Effect of specific trinitrophenylation of the lysine epsilon amino group of glucagon on receptor binding and adenylate cyclase activation

The trinitrophenyl group was specifically introduced into the ?-amino group of glucagon by reaction of N^α-citraconyl glucagon with trinitrobenzenesulfonic acid. The N^α-citraconyl blocking group was subsequently removed by acid treatment yielding N^?-trinitrophenyl glucagon which was purified by anion-exchange chromatography. The derivative showed less secondary structure as measured by circular dichroism than the native hormone at pH 8.0 and at pH 2.0 in the presence of sodium dodecyl sulfate. The analog possessed 4–5% the potency of glucagon in stimulating adenylate cyclase with 90% maximal stimulation and possessed 30% the potency of glucagon in competing for glucagon-specific receptor sites in hepatic plasma membranes. Although the structure of N^?-trinitrophenyl glucagon is very similar to N^?-4-azido-2-nitrophenyl glucagon, the photoaffinity antagonist synthesized by M. D. Bregman and D. Levy [(1977) Biochem. Biophys. Res. Commun., 78, 584–590.], the biological activities of the two are different. Possible explanations for these differences are discussed.     

N-acylation of Aplysia egg-laying hormone with biotin. Characterization of bioactive and inactive derivatives.

Chemical modification of the egg-laying hormone (ELH) of Aplysia by reaction with the N-hydroxysuccinimide ester of biotin, which contained 6-aminohexanoic acid as spacer, yielded seven distinct derivatives that were readily separated by reversed-phase high performance liquid chromatography. The derivatives were chemically characterized by amino acid compositional analysis, sequence analysis, and mass spectrometry. The seven derivatives resulted from combinations of differential modification of the three amino groups in the ELH molecule located at Ile1 (alpha-NH2), Lys8, and Lys36. Of the seven derivatives formed, only one, monobiotinyl Lys36-ELH, was biologically active in eliciting egg-laying activity and altering the electrophysiological activity of the abdominal ganglion neuron R15 and LB and LC cluster neurons. In addition, evaluation of the time course of biotinylation of ELH revealed that the relative rate of amino group reactivity was epsilon-NH2-Lys36 greater than epsilon-NH2-Lys8 much greater than alpha-NH2-Ile1. The slow rate of reaction of the terminal alpha-amino group suggested that it was relatively inaccessible to biotinylation, possibly due to conformational factors or to ion-pair formation with an unidentified carboxyl group. Loss of bioactivity of ELH monobiotinylated on the alpha-amino group, coupled with the unusually low reactivity of the alpha-amino group, provided strong evidence for the importance of the alpha-amino group in ELH function. Furthermore, the development and availability of a bioactive ELH probe should greatly facilitate the isolation, characterization, and localization of the ELH receptor.     

The use of p-fluorobenzenesulfonyl chloride as a reagent for studies of proteins by fluorine nuclear magnetic resonance

The reagent p-fluorobenzenesulfonyl chloride modifies the protein side chains of tyrosine, lysine, and histidine and the alpha-NH2 group. The p-fluorobenzenesulfonyl (Fbs-) group, identified by the 19F nuclear magnetic resonance signal, exhibits a different 19F chemical shift for each functional group modified. The Fourier-transformed spectra of the Fbs- group displayed the expected nine-line multiplet in Fbs- amino acids and simple Fbs- peptides but not in the Fbs- proteins, where the resolution was less. Lysozyme, RNase, DNase, and chymotrypsin react with this reagent and each Fbs- protein exhibits a distinctive pattern of 19F NMR signals due to the label, suggesting that the reaction of the reagent varies with the reactivity of the side chains in a protein. The three major 19F signals of the unfolded Fbs-RNase in 8 M urea are due to the Fbs- label on the imidazolium, alpha-NH2, and epsilon-NH2 groups. Based upon results from amino acid and 19F NMR analyses of the tryptic-chymotryptic peptides of Fbs-RNase, portions of the imidazolium and epsilon-NH2 resonances were assigned to the Fbs- label on His-105 and Lys-41, respectively, while the alpha-NH2 resonance was entirely due to the Fbs- label on the alpha-NH2 of Lys-1. Because Fbs-RNase has an unchanged, near-ultraviolet circular dichroism spectrum and because it retains approximately 80% of the RNase activity, the conformation of Fbs-RNase is probably not altered from the folded conformation of the native enzyme. Upon unfolding in 8 M urea or heating at 70 degrees C, Fbs-RNase gave a 19F NMR spectrum differing from that of the folded Fbs-RNase. In the presence of uridylic acid, Lys-41 was the only residue protected from modification by the reagent with a concomitant reduction of the epsilon-NH2 resonance, and the RNase thus modified was fully active. Hence, 19F NMR analysis of protein, via the reaction with p-fluorobenzenesulfonyl chloride, provided not only information about the protein conformation but also direct measurements of the modification status.     

