Binding of glucagon to lipid bilayers

At physiological pH and temperature, glucagon binds to liposomes composed of egg phosphatidylcholine and cholesterol (2:1 mol/mol) in a highly specific manner. The chemical reactivities of the functional groups were determined over the concentration range of 1.0 X 10(-6)-3.0 X 10(-8) M by the method of competitive labelling with 1-fluoro-2,4-dinitrobenzene as the labelling reagent. At concentrations above 3 X 10(-7) M, the amino terminal histidine and the two tyrosine residues showed a marked decrease in reactivity in the presence of liposomes, but the reactivity of the Lys-12 N epsilon-amino group was unaltered. At lower concentrations the Lys-12 reactivity also decreased markedly, owing to a change in the environment of this group. These results indicated that two different forms of glucagon existed over the concentration range studied. Both in the absence and presence of liposomes the Lys-12 N epsilon-amino groups showed a transition in reactivity at 1.8 X 10(-7) M. In the presence of liposomes the other functional groups also showed a transition in reactivity at 2 X 10(-7) M but the change was much smaller. The pattern of reactivities were consistent with the X-ray crystallographic structure of the type 2 glucagon trimer being the predominant species at 10(-6) M, with free monomeric glucagon occurring at 3 X 10(-8) M. A trimerization constant of 4 X 10(13) M-2 at pH 7.5 and 37 degrees C was determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical properties of the functional groups of insulin.

The method of competitive binding [Kaplan, Stevenson & Hartley (1971) Biochem. J. 124, 289-299] with 1-fluoro-2,4-dinitrobenzene as the labelling reagent [Duggleby & Kaplan (1975) Biochemistry 14, 5168-5175] was used to determine the chemical properties, namely pK and reactivity, of the amino groups, the histidine residues and the tyrosine residues of the dimeric form of pig zinc-free insulin at 20.0 degrees C. The N-terminal glycine residue of the A-chain has a pK of 7.7 and a slightly higher than normal reactivity. The N-terminal phenylalanine residue of the B-chain has a pK of 6.9 and is approximately an order of magnitude more reactive than a corresponding amino group with the same pK value. The lysine epsilon-amino group has an unusually low pK of 7.0 but has approximately the expected reactivity of such a group. In the case of the two histidine and four tyrosine residues only the average properties of each class were determined. The histidine residues have a pK value of approx. 6.6, but, however, their reactivity is at least an order of magnitude greater than that of a free imidazole group. The tyrosine residues have a pK value of approx. 10, but their average reactivities are substantially less than for a free phenolic group. At alkaline pH values above 8 the reactivity of all the functional groups show sharp discontinuities, indicating that insulin is undergoing a structural change that alters the properties of these groups.     

Chemical reactivity of the functional groups of insulin. Concentration-dependence studies.

A modification to the competitive labelling procedure of Duggleby and Kaplan [(1975) Biochemistry 14, 5168-5175] was used to study the reactivity of the N-termini, lysine, histidine and tyrosine groups of insulin over the concentration range 1 X 10(-3)-1 X 10(-7)M. Reactions were carried out with acetic anhydride and 1-fluoro-2,4-dinitrobenzene in 0.1 M-KCl at 37 degrees C using Pyrex glass, Tefzel and polystyrene reaction vessels. At high concentrations all groups had either normal or enhanced reactivity but at high dilution the reactivities of all functional groups became negligible. This behaviour is attributed to the adsorption of insulin to the reaction vessels. The histidine residues show a large decrease in reactivity in all reaction vessels in the concentration range 1 X 10(-3)-1 X 10(-5)M where there are no adsorption effects and where the reactivities of all other functional groups are independent of concentration. With polystyrene, where adsorption effects become significant only below 1 X 10(-6)M, the reactivity of the phenylalanine N-terminus also shows a decrease in reactivity between 1 X 10(-5) and 1 X 10(-6)M. In 1 M-KCl insulin does not absorb to Pyrex glass and under these conditions the histidine reactivity is concentration-dependent from 1 X 10(-3) to 5 X 10(-6)M and the B1 phenylalanine alpha-amino and the B29 lysine epsilon-amino reactivities from 5 X 10(-6) to 1 X 10(-7)M, whereas the reactivities of all other groups are constant. These alterations in reactivity on dilution are attributed to disruption of dimer-dimer interactions for histidine and to monomer-monomer interactions for the phenylalanine and lysine amino groups. It is concluded that the monomeric unit of insulin has essentially the same conformation in its free and associated states.     

