Properties of glutathione peroxidase isolated from human plasma

Human plasma glutathione peroxidase (GPx) was purified to homogeneity by ammonium sulfate fractionation, gel filtration on Sephadex G-150, chromatography on DEAE Sephacel, chromatofocusing with polybuffer, and gel filtration with Sephadex G-75. This isolation resulted in about 5,400-fold purification of the enzyme with a 32% yield in enzyme activity. The final preparation had a specific activity of about 28 units (mmoles NADPH oxidized) per milligram of protein. Determination of selenium on the purified enzyme revealed a content of 3.8 g atoms per mole GPx. Gel electrophoresis using SDS with standard proteins revealed a molecular weight of about 23,000 for the subunits, which would indicate a molecular weight of about 92,000 for the native enzyme. Amino acid analyses of the purified GPx indicated aspartate, glutamate, proline, glycine, alanine, and leucine as the predominant amino acids and cysteine, methionine, tryptophan, and histidine as the minor amino acids.     

Big angiotensin-25: A novel glycosylated angiotensin-related peptide isolated from human urine

The renin–angiotensin system (RAS), including angiotensin II (Ang II), plays an important role in the regulation of blood pressure and body fluid balance. Consequently, the RAS has emerged as a key target for treatment of kidney and cardiovascular disease. In a search for bioactive peptides using an antibody against the N-terminal portion of Ang II, we identified and characterized a novel angiotensin-related peptide from human urine as a major molecular form. We named the peptide Big angiotensin-25 (Bang-25) because it consists of 25 amino acids with a glycosyl chain and added cysteine. Bang-25 is rapidly cleaved by chymase to Ang II, but is resistant to cleavage by renin. The peptide is abundant in human urine and is present in a wide range of organs and tissues. In particular, immunostaining of Bang-25 in the kidney is specifically localized to podocytes. Although the physiological function of Bang-25 remains uncertain, our findings suggest it is processed from angiotensinogen and may represent an alternative, renin-independent path for Ang II synthesis in tissue.     

Calcitermin, a novel antimicrobial peptide isolated from human airway secretions

The human airways are protected from pathogenic colonization by a blanket of fluid impregnated with innate antimicrobial effector molecules. Among several previously uncharacterized components, we isolated a peptide that had activity primarily targeting Gram-negative bacteria. We named the peptide 'calcitermin' since its amino acid sequence and mass were equivalent to the 15 C-terminal residues of the S100 protein, calgranulin C. The antimicrobial activity of calcitermin was enhanced in acidic buffers (pH 5.4) and in the presence of micromolar concentrations of ZnCl(2). Analysis revealed a putative zinc-binding consensus sequence as well as an alpha-helical conformation in structure-promoting solvents.     

Biochemical characterization of plasma membrane isolated from human skeletal muscle

Specific components of ion translocation systems were studied in excitable plasma membranes isolated from normal human muscle. Na+-K+ ATPase and ouabain-sensitive K+ phosphatase activities were 8.9 +/- 1 mumol Pi/h per mg protein and 96 +/- 9 nmol/min per mg protein, respectively. Scatchard analysis of equilibrium binding assays with [3H]ouabain showed non-linear curves consistent with high- and low-affinity sites (estimated Kd 3 nM and 0.22 microM). Two families of receptors with different affinities for a tritiated TTX derivative (estimated Kd 0.4 and 4 nM) were also identified suggesting the existence in human muscle of at least two classes of voltage-dependent Na+ channels. In addition (+)-[methyl-3H]PN200-110, a potent Ca2+ antagonist used for labeling voltage-dependent Ca2+ channels, was observed to bind to a homogeneous population of receptors in the plasma membrane (Kd = 0.2 nM).     