Modification of ovine luteinizing hormone subunits with SMPT and its effect on subunit recombination, immunological activity, receptor binding and steroidogenic activity

The increasing use of heterobifunctional cross-linking agents in the design of defined conjugates for selective targeting and inducing immune response has prompted us to study the role of epsilon-NH2 group modification of oLH subunits, their recombination and effect on immunoreactivity, receptor binding and biological activity. The epsilon-NH2 groups of alpha oLH and beta oLH subunits were separately modified by using SMPT. The alpha oLH-SMPT modified derivatives hybridize to beta oLH. Similarly, the beta oLH-SMPT derivatives recombined with alpha oLH. The recombination was judged by gel filtration chromatography and RP-HPLC analysis. The sequential modification of subunits led to progressive reduction in immunoreactivity and receptor binding activity. The modification of six or more epsilon-NH2 groups in alpha oLH although recombine fully with native beta oLH but failed to react to anti-oLH antibody. Moreover, the steroidogenic activity was also abolished. Introduction upto four SMPT groups in alpha oLH compromised immunological and biological activities but further addition of two or more SMPT groups completely abolished antibody reactivity, receptor binding and steroidogenic activity indicating the importance of later two amino groups in the receptor binding and steroidogenic activity. The present investigation clearly demonstrate that only 1:2-3 molar ratio of oLH subunits:SMPT could generate the site(s) in the subunits of the oLH that retained reasonable immunological, receptor binding and biological activity of the hormone. Therefore, this molar ratio may be used in future for the design and synthesis of bioeffective hormonotoxins.     

Inhibition of electron transport and oxidative phosphorylation in plant mitochondria by gladiolic acid and structurally-related aromatic ortho dialdehydes

Gladiolic acid (GA, 4-methoxy-5-methyl-0-phthalaldehyde-3-carboxylic acid), an antifungal aromatic ortho dialdehyde produced by Penicillium gladioli was found to be a potent inhibitor of electron transport and oxidative phosphorylation reactions in sweet potato and mung bean mitochondria. Similar results were also found with the naturally occurring ortho dialdehydes, cyclopaldic acid, quadrilineatin, and flavipin as well as the synthetic dialdehydes, 3-formyl opianic acid and 0-phthalaldehyde. Because of their highly reactive ortho-diformyl grouping, GA and structurally related dialdehydes apparently act as multisite inhibitors affecting electron transport and oxidative phosphorylation (at each coupling site). Gladiolic acid has no uncoupling effect like 2,4-dinitrophenol and does not have the same point of interaction in the energy transfer process as oligomycin. Several "partial" reactions of phosphorylation (Mg+2-DNP-stimulated ATPase; ATP-Pi exchange) were strongly inhibited by the various dialdehydes. Flavipin and quadrilineatin are potent inhibitors (80% at a concentration of 25 microM) of site III phosphorylation. Gladiolic acid and related ortho dialdehydes inactivate the catalytic activity of native cytochrome c in vitro. Lysyl epsilon-NH2 rich cytochrome c may be a major site of GA action in the intact mitochondrion. In view of the high chemical reactivity of the orthodiformyl group, it is suggested that mitochondrial function may be affected by aromatic ortho dialdehydes through a combination of reactions involving cross-linking of amino groups on membrane polypeptides and monofunctional reaction with free amino groups important for enzyme function, including epsilon-NH2 groups on cytochrome c. Cross-linking in mitochondrial membrane systems might affect function by interfering with molecular motion in the operation of the terminal portion of the electron-transport chain. The primary toxicological mode of action of GA and related dialdehydes appears to be due to inhibition of mitochondrial function.     