Oxidative deamination of epsilon-aminolysine residues and formation of Schiff base cross-linkages in cell envelopes of Escherichia coli.

Oxidative deamination of the epsilon-amino group of lysyl residues to form allysine is the initial reaction in the cross-linking of collagen and elastin in vertebrates. The allysyl residues, generated by lysyl oxidase in this reaction, condense with either other allysyl residues or epsilon-amino groups of lysyl or hydroxylysyl to form aldol or Schiff base cross-links. This paper presents evidence that similar allysyl residues and Schiff base cross-links are synthesized in cell envelopes of Escherichia coli. Acid hydrolysis followed by amino acid analysis of envelopes either reduced with NaB[3H]4 or labeled with [14C]lysine and reduced with NaBH4 yielded allysine and two labeled fragments with elution profiles and molecular weights (250 and 330) consistent with Schiff base products derived at least in part from allysine. When [6-3H]lysine-labeled cell envelopes were incubated at 37 degrees C, gradual release of tritiated water occurred. This suggests that an enzymatic reaction catalyzes the deamination of lysine in E. coli membranes and that the higher molecular weight proteins detected in stationary phase or in log phase cell envelopes after NaBH4 reduction occur as a result of formation of Schiff base cross-links.     

Characterization of the carbamino adducts of insulin.

Carbon-13 (13C) nuclear magnetic resonance spectroscopy (NMR) is performed to characterize the formation of carbamino adducts between insulin and (13C) carbon dioxide over a range of pH values in the presence of a physiological concentration (23 mM) of sodium bicarbonate. The peaks from two of the carbamino adducts resonate at higher frequencies than the signal from bicarbonate, at 164.6 and 165.3 ppm, and are attributed to the adducts with the terminal amino groups of phenylalanine B1 and glycine A1. The intensities of these signals vary with the pH, with unique patterns. Over 6% of each terminal amino group exists as the carbamino adduct at the optimum pH values of 7.8 and 8.3. A unique third adduct resonates at 159.3 ppm, and is attributed to lysine B29. This adduct is present on 2% of the insulin molecules at pH 8.2, but has minimal intensity at pH 7.4. No signals from adducts are detected below pH 6.2, where the amino groups exist predominantly in the protonated form. Creation of the adducts is rapid and they are stable for over 4 wk at 37 degrees C. The narrow bandwidth of the resonance of the adduct (4.0-4.5 Hz) relative to the irreversible cyanate adduct is consistent with molecular forms of the carbamino adduct smaller than the 2-Zn-hexamer which is the preponderate form of clinically utilized U-100 insulin (i.e., 100 U/ml).     

The role of epsilon-amino groups of lysine of human serum albumin in heat-induced protein denaturation. Increased stability of glycosylated and alkylated albumin in aggregation during heating

Human serum albumin was glycosidated by prolonged protein incubation in phosphate buffer, pH 6.8-7.0, with excess glucose at 37 degrees C. epsilon-amino groups of lysine residues of the albumin molecule were alkylated by pyridoxal-5-phosphate in the presence of NaBH4. The solutions of glycosidated and alkylated serum albumin were incubated at different temperature values in the range of 20 to 80 degrees C in phosphate buffer, pH 7.0, over 30 min. The nondenatured monomer and the resulting aggregated were isolated by TSK-HW-55-gel column chromatography and polyacrylamide gel electrophoresis. The stability of modified proteins elevated in parallel to the increase in the number of the ligand molecules covalently bound to albumin amino groups. The 1-3% aqueous solutions of glycosidated serum albumin containing 3-4 glucose residues and those of alkylated albumin containing 6-7 residues of pyridoxal-5-phosphate were stable on heating up to 80 degrees C and did not form aggregates. Under these conditions the initial serum albumin completely aggregated. Preincubation of the aggregated albumin with glucose at 37 degrees C resulted in protein "renaturation" to the monomeric form with a small number of dimers and trimers.     

Reactivity and ionization constants of the lysine residues in apovitellenin i of emu egg yolk low-density lipoprotein by competitive labelling.