A new antimicrobial peptide isolated from Oudneya africana seeds

Oudneya africana R. Br. (Brassicaceae), a wild‐growing plant in the arid region of Tunisia, is used in ethno‐medicinal treatment of microbial infections. Validation of ethno‐therapeutic claims pertaining to the plant was sought by investigating its antimicrobial activity. A proteinaceous extract of the seeds, called AS‐3000, showed activity against various organisms including L. monocytogenes, E. coli, B. subtilis, E. hirae, P. aeruginosa, S. aureus and C. albicans. Extract AS‐3000 exhibited a synergistic effect against L. ivanovii when combined with vancomycin or chloramphenicol. The post‐antibiotic inhibitory effect of the ampicillin/AS‐3000 combination was 2.3‐fold greater than for the antibiotic alone. The mode of action of AS‐3000 on Listeria and Escherichia was visible using SEM. These results support the use of O. africana for treating microbial infections.     

Low molecular weight plasma proteins isolated from preparations of human immunoglobulin

Low molecular weight proteins co-purified with IgG constitute 0.22% of the total protein purified from human plasma by ion-exchange chromatography on DEAE-cellulose. We have found that these low molecular weight proteins were obtained free of immunoglobulin by ultrafiltration in 5 M guanidinium chloride. Electrophoresis and isoelectric focusing in polyacrylamide gels demonstrated that this fraction of low molecular weight proteins is remarkably heterogeneous. Chromatography of an Mr 6000 to 12 000 fraction on hydroxyapatite resolved fourteen discrete protein peaks. Three of the peaks contained proteins which appeared to be homogeneous on acid-urea polyacrylamide gels. Two of these proteins were similar in composition to B2 globulin and may represent degradation products of some larger protein. The third protein was found to have an amino-terminal sequence identical to C3a. This population of low molecular weight plasma proteins has previously been shown to contain the cystic fibrosis mucociliary inhibitor and is here shown to contain two proteins similar to B2 globulin, C3a and many proteins remaining to be characterized. The presence of these low molecular weight proteins in measurable concentrations may be insufficiently appreciated in studies using 'purified' immunoglobulins as biological or chemical probes.     

The amino acid sequence of kinetensin, a novel peptide isolated from pepsin-treated human plasma: homology with human serum albumin, neurotensin and angiotensin

A novel nonapeptide with neurotensin-like immunoreactivity was isolated from pepsin-treated human plasma by dialysis, ion-exchange chromatography and high performance reversed-phase liquid chromatography. The amino acid sequence was determined by automated gas-phase sequence analysis as Ile-Ala-Arg-Arg-His-Pro-Tyr-Phe-Leu. Sequence homology with human serum albumin and with the biologically active peptides neurotensin and angiotensin is demonstrated. The name proposed for this peptide is kinetensin.     

Methylation analysis of the carbohydrate portion of fibronectin isolated from human plasma

Fibronectin was isolated from pooled human plasma by affinity chromatography on Sepharose-coupled gelatin and subjected to methylation analysis. The results indicate that the carbohydrate chains in fibronectin are very similar to those found in other serum glycoproteins, i.e. a common inner core structure consisting of two N-acetylglucosamine and three mannose residues attached to the polypeptide backbone via a N-acetylglucosamine-asparagine linkage. To this inner core are attached two side chains made up of N-acetylglucosamine, galactose and terminated by sialic acid.     

Atrial natriuretic peptide and vasopressin in human plasma

Using a specific radioimmunoassay for atrial natriuretic peptide (ANP), plasma immunoreactive ANP was measured in 17 normal subjects and 83 patients with various diseases. Plasma ANP concentration in normal subjects was 14.1 +/- 1.7 pg/ml (mean +/- S.E.). Relatively high plasma ANP concentrations were detected in patients with diabetes mellitus, hyperthyroidism, atrial fibrillation and liver cirrhosis. Plasma ANP concentrations in the patients correlated positively with mean arterial blood pressure and plasma AVP concentrations. Plasma ANP concentrations in the patients also had positive correlations with left atrial dimension and left ventricular diastolic dimension determined by echocardiography. Another positive correlation was observed in the patients between plasma AVP concentrations and mean arterial blood pressure. These results suggest that ANP is a volume regulatory hormone but also that ANP may be involved in the blood pressure regulating system.     

Identification of novel semenogelin I-derived antimicrobial peptide from liquefied human seminal plasma.

