Immunochemical studies of (Na+ + K+)-ATPase using site-specific, synthetic peptide directed antibodies

Rabbit antisera were raised against a series of synthetic peptides corresponding to regions of the alpha subunit of lamb kidney (Na+ + K+)-ATPase which chemical labeling studies and hydropathy plots of the amino-acid sequence suggest are exposed, accessible regions of the enzyme and may comprise the cation selectivity region, the ATP and cardiac glycoside binding sites, and the phosphorylation site. Five of six peptides tested (11-15 residues in length) were immunogenic and the antisera to four peptides recognized the intact, electroblotted (Western blot analysis) alpha subunit. Immunization with peptides conjugated to keyhole limpet haemocyanin (KLH) produced antipeptide antibodies for seven of nine conjugates. Antisera to four peptide conjugates recognized the native enzyme, confirming predictions that these sequence regions are exposed regions of the holoenzyme. In addition, a collection of four polyclonal antisera and five monoclonal antibodies raised to native holoenzyme were tested for their ability to bind to the peptide conjugates. In this way, two NH2-terminal sequence regions (1-12 and 16-30) and the putative ATP-binding site region (496-506) were identified as epitopes of the native enzyme. These results confirm some aspects of the transmembrane folding models proposed by Shull et al. and Kawakami et al. for the membrane-bound (Na+ + K+)-ATPase.     

Immunochemical studies of pancreatic colipase-lipase interaction employing immobilized synthetic peptides.

In view to study the possible participation of the sequence portions of colipase including or close to the free carboxyl groups at positions 15 and/or 72 to the binding with pancreatic lipase, we have used three synthetic peptides matching portions 8-16, 59-67 and 67-72 of the amino acid sequence. Polyclonal rabbit anticolipase immune serum, which cross-reacts with peptides in ELISA, was fractionated on columns of peptide coupled to Sepharose. Of the three fractions of antibodies, only that interacting with peptide 8-16 had the capacity to inhibit colipase-dependent lipase activity by specifically preventing the association of lipase with its protein cofactor previously bound to lipid. We conclude that the region spanning residues 8-16 of colipase is of importance for colipase-lipase interaction in the active complex formed at interface.     

Immunochemical studies of human erythropoietin using site-specific anti-peptide antibodies. Identification of a functional domain

Anti-peptide antibodies that bind to the amino terminus of human erythropoietin (residues 1-26) do not inhibit the hormone's biological activity, indicating that this region of the protein does not play a role in receptor recognition (Sytkowski, A. J., and Fisher, J. W. (1985) J. Biol Chem. 260, 14727-14731). We have now identified six other regions of the primary sequence that are relatively hydrophilic and, therefore, have a higher probability of being accessible to such antibody probes. Antibodies raised against synthetic peptides homologous to five of these regions, corresponding to residues 40-59, 80-99, 99-118, 111-129, and 131-150 recognize erythropoietin, confirming the prediction based upon relative hydrophilicity. Antibodies to a carboxyl terminal peptide 147-166 failed to bind the hormone, presumably due to steric hindrance imposed by a disulfide bond between Cys161 and one of the other cysteinyl residues. The antibodies were affinity purified on the relevant immobilized peptide and their capacity to inhibit (neutralize) erythropoietin's activity was assessed. Only anti-peptide 99-118 and anti-peptide 111-129 antibodies inhibited erythropoietin. This effect was reversed by excess peptide, demonstrating that the neutralizing action of the antibody was due to its antigen-specific binding. The results strongly suggest that the portion of erythropoietin's amino acid sequence represented by these peptides plays a functional role in the hormone's action, most probably by forming part of the receptor-binding domain.     