Competitive labelling with[14C]acetic anhydride over a range of pH values has been used to explore the surface topography of the apovitellenin I moiety in emu egg yolk low-density lipoprotein. The reaction of the lysine xi-amino groups with acetic anhydride has been related to pH in a set of titration curves; from these, the reactivities relative to alanine and the ionization constants of all but the amino terminal lysines have been determined. All lysines have near normal pKa values around 10, and lower than normal reactivities (except the amino terminal lysine). At pH values above 10, the titration curves show breaks where the epsilon-amino groups become much more reactive, except for lysine 71 which in this regard behaves like a normally ionizing lysine in not showing a discontinuity. Most of the basic residues in this apoprotein may occur clustered at the surface of the molecule. This accounts best for the observed low reactivities and pKa values. The amino terminal lysine residue is presumably completely exposed to the aqueous environment.     

Fluoresceination of FepA during colicin B killing: effects of temperature, toxin and TonB

We studied the reactivity of 35 genetically engineered Cys sulphydryl groups at different locations in Escherichia coli FepA. Modification of surface loop residues by fluorescein maleimide (FM) was strongly temperature-dependent in vivo , whereas reactivity at other sites was much less affected. Control reactions with bovine serum albumin showed that the temperature dependence of loop residue reactivity was unusually high, indicating that conformational changes in multiple loops (L2, L3, L4, L5, L7, L8, L10) transform the receptor to a more accessible form at 37°C. At 0°C colicin B binding impaired or blocked labelling at 8 of 10 surface loop sites, presumably by steric hindrance. Overall, colicin B adsorption decreased the reactivity of more than half of the 35 sites, in both the N- and C- domains of FepA. However, colicin B penetration into the cell at 37°C did not augment the chemical modification of any residues in FepA. The FM modification patterns were similarly unaffected by the tonB locus. FepA was expressed at lower levels in a tonB host strain, but when we accounted for this decrease its FM labelling was comparable whether TonB was present or absent. Thus we did not detect TonB-dependent structural changes in FepA, either alone or when it interacted with colicin B at 37°C. The only changes in chemical modification were reductions from steric hindrance when the bacteriocin bound to the receptor protein. The absence of increases in the reactivity of N-domain residues argues against the idea that the colicin B polypeptide traverses the FepA channel.     

The carbamate reaction of glycylglycine, plasma, and tissue extracts evaluated by a pH stopped flow apparatus.

We have used a stopped flow rapid reaction pH apparatus to investigate the carbamate equilibrium in glycylglycine solutions and in three biological tissues, human plasma, sheep muscle, and sheep brain, as well as to investigate the kinetics of carbamate formation in glyclyglycine solution and in human plasma. The rapid reaction apparatus was equipped with a pH sensitive glass electrode in order to follow the time course of pH from 0.005 to 100 s after rapid mixing of a solution of amine or protein and CO2. Two phases of the pH curve were observed: a fast phase representing carbamate formation, and a slow phase due to the hydration of CO2 which was uncatalyzed since a carbonic anhydrase inhibitor was added to the biological solutions. From the time course of pH change during the fast phase K2, the R-NH2 ionization constant, and Kc, the carbamate equilibrium constant as well as the velocity constant for the formation of carbamate, ka could be calculated from data at different pH and pCO2. The carbamate formed in glycylglycine solutions over a wide range of pH and pCO2 was found consistent with the theory of carbamate formation and with published data. At ionic strength 0.16 and 37 degrees pK is 7.67. pKc 4.58. The heat of the carbamate reaction (deltaH) was calculated to be -3.2 kcal/mol between 20 degrees and 37 degrees. Kt of glycylglycine depends quantitatively on ionic strength as predicted by the Debye-Huckel theory. With ionic strength 0.16 ku was found to be 2,500 M1 S1 at 37 degrees. The activation energy of carbamate formation is 6.7 kcal/mol. Carbamate measurements in human plasma at pCO2 from 38 to 359 Torr. pH from 6.9 to 8.3, temperature 37 degrees, and ionic strength 0.15 provided evidence that two kinds of amino groups participate in carbamate formation. From the equilibrium constants computed for the two species they could be identified as alpha- and epsilon-amino groups. On the basis of a protein molecular weight of 69.000. 0.6 alpha-amino groups/molecule with pKz=7.0 and pKc=4.2, and 5.9 epsilon-amino groups/molecule with pKz=9.0 and pKc=4.3 contribute to carbamate formation. The velocity constant ka was estimated to be 4,950 M1 S1 for the alpha-amino groups and 13,800 M1 S1 for the epsilon-amino groups. Under physiological conditions (pCO2=40 Torr. pH=7.4). The concentration of carbamate in plasma is 0.6 mM and the half-time of carbamate formation is 0.05 s. In extracts prepared from sheep brain at 37 degrees pH=7 and pCO2=35 Torr. the carbamate formation was estimated to be 0.8 mM. With pCO2=70 Torr and the same pH and temperature the carbamate concentration in muscle approximates 0.3 mM and increases to 7 mM as pH rises to 8. It is concluded that, as in plasma, a considerable number of epsilon-amino groups appear to be available for carbamate formation in these tissues.     

