamelogenins. The 89 kDa enamelin-like immunoreactivity over enamel prisms was intense at the outer layer and decreased toward the middle layer. Staining by the anti-89 kDa enamelin serum of prism sheaths was faint. In immature rat incisor enamel, the C-terminal specific 25 kDa amelogenin antiserum demonstrated a staining pattern similar to that in the immature enamel of the pig. Distinct 13–17 kDa nonamelogenin-like and 89 kDa enamelin-like immunoreactivities were found especially in the layer adjacent to the Tomes' process. We conclude that some enamel proteins are degraded soon after their secretion from the secretory ameloblast in the rat and the pig. The specific enamel proteins which reacted with the antiserum to the 13–17 kDa nonamelogenins seem to be involved with the formation of prism sheaths in immature porcine enamel, but not in rat incisor enamel.     

The Evaluation of Dipeptidyl Peptidase (DPP)-IV, α-Glucosidase and Angiotensin Converting Enzyme (ACE) Inhibitory Activities of Whey Proteins Hydrolyzed with Serine Protease Isolated from Asian Pumpkin (Cucurbita ficifolia)

In the present study, whey protein concentrate (WPC-80) and β-lactoglobulin were hydrolyzed with a noncommercial serine protease isolated from Asian pumpkin (Cucurbita ficifolia). Hydrolysates were further fractionated by ultrafiltration using membranes with cut-offs equal 3 and 10 kDa. Peptide fractions of molecular weight lower than 3 and 3–10 kDa were further subjected to the RP-HPLC. Separated preparations were investigated for their potential as the natural inhibitors of dipeptidyl peptidase (DPP-IV), α-glucosidase and angiotensin converting enzyme (ACE). WPC-80 hydrolysate showed higher inhibitory activities against the three tested enzymes than β-lactoglobulin hydrolysate. Especially high biological activities were exhibited by peptide fractions of molecular weight lower than 3 kDa, with ACE IC50 <0.64 mg/mL and DPP-IV IC50 <0.55 mg/mL. This study suggests that peptides generated from whey proteins may support postprandial glycemia regulation and blood pressure maintenance, and could be used as functional food ingredients in the diet of patients with type 2 diabetes.     

Mixed-Type Inhibition of Pulmonary Angiotensin I-Converting Enzyme by Captopril,Enalaprilat and Ramiprilat

Abstract

We have compared at the enzymological level pulmonary angiotensin I-converting enzymes (ACE) purified to electrophoretic homogeneity from four mammalians species: pig, rat, monkey and human. Using both substrates hippuryl-histidyl-Ieucine and furylacryloyi-phenylal-anyl-glycyi-glycine in steady-state conditions, all the ACES exhibited Michaelis kinetics with identical Michaelis constants, maximal velocities, optimal pH and optimal activating chloride-concentrations. The apparent inhibitory constant was higher for Captopril than for Enalaprilat and even more so for Ramiprilat irrespective of the origin of ACE and the substrate used. Although these inhibitors have been described as competitive inhibitors, Lineweaver-Burk plots were not in accordance with a simple competitive model; moreover, Dixon plots were rather characteristic of non-competitive inhibition. These data emphasize the hypothesis that ACE inhibitors act with mixed-type inhibition, which is consistent with their slow-tight binding to the ACE active center, also with binding of chloride on a critical lysine residue leading to a potential conformational change, and finally with the fact that ACE has two domains, each bearing one catalytic site. On the other hand, as identical kinetic parameters were obtained on the different ACE preparations, results from animal models should allow the extrapolation to humans, in particular for investigations on both renin-angiotensin and kallikrein-kinin systems, and on their inhibition.     

Use of the polymerase chain reaction for homology probing of butyrylcholinesterase from several vertebrates

Genomic blots from man, monkey, cow, sheep, pig, rabbit, dog, rat, mouse, guinea pig, and chicken DNA were hybridized with probes derived from the four exons of the human butyrylcholinesterase gene (BCHE) (Arpagaus, M., Kott, M., Vatsis, K. P., Bartels, C. F., La Du, B. N., and Lockridge, O. (1990) Biochemistry 29, 124-131). Results showed that the BCHE gene was present in a single copy in the genome of all these vertebrates. The polymerase chain reaction was used to amplify genomic DNA from these animals with oligonucleotides derived from the human BCHE coding sequence. The amplified segment contained 423 bp of BCHE sequence including the active site serine of the enzyme (amino acid 198) and a component of the anionic site, aspartate 70. Amplification was successful for monkey, pig, cow, dog, sheep, and rabbit DNA, but unsuccessful for rat, guinea pig, mouse, and chicken DNA. Amplified segments were cloned in M13 and sequenced. The mouse sequence was obtained by sequencing a genomic clone. The highest identity of the human amino acid sequence was found with monkey (100%) and the lowest with mouse (91.5%). The sequence around the active site serine 198, Phe-Gly-Glu-Ser-Ala-Gly-Ala, was conserved in all eight animals as was the anionic site component, aspartate 70. A phylogenetic tree of mammalian butyrylcholinesterases was constructed using the partial BCHE sequences.     

Characterization of neuromedin U like immunoreactivity in rat, porcine, guinea-pig and human tissue extracts using a specific radioimmunoassay

Two novel bioactive peptides termed neuromedin U-8 and neuromedin U-25 have recently been isolated from porcine spinal cord but nothing is known of their occurrence and molecular forms in other species. Following gel permeation chromatography, a specific radioimmunoassay detected only a single molecular form of neuromedin U-like immunoreactivity (NmU-LI) in rat, porcine and human central nervous system and gastrointestinal tract. Only guinea pig tissue extracts revealed two molecular forms of NmU-Li. Reverse phase high performance liquid chromatographic (HPLC) analysis demonstrated that porcine NmU-LI co-eluted with synthetic neuromedin U-25 standard. Human and rat NmU-LI however, was more hydrophobic on HPLC thus indicating species differences.