Two cytoplasmic loops of the glucagon receptor are required to elevate cAMP or intracellular calcium.

The glucagon receptor is a member of a distinct class of G protein-coupled receptors (GPCRs) sharing little amino acid sequence homology with the larger rhodopsin-like GPCR family. To identify the components of the glucagon receptor necessary for G-protein coupling, we replaced sequentially all or part of each intracellular loop (i1, i2, and i3) and the C-terminal tail of the glucagon receptor with the 11 amino acids comprising the first intracellular loop of the D4 dopamine receptor. When expressed in transiently transfected COS-1 cells, the mutant receptors fell into two different groups with respect to hormone-mediated signaling. The first group included the loop i1 mutants, which bound glucagon and signaled normally. The second group comprised the loop i2 and i3 chimeras, which caused no detectable adenylyl cyclase activation in COS-1 cells. However, when expressed in HEK 293T cells, the loop i2 or i3 chimeras caused very small glucagon-mediated increases in cAMP levels and intracellular calcium concentrations, with EC50 values nearly 100-fold higher than those measured for wild-type receptor. Replacement of both loops i2 and i3 simultaneously was required to completely abolish G protein signaling as measured by both cAMP accumulation and calcium flux assays. These results show that the i2 and i3 loops play a role in glucagon receptor signaling, consistent with recent models for the mechanism of activation of G proteins by rhodopsin-like GPCRs.     

Kinetic study of the reaction between trinitrobenzenesulfonic acid and amino acids with a trinitrobenzenesulfonate ion-selective electrode

Potentiometric studies of the reaction between trinitrobenzenesulfonic acid (TNBS) and several amino acids with the TNBS electrode indicate that the reaction is first-order with respect to TNBS and amino acid concentration. The reaction is zero-order with respect to hydroxide concentration at pH greater than 10.5, indicating that the nonprotonated amino group is the reactive species. Rate constants were calculated for each amino acid and a simple mechanism of the reaction is proposed.     

The essential role of the imidazole group of glucagon in its biological function

Guanidinated, carbamylated, and acetylated glucagon largely retains the ability to stimulate the formation of cyclic AMP in rat liver homogenates, while with N-ethoxyformyl-acetylated glucagon this ability is completely lost. This latter derivative can be reconverted to a biologically active peptide by treatment with hydroxylamine. These results indicate that the imidazole group of the amino terminal histidine, but not the α or ? amino groups of glucagon, is essential for activity. Histidine amide does not stimulate the activity of adenyl cyclase even at 0.2 m concentration. The titration behavior of glucagon shows a normal pK for the amino terminal histidine.     

The role of nonspecific hydrophobic interactions in the biological activity of N epsilon-acyl derivatives of glucagon. Studies of conformation, receptor binding, and adenylate cyclase activation

Glucagon was acylated at position 12 using conditions favoring reaction with the epsilon-amino group of lysine. The N epsilon-acetyl, N epsilon-hexanoyl, and N epsilon-decanoyl derivatives were prepared and purified. Secondary structure as measured by circular dichroism was lower in all derivatives than in glucagon, both in 95% methanol and in 25 mM sodium dodecyl sulfate at pH 2 and pH 12. N epsilon-Acetyl glucagon was less active than the native hormone in a radioreceptor assay and higher concentrations of this derivative were required to stimulate the adenylate cyclase activity of rat liver plasma membranes. The maximal extent of cyclase activation by this derivative was less than that found with the native hormone. N epsilon-Hexanoyl glucagon and N epsilon-decanoyl glucagon had greater activity than N epsilon-acetyl glucagon in receptor binding as well as in adenylate cyclase activation, although these two derivatives were not as active as the native hormone. N epsilon-hexanoyl glucagon and N epsilon-decanoyl glucagon were more potent in receptor binding than in adenylate cyclase activation. From these results it appears that the positive charge of the epsilon-amino groups may have a specific role in obtaining maximal biological activity, while the acyl groups contribute to the nonspecific hydrophobic interactions between the hormone and its receptor. In addition, a possible relationship between stabilization of the amphipathic helix in solution and the activity of these and other N epsilon-derivatives of glucagon is discussed.     

Hemoglobin-binding site on human haptoglobin. Identification of lysyl residues participating in the binding.