A chemical approach to the fine structure of biomolecular complexes: the amino terminal region of the 50S ribosomal "A" protein from Bacillus stearothermophilus.

An experimental approach and methodology are described for determining the reactive properties and ionization constants of individual functional groups of proteins within biomolecular complexes. The ionization constants and reactivities of the methionyl-l amino terminus and the lysyl-3 residue of the alanine rich 50S ribosomal "A" protein from Bacillus stearothermophilus have been determined by an extension of the competitive labeling technique used by H. Kaplan, K. J. Stevenson, and B. S. Hartley ((1971), Biochem. J. 124, 289-299). This approach employs (1-14C)- and (3H)acetic anhydride in a double-labeling procedure. In 0.1 M KCl-0.02 M Mg2+-0.05 M Veronal at 10 degrees the methionyl-l amino terminus has a pKa of 7.5 and is exposed on the surface of the ribosome. The lysyl-3 has a pKa of 10 and is also exposed to solvent at the surface of the 50S subunit. Based on a linear free energy relationship (Bronsted plot) obtained with a series of standard amines the methionyl amino terminus has a substantially higher reactivity than expected from its ionization constant. The lysyl epsilon-amino group has the expected reactivity. The abnormally high reactivity of the methionyl amino terminus can only be accounted for by a specific interaction with other functional groups in the ribosome. These data support the proposal that the charged state of this residue is important in the structure and function of the "A" protein at the surface of the ribosome.     

Reactivity of the imino acids formed in the amino acid oxidase reaction.

The reactivity of the imino acids formed in the D- or L-amino acid oxidase reaction was studied. It was found that: (1) When imino acids reacted with the alpha-amino group of glycine or other amino acids, transimination yielded derivatives less stable to hydrolysis than the parent imino acids. In contrast, when imino acids reacted with the epsilon-amino group of lysine or other primary amines, transimination yielded derivatives more stable to hydrolysis than the parent imino acids. (2) Imino acids react rapidly with hydrazine and semicarbazide, forming stable hydrazones and semicarbazones. At pH 7.7, the rate of reaction of the imino acid analogue of leucine with semicarbazide was 10(4) times greater than that of the corresponding keto acid. The reaction of imino acids with these reagents is rapid enough to permit one to follow spectrophotometrically the amino acid oxidase reaction. Imino acids also reacted with cyanide to yield stable adducts. (3) The rate of hydrolysis of the imino acid analogue of leucine was independent of pH above pH 8.5. At lower pH values, the rate of hydrolysis increased with decreasing pH. At 25 degrees C and in the absence of added amino compounds, this imino acid had a half-life of 22 s at pH 8.5. Its half-life was 9.9 s at pH 7.9.     

