Theoretical prediction of antigenic sites in the Β-subunits of human choriogonadotropin and luteinizing hormone

Using hydrophilicity and recognition values of amino acids, the antigenic sites of theΒ-subunits of human choriogonadotropin and luteinizing hormone were computed from their amino acid sequences. Six antigenic sites were calculated for human choriogonadotropinΒ-subunits: residues 3–8, 17–22, 59–65,100–106,110–116 and 134–139. For luteinizing hormoneΒ-chain three antigenic sites were calculated: residues 17–22,59–65, and 100–106; all these three sites of luteinizing hormoneΒ being identical to the corresponding sites in human choriogonadotropinΒ. There was no antigenic site in luteinizing hormone that was also not found in human choriogonadotropin. On the other hand, there were unique determinants in human choriogonadotropin that were not found in luteinizing hormone; these determinants were residues 3–8, 110–116 and 134–139     

Synthesis of cystine peptides 21-25/70-73 and 35-39/56-59 of the beta-subunit of human choriogonadotropin.

Syntheses of two asymmetrical cystine peptides with the amino acid residues 21-25/70-73 and 35-39/56-59, based on the linear amino acid sequence and the disulfide bond assignment in the beta-subunit of human choriogonadotropin (hCG-beta), are described. S-trityl and S-acetamidomethyl peptide fragments of each cystine peptide were prepared in solution phase and were subjected to oxidation with I2/MeOH to form the disulfide bridge. The cystine peptides were characterized by their amino acid analyses and fast atom bombardment mass spectrometry. Immunological characterization by several homologous radioimmunoassay systems showed that peptide 21-25/70-73 had significant hCG, hCG-beta, and hLH activities while peptide 35-39/56-59 failed to reveal any immunoreactivity.     

Selenomethionyl analog of recombinant human choriogonadotropin

Selenomethionyl and high mannose type analog of recombinant human choriogonadotropin (hCG) to solve the crystallization and phase problems has been obtained by gene transfer methodology. SF9 insect cells were infected with the recombinant viruses containing hCG alpha and hCG beta cDNAs in selenomethionine containing methionine-free Grace's medium. The selenomethionyl hCG (SehCG) was purified from the culture medium by one step immunoaffinity chromatography using an immobilized monoclonal antibody against hCG beta. The presence of selenomethionine was demonstrated by amino acid analysis of SehCG. The amino acid composition indicated that more than 84% of methionine residues were substituted by selenomethionine. Its sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis yielded a single 38-kDa protein band under nonreducing conditions. The carbohydrate analysis of SehCG was consistent with the presence of four N-linked high mannose type carbohydrates and four O-linked simple disaccharide chains. The in vitro immunological and biological studies of SehCG indicated that selenomethionine substitution had no effect on the immunopotency, receptor binding, and steroidogenic activities of the hormone.     

Purification and characterization of a ribonuclease from human spleen. Immunological and enzymological comparison with nonsecretory ribonuclease from human urine

A ribonuclease has been isolated from human spleen (RNase HS) by means of acid extraction, ammonium sulphate fractionation, successive column chromatographies on CM-cellulose, heparin-actigel, and poly(G)-agarose, and double gel-filtration on Sephadex G-75. The purified preparation was homogeneous as judged by SDS/PAGE. RNase HS was found to be a glycoprotein, containing three fucose, one mannose and five glucosamine residues/molecule, with a molecular mass of 17 kDa as determined by both SDS/PAGE and gel filtration. The catalytic properties and structural features, including its amino acid composition and the amino acid sequence of the N-terminal 35 residues, indicated that the enzyme was strictly related to nonsecretory RNase isolated from human urine and liver. In particular, the amino acid sequence of the N-terminal was identical with that of urine nonsecretory RNase and eosinophil-derived neurotoxin. Furthermore, analyses using three different antibodies specific to RNase HS, urine nonsecretory RNase and urine secretory RNase, indicated that RNase HS was not immunologically distinguishable from urine nonsecretory RNase, but clearly so from urine secretory RNase. However, the carbohydrate compositions of RNase HS and urine nonsecretory RNase were found to differ. It therefore remains to be resolved whether or not the tissue of origin of nonsecretory RNase in urine is the spleen.     