Semenogelin I (SgI) is one of the most abundant proteins in human seminal plasma. SgI plays a key role in sperm coagulation and spermatozoon immobilization. In addition, SgI and/or its proteolytic fragments are involved in regulating spermatozoon motility, capacitation and inhibin-like activity. However, little is known about the antibacterial activity of SgI-derived peptides. By a combination of ion-exchange, gel filtration and high-performance liquid chromatography, peptides from liquefied human seminal plasma from 40 healthy donors were isolated and characterized. N-terminal amino-acid sequencing and fast atom bombardment mass spectrometry revealed that four isolated peptides were SgI-derived, namely SgI-29 (85-113), SgI-46 (85-130), SgI-47 (85-131) and SgI-52 (85-136). Interestingly, SgI-29, SgI-46 and SgI-47 are newly identified SgI-derived peptides. Antimicrobial activity assay results indicated that synthesized SgI-29 had strong antibacterial activity toward various bacterial strains. Our results indicate that SgI can be digested into small fragments like newly identified SgI-29, SgI-46 and SgI-47 and may have diversified functions.     

A proenzyme from chicken plasma similar to human plasma prekallikrein

We report the isolation of a specific protease zymogen from chicken plasma. The purification procedure involves barium citrate precipitation, ammonium sulfate fractionation, removal of plasminogen and plasmin on lysine-Sepharose, followed by anion and cation exchange, and gel permeation chromatography. Based on quantitative radioimmunoassay the zymogen is present in plasma at a concentration of 160 mg/liter, and it is obtained by our procedure in highly purified form with a yield of 1.4%. The single polypeptide chain contains an NH2-terminal alanine residue. The native molecule migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 84,000 under reducing conditions. It can be identified as an inactive proenzyme because it has very low amidolytic activity, does not react with the fluorescent active site titrant 4-methyl-lumbelliferyl p-guanidinobenzoate, and does not incorporate radioactive [3H]diisopropylfluorophosphate. It is very susceptible to limited proteolysis which converts it to an active enzyme with trypsin-like specificity. The active enzyme, likewise a single polypeptide chain, migrates as a doublet with apparent molecular weights of 39,000 and 40,000. Its amidolytic activity with synthetic peptide substrates is at least 40-fold higher than that of the proenzyme, it reacts efficiently with 4-methylumbelliferyl p-guanidinobenzoate, and incorporates [3H]diisopropylfluorophosphate while undergoing irreversible inactivation. The enzyme appears to be a reasonably efficient plasminogen activator in zymographic gels, but not in solution. With human high molecular weight kininogen as substrate the enzyme was about 25% as efficient as human plasma kallikrein. It lacks any plasminogen-independent proteolytic activity with other protein substrates, and it hydrolyzes small peptide substrates designed for both human kallikrein and urinary urokinase, respectively. Inhibition studies with peptide chloromethyl ketones indicate enzymatic properties closer to human plasma kallikrein than to the human plasminogen activator urokinase (EC 3.4.21.31). The chicken plasma enzyme and the plasminogen activator from the conditioned media of Rous sarcoma virus-transformed chick embryo fibroblasts treated with tumor promoter are different by criteria of tryptic peptide maps, and amino acid composition and enzymatic specificity. The designations chicken plasma prekallikrein plasminogen proactivator and chicken plasma kallikrein plasminogen activator are proposed for the zymogen and enzyme forms, respectively. Using rabbit antibodies against the proenzyme we developed a solid phase immunoadsorption procedure that allowed us to isolate the protein with an overall yield of 11.4%.     

A bromine compound isolated from human cerebrospinal fluid

A new bromine compound with properties characteristic of a ketone was isolated from human cerebrospinal fluid. It was degraded by alkali treatment producting acetic acid, glycoli acid, oxalic acid, HBr and 1-methylheptanol. By this treatment, a part of the compound was converted to di-1-methylheptyl-2,5-dioxocyclohexane-1,4-dicarboxylate. These degradation products were also obtained from synthetic 1-methylheptyl-γ-bromoacetoacetate by alkali treatment. 1-methylheptyl-γ-bromoacetoacetate and realted substances were synthesized and compared with the isolated bromine compound in chemical properties, infrared spectra, spectra of nuclear magnetic resonance, and elemental analysis, etc. These results showed that the isolated bromine compound corresponded well to 1-methylheptyl-γ-bromoacetoacetate (synonym of 2-octyl-γ-bromoacetoacetate).     