1-Deoxyglycitolation of protein amino groups and their regeneration by periodate oxidation

Reductive alkylation of lysyl epsilon-amino groups with sugars (1-deoxyglycitolation) using pyridine borane as the reducing agent has been recently described [Wong, W.S.D., Osuga, D.T. & Feeney, R.E. (1984) Anal. Biochem. 139, 58-67]. The regeneration of the lysines has now been achieved by oxidation with approximately 10 mM periodate. In experiments with glycitolated bovine serum albumin, reactions using 5, 20, and 80 mM periodate were essentially complete in the first 10 min. Oxidations of methionine to the sulfoxide were evident even at this lower concentration of periodate, while higher concentrations and prolonged reaction times only caused unnecessary destruction of amino acids. The removal was shown to occur in a wide pH range with little variation in the recovery of the lysines. The degree of glycitolation, or the nature of the attached carbohydrate residues, had no effect on the yield of products. Reductive 1-deoxygalactitolation of approximately 55% of the lysyl epsilon-amino groups of lysozyme caused no loss in enzymatic activity; when 5 mM periodate was used to remove the 1-deoxygalactitol moiety, approximately 95% of the amino groups were regenerated, concomitant with destruction of approximately 16% of the activity. On reductive 1-deoxygalactitolation of the amino groups of turkey ovomucoid, 65% of the lysyl epsilon-amino groups were modified with 70% loss of the inhibitory activity against trypsin. On treatment with 5 mM periodate, approximately 80% of the amino groups were regained with a similar recovery of the inhibitory activity.     

Complete inactivation and labeling of methionyl-tRNA synthetase by periodate-treated initiator tRNA in the presence of sodium cyanohydridoborate.

Methionyl-tRNA synthetase from Escherichia coli can react with periodate-treated tRNA to form a Schiff's base through the epsilon-amino group of a lysine within the enzymic active center and the 2',3'-aldehyde groups created at the 3'-terminal ribose of tRNA. At alkaline pH, the Schiff's base equilibrium can be continuously and specifically displaced by reduction in situ with sodium cyanohydridoborate, which on the other hand leaves intact the reacting aldehyde groups of oxidized tRNA. The effects of temperature, pH and of reducing agent concentration on the rate and extent of reduction of the Schiff's base are analysed. Conditions are described (37 degrees C, pH 8.0, in the presence of 1 mM cyanohydridoborate) which allowed rapid and complete conversion of the monomeric trypsin-modified methionyl-tRNA synthetase into its 1:1 covalent complex with tRNAfMet.     

Unfolding and breakdown of insulin in the presence of endogenous thiols

Native insulin denatures and unfolds in the presence of thiol catalyst via disulfide scrambling (isomerization). It undergoes two transient non-native conformational isomers, followed by an irreversible breakdown of the protein to form oxidized A- and B-chain. Denaturation and breakdown of native insulin may occur under physiological conditions. At 37 degrees C, pH 7.4, and in the presence of cysteine (0.2 mM), native insulin decomposes with a pseudo first order kinetic of 0.075 h(-1). At 50 degrees C, the rate increases by 5-fold. GdnCl and urea induced denaturation of insulin follows the same mechanism. These results demonstrate that stability and unfolding pathway of insulin in the presence of endogenous thiol differ fundamentally from its reversible denaturation observed in the absence of thiol, in which native disulfide bonds of insulin were kept intact during the process of denaturation.     

Reductive methylation and 13C NMR studies of the lysyl residues of fd gene 5 protein. Lysines 24, 46, and 69 may be involved in nucleic acid binding

We have examined the role of lysyl residues in the binding of fd gene 5 protein to a nucleic acid polymer. The lysyl residues of the protein were chemically modified to form N epsilon, N epsilon-dimethyllysyl derivatives containing 13C-enriched methyl groups. The 13C NMR spectrum of the modified protein was studied as a function of pH and salt concentration. Differences in the local magnetic environment of the six dimethyllysyl amino groups allowed all six 13C resonances to be resolved for samples in the pH range 8.5-9.0 at less than 50 mM ionic strength. One of the dimethylamino resonances was split at low pH, indicating that the two methyl groups were nonequivalent and that the corresponding lysyl residue (either Lys-3 or Lys-7) might be involved in an ion-pairing interaction. Specific lysyl residues were protected from methylation when the protein was bound to poly(rU). The level of protection of individual lysyl residues was quantitated using peptide mapping and sequencing of gene 5 protein labeled with 3H and 14C radioactive labels. Lysines 24, 46, and 69 showed significant protection (33-52%) from methylation in the protein-polynucleotide complex, suggesting that these 3 residues form part of the nucleic acid-binding site. The alpha-amino group of Met-1 was relatively unreactive in both the free and bound protein, which indicated that the amino terminus is not as exposed in solution as in the crystal structure (Brayer, G.D., and McPherson, A. (1983) J. Mol. Biol. 169, 565-596).     