Immunochemical studies of human urinary trypsin inhibitor

Antisera against purified urinary trypsin inhibitor (UTI-I, molecular weight 67,000) and UTI-III (molecular weight 23,000) were first produced in rabbits. Both anti-UTI-I and anti-UTI-III sera formed a single immunoprecipitin line with human plasma inter-alpha-trypsin inhibitor (I alpha TI), whereas two immunoprecipitin lines were formed with crude urine. It was speculated that both UTI-I and UTI-II might be present in normal human urine. In the present study, the inhibitory effects of anti-UTI sera on UTI activity were examined by three different assay methods. The results indicated that the inhibitory effect was almost immediate. Although the inhibitory effect of anti-UTI-III serum on UTI-III was almost of the same degree of completeness for the three assay methods. UTI-I was partially inhibited by the anti-UTI-I serum when residual trypsin activity was measured by the caseinolytic or fibrinolytic assay method. This discrepancy was considered to be due to the difference in conformational change between UTI-I and UTI-III by antigen-antibody reaction.     

Immunochemical analysis of Lewis rat antisera to the synthetic encephalitogenic peptide S49

Discrete populations of anti-S49 antibodies were found in the antisera of Lewis rats recovered from S49-induced experimental allergic encephalomyelitis (EAE). A potent inducer of EAE in Lewis rats, S49 is a synthetic peptide representing residues 69–84 of bovine myelin basic protein but with deletions at Gly-77 and His-78 to form an analogue of guinea pig or rat 69–84, GSLPQKAQRPQDENG. Each population within a given antiserum, as identified by Scatchard and Sipsian window analysis, was found to exhibit reactivity for a different S49 determinant, and the affinities of each population were relatively restricted and discontinuous. The high affinity populations (10⁷–10⁸ M^–1) were cross-reactive with YS8 (YGSLPQKAQGHRPQDENG) in equilibrium competitive inhibition reactions whereas the low affinity populations (10⁵–10⁶ M^–1) were reactive only with S49 and YS49 among a panel of peptide analogues. Of the YS8 cross-reactive antibodies the highest affinity (10⁸ M^–1) were also cross reactive with S81 (YGSLPQKAQGHRPQDEG) but not S49 (69-84-Gly), thus emphasizing the need for Tyr-68 for format stability of the determinant involved. The other YS8 cross-reactive population (10⁷ M^–1) was completely reactive with S49 but totally unreactive with S81 in equilibrium reactions, thus emphasizing the requirement for Asn-84 but not Tyr-68 for the determinant's topographic stability. Peptides shorter than S49 from the N-terminal end, but retaining the sequences AQRPQDEN or SQRSQDEN (suspected residence of minimal encephalitogenic determinants), reacted only under conditions of two-step non-equilibrium competitive inhibition assays. Such reactions would occur only at very low affinity (<10⁵ M^–1) with the anti-S49 antibodies. It was hypothesized that the encephalitogenic T-cell determinant for Lewis rats, although permitting B-cell responses at very low affinity, may exclude high affinity responses in susceptible animals.This work supported at Duke University Medical Center by research grant NS-10237 from the National Institutes of Health of the U.S. Public Health Service, Immunology Training Grant #5-T32-CA-09058-10, and Medical Scientist Training Program #5-T32-OM-07171-08, and at St. Luke's Hospital Center by NS-21466 from the National Institutes of Health and RG1197-B7 from the National Multiple Sclerosis Society.     

Immunochemical studies of isolated human brain ganglioside components

Abstract— Gangliosides G₁ to G₅ were isolated from human brain by means of TLC and tested with respect to their specificity to antisera against normal brain and Tay-Sachs brain gangliosides by agar double diffusion analysis. Gangliosides G₂ and G₄ gave precipitation reactions with antisera to normal human gangliosides (NHG) while only ganglioside G₆ reacted with antisera to Tay-Sachs gangliosides (TSG). Additional specificity information was also obtained by use of the enzyme neuraminidase for the removal of specific sialic acid (NANA) residues. It was concluded from these data that the specificity of the anti-NHG antibodies is determined by the presence of a galactose (β1, 3) N-acetyl galactos-amine–while that of anti-TSG antibodies is due to a N-acetyl galactosamine (β1, 4) galactose-end sequence. By means of natural compounds of known structure it was found that both the sequence of carbohydrate residues and position of NANA residues in the molecule played a critical role in the formation of precipitation bands with NHG-antisera. This information was utilized to distinguish one isomeric form of disialoganglioside from another, i.e. G₂ from G₃ and to confirm the structure of the trisialoganglioside, G₁. The immunochemical method appears to be a useful one for elucidating structural differences in ganglioside molecules.     