The locus of human haptoglobin (Hp) molecules that interacts with human hemoglobin (Hb) was examined by the differential labeling technique. First, amino groups of Hp were extensively labeled with ethyl acetimidate (EAI) either in the free state (experiment A) or in the equimolar complex with Hb (experiment B). Each of the labeled Hp samples was reduced and S-carboxymethylated, and the remaining amino groups were then allowed to react with trinitrobenzenesulfonic acid (TNBS) under denatured conditions. Only 0.6 mol/mol of the trinitrophenyl (TNP) group was introduced into the heavy chain of Hp in experiment A, whereas 2.5 mol/mol of TNP groups were into the same chain in experiment B. The extent of TNP modification for the light chain was very low in either of the experiments. The amino acid residues carrying TNP groups in the heavy chain were identified by the peptide mapping procedure. They were Ile1, Lys136, and Lys218 in experiment B, and only Ile1 in experiment A. These findings suggest that epsilon-amino groups of Lys136 and Lys218 in the heavy chain are both situated within the Hb-binding locus of Hp, and that the alpha-amino group of Ile1 is buried inside the molecule either in the presence or absence of Hb.     

Study of the modification of histidine residues, SH- and epsilon-NH2-groups of rat sarcoma-45 myosin by specific reagents

Modification of histidine residues, SH- and epsilon-NH2-groups of myosin from rat sarcoma-45 by specific reagents was studied. It was shown that diethylpyrocarbonate modifies histidine residues essential for the ATPase activity. A kinetic analysis of myosin epsilon-NH2-groups modification by 2,4,6-trinitrobenzene sulfonate revealed that myosin trinitrophenylation and its inactivation by Ca2(+)-ATPase occurs in two steps: a fast and a slow (Km = 2400 and 1.7 s-1 M-1, respectively). Two essential epsilon-NH2-groups of tumour myosin active sites react in the fast reaction. The relatively low concentrations of p-chloromercuribenzoic acid activate rat sarcoma-45 myosin Ca2(+)-ATPase and Mg2(+)-ATPase, whereas higher ones inhibit the enzyme. The data obtained suggest that two SH-groups, SH1 and SH2 are essential for the tumour myosin ATPase function.     

Amino group modification inhibits ristocetin cofactor activity of human von Willebrand factor

Purified von Willebrand factor rapidly loses activity when treated under mild conditions with the highly specific amino group reagent trinitrobenzenesulfonic acid. Greater than 90 percent inhibition of activity is achieved by modification of only 7 percent of the amino groups. Other modifications such as acetylation and succinylation also abolish activity. It is unlikely that the essential rapidly reacting amino groups function simply in an electrostatic manner since modifications such as amidination and methylation which produce derivatives which retain positive charge are also inactive or nearly so.     

Reactions of the amino groups in ribonuclease A: II. Identification of reactive amino groups in ribonuclease

Ribonuclease A has been trinitrophenylated to varying degrees by reaction with trinitrobenzenesulfonic acid. The reactive amino groups were identified by use of the peptides obtained from the oxidized TNP-RNase by tryptic and chymotryptic hydrolysis. From a quantitative study of the TNP-peptides it was possible to associate each amino group with values of pK_ma. It was shown that the lys-41 amino group had a pK_a of 9.03 in TEA buffer. The pK_a values of all of the other amino groups were dependent on the nature of the buffer (triethanolamine and phosphate) and on the pH.     

Effect of modification of cytochrome c on its reactions with superoxide and NADPH:cytochrome P-450 reductase

Acetylation and succinylation of cytochrome c decrease its rate of reaction with superoxide. The effect of succinylation is greater than that of acetylation. As predicted by the Brönsted-Debye-Hückel relationship, the effect of modification of cytochrome c is more pronounced at low ionic strength. Modification of cytochrome c causes a much greater decrease in its reaction with NADPH-cytochrome P-450 reductase, compared to its reaction with superoxide. This data forms the quantitative basis for the enhanced specificity of modified cytochrome c for superoxide detection previously described by other investigators. Additionally, a greatly simplified version of the trinitrobenzenesulfonic acid method for estimation of free amino groups is presented.     