Chemical properties of the histidine residue of secretin: evidence for a specific intramolecular interaction

Secretin has a single histidine residue located at the amino terminus which plays a crucial role in its biological activity. The chemical properties, viz. pK and reactivity, of the alpha-amino and imidazole groups of this residue were determined at a secretin concentration of 10(-6) M in 0.1 M KCl at 37 degrees C. Competitive labelling using tritiated 1-fluoro-2,4-dinitrobenzene (DNP-F) as the labelling reagent was the experimental approach employed. The alpha-amino group was found to have a pK value of 8.83 and a reactivity 5-times that of the alpha-amino group in the model compound, histidylglycine. For the imidazole function a pK value of 8.24 and a reactivity 26-times that of the imidazole function in histidylglycine was found. Both these groups in secretin had pK values which were shifted one pK unit higher than in histidylglycine, but like the model compound the reactivity of the imidazole function was still linked to the state of ionization of the alpha-amino group. These observations are interpreted as evidence for the existence of a major conformational state in dilute aqueous solution in which the amino-terminal histidine of secretion is interacting with a negatively charged carboxyl group.     

The determination of epsilon-amino groups in soluble and poorly soluble proteinaceous materials by a spectrophotometric method using trinitrobenzenesulfonic acid.

A procedure using 2,4,6-trinitrobenzenesulfonic acid (TNBS) for the determination of epsilon-amino groups in soluble and poorly soluble proteinaceous materials is presented. The major modification from previous procedures is an extended TNBS reaction time to allow a stoichiometric reaction with amino groups. In addition, autoclave hydrolysis is used to assure sample dissolution for spectrophotometric measurements. The assay accuracy was evaluated by determining epsilon-amino groups of insulin and bovine albumin. The determinations differed from literature values by < or = 3.3%. The epsilon-amino group content of Type B gelatin was found to be 33.0 mol/gelatin molecule of 1000 residues and is in agreement with similar source gelatins and collagen. The coefficient of variation for determinations on all three materials was < or = 5.3%. The assay should be applicable to a broad range of proteinaceous materials.     

Determination of the microscopic and macroscopic acid dissociation constants of glycyl-L-histidyl-L-lysine and related histidine peptides.

Proton magnetic resonance studies of the acid-base chemistry of the glycyl ammonium, histidyl imidazolium, and lysyl ammonium groups of glycyl-L-histidyl-L-lysine and of the glycyl ammonium and histidyl imidazolium groups of glycyl-L-histidine and glycyl-L-histidylglycine are described. Chemical-shift data indicate that, at the molecular level, the glycyl ammonium and the histidyl imidazolium groups are titrated over the same pH range, with the acidity of the imidazolium group some 8 to 10 times that of the glycyl ammonium group, depending on the peptide. The lysyl ammonium group of Gly-His-Lys is much less acidic and is titrated over a higher pH range. Microscopic and macroscopic acid-dissociation constants were determined from chemical-shift data for each of the peptides. It is shown how microscopic formation constants for protonated metal complexes of these ligands, which are being used increasingly as models for the binding of metal ions by proteins, can be calculated from the macroscopic formation constants and the microscopic acid-dissociation constants. The acid-base chemistry of Gly-His-Lys is discussed with respect to its recently discovered biological activity.     

Guanidination and nitroguanidination of staphylococcal enterotoxin B

Guanidination of the free amino groups of staphylococcal enterotoxin B with 3,5-dimethyl-1-guanylpyrazole converted 31-32 of 33 epsilon-amino groups and 30% of the N-terminal residue. This product, although markedly reduced in solubility, suffered no gross change in conformation and retained full biological activity. A derivative prepared by reaction with O-methylisourea with only one lysyl residue unaltered lost most of its emetic activity. Nitroguanidination with 3,5-dimethyl-1-nitroguanylpyrazole converted up to 28 of the epsilon-amino groups and essentially all of the N-terminus. This material was greatly reduced in ability to produce emesis and like the O-methylisourea prepared guanidinated enterotoxin, gave only a line of partial identity in double diffusion. The loss of activity is attributed to unfolding and it is concluded that the free amino groups of enterotoxin B do not critically participate in either its antigenic determinants or its active center for emesis.