Immunochemical studies on lactate dehydrogenase A subunit deficiencies.

In lactate dehydrogenase (LDH) A subunit deficiency, is there not only a lack of activity but also a lack of subunit production? We demonstrated three remarkable points to answer this question: There are no proteins that immunologically react with anti-A subunits. There are no heterotetramers that react with anti-B subunits. B subunits seem to be genetically produced at normal level, and all of them form only one isoenzyme, LDH-B4. From these points, we concluded that there is a complete lack of A subunit production or production of incomplete A subunits that can neither react with anti-A subunits nor form heterotetramers.     

A synthetic peptide of human apoprotein E with antibacterial activity

In recent years, several endogenous mammalian antibacterial peptides have been described. An amphipathic cationicalpha-helix is a common feature in many cases; therefore, other peptides with this characteristic might also possess antibiotic activity. In fact, a 30-mer peptide of apoprotein E 133-162 (LRVRLASHLRKLRKRLLRDADDLQKRLAVY) was found to have antibiotic activity comparable to those of a classic antibiotic (Gentamicin) and a neutrophil-derived antibiotic peptide (CAP18). Calculation of cationicity, hydrophobicity, and hydrophobic moment and the helical wheel diagram of apoprotein E 133-162 revealed close similarities to CAP18.     

Cleavage specificity on synthetic peptide substrates of human rhinovirus 2 proteinase 2A.

Proteinase 2A of human rhinovirus serotype 2 (HRV2 2A) was expressed in Escherichia coli and partially purified; the preparation was used to study various enzymatic parameters. Using a 16-amino acid peptide representing the native cleavage region of HRV2 2A, an apparent Km value of 5.4 x 10(-4) mol/liter was determined. A minimum of 9 amino acids (comprising residues P8 to P1') was necessary for cleavage to occur. Proteolysis of substituted peptides was highly tolerant toward changes at P1, P2', and P3' but an absolute requirement for glycine P1' and a high preference for threonine P2 was found. Furthermore, HRV2 2A only cleaved peptide substrates derived from other rhinovirus serotypes and poliovirus that possessed P2 Thr and P1' Gly. Thus, the sequence Thr-X-Gly may form the basis of the cellular cleavage site processed by rhinoviral 2As during viral replication. Studies with various inhibitors support the hypothesis that HRV2 2A belongs to a new class of cysteine proteinases.     

Complex formation in plant thylakoid membranes. Competition studies on membrane protein interactions using synthetic peptide fragments

Thylakoid membranes of pea were used to study competition between extra-membrane fragments and their parental membrane-bound proteins. Phosphorylated and unphosphorylated fragments of light harvesting complex II (LHC II) from higher plants were used to compete with LHC II for interactions with itself and with other thylakoid protein complexes. Effects of these peptide fragments of LHC II and of control peptides were followed by 80 K chlorophyll fluorescence spectroscopy of isolated thylakoids. The phosphorylated LHC II fragment competes with membrane-bound phosphoproteins in the phosphatase reaction. The same fragment accelerates the process of dark-to-light adaptation and decreases the rate of the light-to-dark adaptation when these are followed by fluorescence spectroscopy. In contrast, the non-phosphorylated LHC II peptide does not affect the rate of adaptation but produces results consistent with inhibition of formation of a quenching complex. In this quenching complex we propose that LHC II remains inaccessible to the LHC II kinase, explaining an observed decrease in LHC II phosphorylation in the later stages of the time-course of phosphorylation. The most conspicuous protein which is steadily phosphorylated during the time-course of phosphorylation is the 9 kDa (psbH) protein. The participation of the phosphorylated form of psbH in the quenching complex, where it is inaccessible to the phosphatase, may explain its anomalously slow dephosphorylation. The significance of the proposed complex of LHC II with phospho-psbH is discussed.Abbreviations LHC II light harvesting complex II - PS II Photosystem II - PS I Photosystem I     

Immunochemical analysis of substance P using its synthetic fragments and analogs

Synthetic fragments and analogs were used to characterize specificity of antisera to substance P. Both, the C-terminal hexapeptide and the pentapeptide completely inhibited binding of 125I-[Tyr8]substance P by these antisera, showing the antigenic identity with substance P. Synthetic fragments shorter than peptide (7-11) did not react with anti-substance P antisera in this system. Substitution of amino acids in different positions in the fragments (6-11) or (7-11) by histidine or glycine revealed that all five amino-acid residues take part in a structure of the antigenic determinant.     