Forms of Aspartic Acid in Human Urine

It was found by amino acid analysis before and after acid hydrolysis of human urine that most glutamic and aspartic acid was in bound form, while glycine, glutamic and aspartic acids accounted for about 70% of bound amino acids. Fractions rich in peptides containing aspartic acid were obtained by chromatography on various columns, and 7 peptides containing aspartic acid were isolated from these fractions. It may be inferred from these results and from the literatures that there are numerous oligopeptides containing aspartic acid in human urine.     

Recombinant carbohydrate and selenomethionyl variants of human choriogonadotropin.

Recombinant human choriogonadotropin and selenomethionyl human choriogonadotropin (rhCG and SehCG) were expressed in baculovirus expression system by coinfection of SF9 insect cells by recombinant viruses, AcMNPV-hCG alpha and AcMNPV-hCG beta containing hCG alpha and hCG beta cDNAs. The expression efficiency of both rhCG and SehCG was quite high. The association of the alpha and beta subunits into a dimer was apparently complete since no detectable amount of rhCG beta was found in the rhCG eluate from the monoclonal hCG beta antibody immunoaffinity column. Both rhCG and SehCG preparations were homogeneous as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high performance liquid chromatography. The apparent molecular mass of rhCG and SehCG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions was about 38 kDa while under reducing conditions the heterodimer dissociated to yield beta and alpha subunits with molecular masses of 22.5 and 18 kDa, respectively. The carbohydrate analysis of rhCG showing the presence of 2.1, 3.3, 7.38, 4.2, and 27.8 residues of Fuc, GalNAC, GlcNAC, Gal, and Man, respectively, per mole of the hormone was consistent with the presence of 4 N-linked high mannose type carbohydrate hydrate and 4 O-linked simple carbohydrate chains, probably made up of Gal-GalNAC. Despite the altered glycosylation, rhCG demonstrated close similarity to the native urinary hCG in amino acid composition, receptor binding, and in its ability to stimulate cAMP and steroidogenesis. This indicates that there is no specificity of carbohydrate required for biological activity. Furthermore, it implies that the alteration from the complex to high mannose type carbohydrates in rhCG does not affect its proper folding. Finally, amino acid analysis of SehCG showed that 84% of methionine residues in rhCG were replaced by selenomethionine.     

Direct extract derivatization for determination of amino acids in human urine by gas chromatography and mass spectrometry

The purpose of this study was to develop a simple and accurate analytical method to determine amino acids in urine samples. The developed method involves the employment of an extract derivatization technique together with gas chromatography-mass spectrometry (GC-MS). Urine samples (300 microl) and an internal standard (10 microl) were placed in a screw tube. Ethylchloroformate (50 microl), methanol-pyridine (500 microl, 4:1, v/v) and chloroform (1 ml) were added to the tube. The organic layer (1 microl) was injected to a GC-MS system. In this proposed method, the amino acids in urine were derivatized during an extraction, and the analytes were then injected to GC-MS without an evaporation of the organic solvent extracted. Sample preparation was only required for ca. 5 min. The 15 amino acids (alanine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tyrosine, tryptophan, valine) quantitatively determined in this proposed method. However, threonine, serine, asparagine, glutamine, arginine were not derivatized using any tested derivatizing reagent. The calibration curves showed linearity in the range of 1.0-300 microg/ml for each amino acid in urine. The correlation coefficients of the calibration curves of the tested amino acids were from 0.966 to 0.998. The limit of detection in urine was 0.5 microg/ml except for aspartic acid. This proposed method demonstrated substantial accuracy for detection of normal levels. This proposed method was limited for the determination of 15 amino acids in urine. However, the sample preparation was simple and rapid, and this method is suitable for a routine analysis of amino acids in urine.     