In vitro mutagenesis of a mouse MHC class I gene for the examination of structure-function relationships

Oligonucleotide-directed, site-specific mutagenesis has been employed to elucidate the role of individual amino acids on the expression and function of a MHC class I antigen. Two oligonucleotides were synthesized to introduce single amino acid substitutions in the murine H-2Ld gene. The highly conserved glycosylation site at amino acid position 86 was changed from asparagine to lysine to remove the carbohydrate moiety from the first external domain of the H-2 molecule, and the phenylalanine at position 116 was changed to tyrosine, replacing the Ld residue with the Kb type amino acid analogous to Kb mutants: bm5 and bm16 mutants derived from the Kb antigen have the Ld-type residue at this position. The mutant genes were constructed by annealing the mutagenic oligomers to the single stranded H-2Ld gene, followed by chain elongation reaction. The expected mutations were confirmed by DNA sequence determination. The mutant genes were introduced into mouse L cells by DNA-mediated gene transfer. Both mutant genes expressed the antigens on the cell surface, as detected by antibody binding; these antigens were reactive with the cytotoxic T cells specific for the H-2Ld antigen. Detailed examination with 16 monoclonal anti-H-2Ld antibodies revealed that the binding of some antibodies was significantly reduced in the glycosylation mutant, implying a certain contribution of the carbohydrates to the antigenic activity of some determinants. No detectable changes have been observed in the mutant of the substitution at position 116 by the parameters we tested.     

Conformational and biological properties of glucagon fragments containing residues 1-17 and 19-29

The 29 amino acid polypeptide hormone glucagon was cleaved into two large fragments by the enzyme clostripain. The conformational properties of these two fragments were monitored by circular dichroism at pH 2 and 12 in both the presence and absence of sodium dodecyl sulfate. Both glucagon (1-17) and glucagon (19-29) have reduced abilities to fold in aqueous solution. However, both fragments can take on structure of higher apparent helical content in acidic solution in the presence of sodium dodecyl sulfate but only the glucagon (19-29) retains this conformation at high pH. Neither of the two fragments react with dimyristoylphosphatidylcholine as the intact peptide does. Only the carboxyl terminal fragment was capable of reacting with an antibody specific for glucagon. The glucagon (1-17) has markedly reduced affinity for binding to the glucagon receptor as well as markedly reduced ability to stimulate adenylate cyclase activity which is not affected by the presence of glucagon (19-29). It is proposed that the intact sequence provides specific groups required for activity as well as the potential for forming a stable amphipathic helix, both of which are necessary for full biological activity at low peptide concentrations.     

The reactivity of the thiol groups of the adenosine triphosphatase of sarcoplasmic reticulum and their location on tryptic fragments of the molecule.

The ATPase (adenosine triphosphatase) from sarcoplasmic reticulum contains 20 thiol groups/115000 daltons, measured by using either N-ethyl[(14)C]maleimide or 5,5'-dithiobis-(2-nitrobenzoate) in sodium dodecyl sulphate. After reduction there were 26 thiol groups, in good agreement with 26.5 residues of cysteic acid found by amino acid analysis. The difference between this and the 20 residues measured before reduction implies the presence of three disulphide residues. The same number of disulphide residues was found by direct measurement. Three to six fewer thiol groups were found in preparations made in the absence of dithiothreitol. The missing residues were accounted for as cysteic acid. The distribution of disulphide bonds and of exposed and buried thiol groups among the tryptic fragments of the molecule was measured after labelling with N-ethyl[(14)C]-maleimide. The disulphides were confined to fragment B (mol.wt. 55000), whereas several thiol groups were present on each of the fragments (A, B, A(1) and A(2)). The kinetics of the reaction of the ATPase with 5,5'-dithiobis-(2-nitrobenzoate) showed that four or five of the thiol groups were unreactive in the absence of detergent and that 13 of the remainder reacted with a single first-order rate constant. In the presence of ATP and Ca(2+) the reaction rate of all but two groups of this class was uniformly decreased. In the presence or absence of ATP and Ca(2+) the rate constant for inactivation was close to the rate constant for this class, but was not identical with it. No selective protection of a specific active-site-thiol group was observed. Parallel experiments with sarcoplasmic reticulum gave similar results, except that the reaction rates were a little lower and there were two more buried groups. Solution of ATPase of sarcoplasmic reticulum in detergent greatly increased the reactivity of all thiol groups. The effects of low concentrations of deoxycholate were reversible. EGTA or low concentrations (0.02mm) of Ca(2+) of Mg(2+) had very little effect on the reactivity.     