Immunochemical studies on the interaction between synthetic glycoconjugates and alpha-L-fucosyl binding lectins

Evonymus europaea lectin precipitated with alpha DGal(1----3) beta DGal(1----4)beta DGlcNAc-bovine serum albumin (BSA), alpha LFuc(1----2)beta DGal(1----3)beta DGlcNAc-BSA, alpha LFuc(1----2)beta DGal(1----4)DGlcNAc, and alpha DGal(1----3)[alpha LFuc(1----2)]beta DGal-BSA. However, the lectin neither precipitated with alpha LFuc(1----2)-beta DGal-BSA, alpha DGal(1----3)beta DGal-BSA, or beta DGal(1----4)beta DGlcNAc-BSA nor agglutinated erythrocytes of Oh phenotype having multiple terminal beta DGal(1----4)beta DGlcNAc residues. These results indicate that the minimal structural requirement for glycoprotein precipitation or cell agglutination by the lectin includes any of the three trisaccharides (fucosylated or nonfucosylated) derived from the blood group B tetrasaccharide. The monosaccharides linked to the beta-D-galactosyl residue in the blood group B tetrasaccharide, namely, alpha-D-galactose, alpha-L-fucose, and N-acetyl-beta-D-glucosamine, participate almost equally in binding to the lectin in as much as removal of any one of these sugars reduces the inhibiting potency of the resulting trisaccharide. alpha LFuc(1----2)beta DGal(1----3)beta DGlcNAc-BSA (H type 1) and alpha LFuc(1----2)beta DGal(1----4)beta DGlcNAc (H type 2) were precipitated to the same extent. The E. europaea lectin neither precipitated alpha DGal(1----4)-beta DGal(1----4)beta DGlcNAc-BSA, Lea-BSA, Leb-BSA, or beta DGlcNAc(1----4)[alpha LFuc(1----6)]beta DGlcNAc-BSA nor agglutinated Oh,Lea and Oh,Leb erythrocytes, demonstrating that terminal D-galactose linked alpha-(1----4) to subterminal beta-D-galactose, or alpha-L-fucose linked to N-acetylglucosamine, prevents lectin binding. Corey-Pauling-Koltun molecular models, built on the basis of data from 1H NMR and hard-sphere exo-anomeric (HSEA) calculations provided by Lemieux and co-workers [Lemieux, R. U., Bock, K., Delbaere, L. T. J., Koto, S., & Rao, V. S. (1980) Can. J. Chem. 58, 631-653], show that these alpha-D-galactosyl and alpha-L-fucosyl groups act to sterically hinder lectin binding to these oligosaccharides; these observations also suggest that the lectin binds to the beta-side of these oligosaccharides. These sides, on both blood group H type 1 and blood group H type 2 oligosaccharides, provide a similar contour which can fully account for their equal reactivity with E. europaea lectin. The only difference found between Lotus and Ulex I lectins in precipitating ability was that only Lotus precipitated with beta DGlcNAc(1----4)[alpha LFuc(1----6)]beta DGlcNAc-BSA.(ABSTRACT TRUNCATED AT 400 WORDS)     

A synthetic peptide with morphine-like pharmacologic action.

On purely theoretical grounds an oligopeptide was synthesized, which produced typical morphine-like effects on the guinea pig myenteric plexus-longitudinal muscle preparation and in the opiate receptor binding assay. It is a linear heptapeptide with structure HTyrGlyGlyLysMetGlyOH. This is the first opioid peptide of known structure.