Partial amino acid sequence of the preprotein form of the alpha subunit of human choriogonadotropin and identification of the site of subsequent proteolytic cleavage

Messenger RNA isolated from first trimester placentae was translated using radiolabeled amino acids in both the wheat germ and the ascites cell-free systems. The choriogonadotropin α subunit product was purified by immunoprecipitation with a subunit specific antiserum. Its amino acid sequence was partially determined by automated Edman degradation analysis. An NH₂-terminal extension of 24 amino acids was found and its partial sequence is:

The preprotein form of the subunit was cleaved by the addition of microsomal membranes resulting in a homogeneous NH₂-terminal product. Hence, it is unlikely that this processing step accounts for the heterogeneity that has been observed previously in the structure of this region of the subunit.     

Induction of human choriogonadotropin in HeLa-cell cultures by aliphatic monocarboxylates and inhibitors of deoxyribonucleic acid synthesis

The ectopic production of the glycopeptide hormone human placental choriogonadotropin by HeLa₆₅ cells was measured by radioimmunoassay with antiserum against the β-subunit of choriogonadotropin and with the ¹²⁵I-labelled β-subunit as a tracer antigen. Choriogonadotropin synthesis was markedly (500-fold) stimulated by sodium butyrate. Kinetic studies and the use of an inhibitor of protein synthesis, cycloheximide, indicated that protein synthesis was required for this induction. Investigation of the efficiency of 22 aliphatic short-chain fatty acids and derivatives in causing increased choriogonadotropin synthesis by HeLa cells showed stringent structural requirements. Induction of choriogonadotropin synthesis in HeLa cells was not restricted to butyrate. Other aliphatic acids (propionate, isobutyrate, valerate and hexanoate) were also capable of inducing choriogonadotropin synthesis at 10–50% of the efficiency of butyrate. Hydroxy derivatives of monocarboxylate inducers, related mono- and di-carboxylic acids, alcohols, amines, ketones, esters and sulphoxide were ineffective in increasing choriogonadotropin production by HeLa cells. A saturated C₄ straight-chain acid without substituent hydroxyl groups but with a methyl group at one end and a carboxyl moiety at the other appeared to be most efficient in activating choriogonadotropin production. A second clonal line of HeLa cells, HeLa₇₁, showed a higher constitutive synthesis of choriogonadotropin than HeLa₆₅ cells, which was also markedly increased by butyrate. Butyrate and other aliphatic monocarboxylate inducers of choriogonadotropin synthesis inhibited HeLa-cell growth and DNA synthesis. This inhibition of DNA replication may be related to the mechanism of choriogonadotropin synthesis, since two well-characterized anti-neoplastic inhibitors of DNA synthesis, hydroxyurea and 1-β-d-arabinofuranosylcytosine, also stimulated a 300-fold increase in choriogonadotropin synthesis in HeLa cells and were synergistic with butyrate in promoting choriogonadotropin synthesis. Thus activation in tumour cells of genes normally expressed by foetal tissue and the consequent ectopic synthesis of polypeptide hormones may require neither cell division nor DNA synthesis.     

alpha-Hydroxy-beta-keto-gamma-aminobutyric acid in human urine.

A new amino acid has been isolated from the normal human urine. The chemical structure of the amino acid was determined to be alpha-hydroxy-beta-keto-gamma-aminobutyric acid based on its physical properties involving NMR, infrared and mass spectra, as well as chemical degradation and synthesis. In six healthy adults the urinary contents of the new amino acid were 3.2--4.5 mumol/24 h.     

Characterization of a trypsin inhibitor from equine urine.

A trypsin inhibitor was isolated from pregnant mares' urine by adsorption on bentonite and elution with aqueous pyridine followed by batch DEAE-cellulose treatment and column chromatography. Final purification to an electrophoretically homogenous glycoprotein was achieved by gel permeation chromatography. This equine urinary trypsin inhibitor (E-UTI) is acid- and heat-stable, has a molecular weight of 22 to 23 kDa, an isoelectric point of 4.55, forms a 1:1 molar complex with trypsin and has serine as its N-terminal amino acid. The N-terminal amino acid sequence of this protein is almost identical with that of EI-14, the inhibitor obtained from horse serum by tryptic treatment, except for two extra amino acid residues, Ser-Lys- on the N-terminal end of E-UTI. In its isoelectric point E-UTI differs from EI-14 and the inhibitor from human urine.     