A glycopeptide isolated from human gastric juice.

A glycopeptide containing 69% carbohydrate was isolated from human gastric juice. The complex was found to be homogeneous and to have mol.wt. 9600. The glycopeptide consisted of a protein core to which were linked, by O-glycosidic linkages to threonine and N-glycosidic linkages, carbohydrate side chains composed of N-acetylgalactosamine, N-acetylglucosamine, galactose, mannose, fucose and sialic acid, in the proportions 2:10:7:4:12:1.     

Lipidomic characterization of exosomes isolated from human plasma using various mass spectrometry techniques

Ultrahigh-performance supercritical fluid chromatography - mass spectrometry (UHPSFC/MS), ultrahigh-performance liquid chromatography - mass spectrometry (UHPLC/MS), and matrix-assisted laser desorption/ionization (MALDI) - MS techniques were used for the lipidomic characterization of exosomes isolated from human plasma. The high-throughput methods UHPSFC/MS and UHPLC/MS using a silica-based column containing sub-2 μm particles enabled the lipid class separation and the quantitation based on exogenous class internal standards in <7 minute run time. MALDI provided the complementary information on anionic lipid classes, such as sulfatides. The nontargeted analysis of 12 healthy volunteers was performed, and absolute molar concentration of 244 lipids in exosomes and 191 lipids in plasma belonging to 10 lipid classes were quantified. The statistical evaluation of data included principal component analysis, orthogonal partial least square discriminant analysis, S-plots, p-values, T-values, fold changes, false discovery rate, box plots, and correlation plots, which resulted in the information on lipid changes in exosomes in comparison to plasma. The major changes were detected in the composition of triacylglycerols, diacylglycerols, phosphatidylcholines, and lysophosphatidylcholines, whereby sphingomyelins, phosphatidylinositols, and sulfatides showed rather similar profiles in both biological matrices.     

Inter-relationship of lipids transferred by the lipid-transfer protein isolated from human lipoprotein-deficient plasma

In a previous study we demonstrated that highly purified lipid-transfer protein facilitated the transfer of triglyceride, cholesteryl ester, and phosphatidylcholine between plasma lipoproteins. It remained unclear, however, whether these lipids were transferred by independent sites on the lipid-transfer protein. To address this point, we have studied the protein-mediated transfer of triglyceride, cholesteryl ester, and phosphatidylcholine as a function of the concentration and lipid composition of donor and acceptor lipoproteins. Lipoproteins labeled in vitro, reconstituted lipoproteins of defined lipid composition, and phosphatidylcholine liposomes with or without triglyceride and/or cholesteryl ester have been used to investigate the inter-relationships of lipids transferred by the lipid-transfer protein. In studies of initial (less than or equal to 10-13%) transfer, we found that, although absolute transfer rates were affected, the ratio of cholesteryl ester to triglyceride transferred was independent of donor and acceptor lipoprotein concentrations and acceptor lipoprotein lipid composition. With reconstituted lipoproteins as donor, we demonstrated that this ratio was linearly related to the ratio of cholesteryl ester to triglyceride in the donor particle; the sum of triglyceride and cholesteryl ester transferred remained constant and independent of the lipid composition of the donor. Experiments with intact lipoproteins labeled in vitro and with small unilamellar vesicles in the presence and absence of p-chloromercuriphenylsulfonate, confirmed the interdependence of triglyceride and cholesteryl ester transfer. In contrast, under all assay conditions, no correlation was found between the amount of phosphatidylcholine transferred and the transfer of triglyceride and/or cholesteryl ester. We conclude that triglyceride and cholesteryl ester compete for transfer and that the extent of transfer for each lipid is determined by its relative concentration in the donor particle, whereas phosphatidylcholine transfer is independent of triglyceride and cholesteryl ester transfer. The data also strongly support the conclusion that lipid transfer protein promotes both the exchange and net transfer of triglyceride and cholesteryl ester and that the net transfer process proceeds by a reciprocal exchange of triglyceride and cholesteryl ester without net transfer of core lipid between lipoproteins.