A method for the determination of the molar extinction coefficient of structure-linked chromophores

Synopsis This paper describes a general method for the determination of the molar extinction ceefficient of a chromophore covalently bound to structure-linked groups, without isolating the compound formed. The method is illustrated by the determination of the molar extinction coefficient of the reaction product of 2,4-dinitro-1-fluorobenzene (DNFB) with films of aminoethyl-cellulose (AE-cellulose). The method is based on the relation between the decrease in extinction of a DNFB staining solution and the increase in extinction of the AE-cellulose after staining as measured in a film-spectrophotometer. In addition, the value of the molar extinction coefficient was used in establishing reaction conditions for a quantitative staining procedure for determining amino groups with DNFB and picric acid. Conditions of optimum DNFB staining were determined and the measured extinction was converted into concentration of amino groups using the molar extinction coefficient. The amino group concentration of the same batch of AE-cellulose was also determined, after finding the optimum reaction conditions, by staining with picric acid. The results, when compared, showed a linear relationship with a slope of unity for batches of AE-cellulose of varying amino group concentrations. This is consistent with the same stoichiometry in both cases and indicates that in both procedures one chromophore molecule has reacted with one amino group and that this reaction has proceeded to completion. The general applicability of the method is discussed.     

A tyrosine motif in the cytoplasmic domain of mason-pfizer monkey virus is essential for the incorporation of glycoprotein into virions

Mason-Pfizer monkey virus (M-PMV) encodes a transmembrane (TM) glycoprotein with a 38-amino-acid-long cytoplasmic domain. After the release of the immature virus, a viral protease-mediated cleavage occurs within the cytoplasmic domain, resulting in the loss of 17 amino acids from the carboxy terminus. This maturational cleavage occurs between a histidine at position 21 and a tyrosine at position 22 in the cytoplasmic domain of the TM protein. We have demonstrated previously that a truncated TM glycoprotein with a 21-amino-acid-long cytoplasmic tail showed enhanced fusogenicity but could not be incorporated into virions. These results suggest that postassembly cleavage of the cytoplasmic domain removes a necessary incorporation signal and activates fusion activity. To investigate the contribution of tyrosine residues to the function of the glycoprotein complex and virus replication, we have introduced amino acid substitutions into two tyrosine residues found in the cytoplasmic domain. The effects of these mutations on glycoprotein biosynthesis and function, as well as on virus infectivity, have been examined. Mutation of tyrosine 34 to alanine had little effect on glycoprotein function. In contrast, substitutions at tyrosine 22 modulated fusion activity in either a positive or negative manner, depending on the substituting amino acid. Moreover, any nonaromatic substitution at this position blocked glycoprotein incorporation into virions and abolished infectivity. These results demonstrate that M-PMV employs a tyrosine signal for the selective incorporation of glycoprotein into budding virions. Antibody uptake studies show that tyrosine 22 is part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein that can also be positively and negatively influenced by changes at this site.     

Effect of lysine residue modification of ovine luteinizing hormone by heterobifunctional crosslinking reagent SPDP on subunit-subunit association, receptor binding and biological activity.

The increasing use of heterobifunctional crosslinking agent in the design of hormone-carrier conjugates for selective targeting or inducing immune response against the hormone has prompted us to study the effect of epsilon-NH2 group modification of oLH-subunit, their recombination, immunoreactivity, receptor binding and biological activity. The epsilon-NH2 groups of oLH alpha and oLH beta subunits were modified by using SPDP. The SPDP modified oLH alpha derivatives hybridize to native OLH beta as judged by RP-HPLC analysis. The sequential modification of alpha and beta subunits led to progressive reduction in immunoreactivity and receptor binding activities. The steroidogenic potential of oLH beta.SPDP.alpha oLH recombinant was relatively comparable. The modification of six or more epsilon-NH2 groups in oLH alpha although recombine fully with native oLH beta but failed to react to anti-oLH antibody. Moreover, steroidogenic activity was also abolished. Introduction up to four SPDP groups in oLH alpha compromised immunological and biological activities but further addition of two more SPDP groups completely abolished antibody reactivity, receptor binding and steroidogenic activity indicating the importance of later two -NH2 groups in the receptor recognition and steroidogenic potential.