α-Hydroxy-β-keto-γ-aminobutyric acid in human urine

A new amino acid has been isolated from the normal human urine. The chemical structure of the amino acid was determined to be α-hydroxy-β-keto-γ-aminobutyric acid based on its physical properties involving NMR, infrared and mass spectra, as well as chemical degradation and synthesis. In six healthy adults the urinary contents of the new amino acid were 3.2–4.5 μmol/24 h.     

Whole-cell response characteristics of ciliated and microvillous olfactory receptor neurons to amino acids, pheromone candidates and urine in rainbow trout.

Olfactory lamellae of teleosts contain two morphologically different types of olfactory receptor neurons (ORNs): ciliated ORNs (cORNs) and microvillous ORNs (mORNs). However, little is known about the functional difference between these two types of ORNs in fish olfaction. We isolated cORNs and mORNs using a Ca(2+)-free solution method from olfactory organs of the rainbow trout and examined their response characteristics to various odorants including fish pheromone candidates by whole-cell voltage-clamp techniques. Quadruple mixture of amino acids, single amino acids, steroids (analogues of DHP; 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one and ECG; etiocholan-3 alpha-ol-17-one glucuronide), prostaglandins (PGFs) and urine samples collected from immature and mature female fish were applied focally to olfactory cilia or microvilli using a multi-barreled stimulation pipette with a pressure ejection system. Inward current responses to odorants were recorded from both cORNs and mORNs at a holding potential of -60 mV. cORNs responded to the amino acid mixture, single amino acids, urine samples and ECG, whereas mORNs responded specifically either to the amino acid mixture or single amino acids. The response profiles of both cORNs and mORNs to various odorants varied widely. None of cORNs and mORNs responded to fish pheromone candidates, PGFs and DHPs. Androgen treatment of immature fish did not influence olfactory sensitivity of both cORNs and mORNs to the amino acid mixture and both urine samples. Amino acid and bile acid analyses by HPLC showed that both urine samples contained 35 amino acids (1-40 mM) and trace amounts of taurocholic acid and glycoursodeoxycholic acid. Our results suggest that cORNs are 'generalists' that respond to a wide variety of odorants, including pheromones, whereas mORNs are 'specialists', specific to amino acids, and also suggest that PGFs and DHPs are not pheromones for the rainbow trout.     

A new type of carbohydrate-protein linkage in a glycopeptide from normal human urine.

A glycopeptide, 3-O-beta-D-glucopyranosyl-alpha-L-fucopyranosyl-L-threonine, has been isolated from normal human urine. The glycopeptide was isolated by gel chromatography, preparative zone electrophoresis, paper chromatography, and high voltage electrophoresis. The average yield of the glycopeptide was in the range of 0.2 to 0.3 mg/liter of urine. Sugar analysis and amino acid analysis gave equimolar amounts of glucose, fucose, and threonine. Linkages and sequential order were established by methylation analysis of the glycopeptide after degradation of the amino acid residue with ninhydrin. The permethylated product was analyzed on gas liquid chromatography and mass spectrometry. Anomeric configuration was deduced from optical rotation.     

Isolation and identification of hydroxyproline analogues of bradykinin in human urine

Hydroxyproline (Hyp) analogues of bradykinin and lysyl-bradykinin, in which the third residue of bradykinin, proline, is replaced by hydroxyproline, were isolated from human urine. Their amino acid sequences were confirmed by both amino acid and sequence analyses, and also by comparison of their chromatographic behavior with that of synthetic peptides. The possibility that Lys-Ala3-bradykinin, isolated by Mindroiu et al. [(1986) J. Biol. Chem. 261, 7407-7411] from human urine, was actually Lys-Hyp3-bradykinin is discussed.     

Consequences of renal mass reduction on amino acid and biogenic amine levels in nephrectomized mice

Summary. Amino acid and biogenic amine changes were investigated in nephrectomized mice ten days postsurgery. Uremic mice exhibited changes in amino acid concentrations in plasma, urine and brain. Particularly plasma methionine, citrulline and arginine levels were significantly enhanced in nephrectomized mice compared to controls whereas serine was decreased. Urinary excretion of methionine, citrulline and alanine was higher in nephrectomized mice compared to controls whereas many amino acids were increased in brain of nephrectomized mice. Brain and urinary amino acid changes were more pronounced in the 75% than in the 50% nephrectomized mice. Brain norepinephrine and dopamine and its metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid were significantly increased whereas serotonin was decreased comparing the 75% nephrectomized mice to the sham-operated mice. This study demonstrates that at very early stages of renal insufficiency, specific amino acid and biogenic amine changes occur in plasma, urine and brain. These alterations might depend qualitatively and quantitatively on the degree of functional renal mass reduction. Received April 5, 1999     

Purification of N-terminal hexapeptide of big gastrin from human urine

We previously demonstrated that extremely high amounts of N-terminal big gastrin (G-34) fragments are excreted in human urine and three of them are N-terminal octa-, nona-, and decapeptide of G-34. Our subsequent examination revealed that there exists a considerable amount of another N-terminal G-34 fragment in urine, less hydrophobic than the three peptides. We purified this fragment from urine of an achlorhydric patient and determined the structure: less than Glu-Leu-Gly-Pro-Gln-Gly. The purification was carried out by Sep-Pak C18 cartridges, Sephadex G-25, and reverse phase HPLC. The structure was determined by a combination of amino acid analysis, amino acid sequence analysis, and mass spectral analysis. N-terminal hexapeptide of G-34 is the second richest component of urinary N-terminal G-34 fragments next to N-terminal octapeptide of G-34 in normal subjects.     

Retinol-binding protein from human urine and its interaction with retinol and prealbumin

In freshly collected urine from a patient with glomerulotubular proteinuria there were two bands which contained retinol-binding proteins. The cathodal band showed fluorescence in the ultraviolet. After extraction with organic solvents only the anodal non-fluorescent band remained. After addition of an excess retinol only one band remained which by mobility corresponded to the cathodal band.The anodal of the two bands was therefore probably the apo form and the cathodal the holo form of the same retinol-binding protein. Their proportions, determined by densitometric scanning were approximately 4/1 (anodal/cathodal band). More than 85% of the retinol-binding protein in the urine bound to prealbumin-Sephrose. The apo retinol-binding protein from urine had the same electrophoretic mobility on agarose gel el-ctrophoresis and the same pattern on isoelectric focusing as an retinol-binding protein prepared from serum. The carboxy-terminal amino acid sequence of the retinol-binding protein from freshly collected urine that bound to prealbumin-Sepharose, was -Arg-Leu. The amino-terminal sequence was Glu-Arg-Asp-Cys-Arg-Val-Ser-X-Phe-Arg-Val-Lys-Glu-Asn-Phe-Asp-Lys-Ala-Arg-Phe-X-Gly-Thr-Trp-Tyr-. This sequence and the amino acid composition are compatible with the view that the retinol-binding protein in urine is the same as in plasma.     

Variation of hydrophobicity of human urinary epidermal growth factor

Epidermal growth factor is present in human urine in large amounts, but its biological significance is not known. The results of this study indicate that the predominant 6000-dalton form of epidermal growth factor in human urine is divided by hydrophobic interaction chromatography into four fractions; only 3% of the total 6000-dalton epidermal growth factor coeluted with the biosynthetic epidermal growth factor and the rest was separated into three different peaks. These different forms may lack one or two amino or carboxy terminal amino acids from the 53 amino acids present in epidermal growth factor, or they may be products of deamidation or oxidation of amino acid(s). Further knowledge of these micromodifications of epidermal growth factor secreted in urine may reveal the origin and function of epidermal growth factor in humans.     

Purification and Primary Structure of a Porcine Kidney Non-secretory Ribonuclease

A non-secretory ribonuclease (RNase PK₃) was isolated from porcine kidney, and its primary structure was analyzed. RNase PK₃ consisted of 126 amino acid residues. The amino acid sequence of RNase PK₃ has high sequence homology with non-secretory RNases from human urine and bovine kidney.