Exposed Carboxyl Groups Research Articles

Calcium binding to the chloroplast andE. coli (CF0) F0 subunit III (c) of the ATP-synthase

Subunit III and c, the 8 kDa components of the chloroplast CF0, andE. coli H+ channel complexes respectively, were isolated and purified for the purpose of studying their Ca++-binding properties. Purified subunit III or c as well as the unfractionated organic-solvent soluble preparation from chloroplasts were used in a45Ca++-ligand blot assay known to detect high affinity Ca++-binding sites in proteins. Both subunit III and c showed strong45Ca++-binding. None of the other CF0 subunits bound Ca++ and of the CF1 only a weak binding was detected in the region of the α,β subunits. The Ca++-binding was inhibited after treating the proteins in solution by derivatizing aqueously exposed carboxyl groups with a water soluble carbodiimide plus a nucleophile, after de-formylation of the N-terminal methionine, or with a subsequent treatment with La3+. Dicyclohexylcarbodiimide treatment (no nucleophile was added) of thylakoid membranes, which derivatizes the hydrophobically located Glu 61 (Asp 61 inE. coli), did not inhibit the Ca++-binding in either protein. The data indicate that for both proteins the carbonyl group of the formylated N-terminal Met-1 and probably the carboxyl group of the subunit III (or c) C-terminal provide some of seven essential oxygen ligands normally required for defining a Ca++-binding site in proteins. Based on the accepted models for the hairpin conformation of the subunit III (c), it seems clear that the Ca++-binding site can form on the lumenal side of the membrane in the functional CF0 structures or on the periplasmic side of theE. coli membrane. A working hypothesis we are testing is that Ca++-binding to the CF0 (or F0) can form an easily reversible gating site such as to enhance the probability for membrane-localized H+ gradients being coupled to ATP formation under moderate energization loads, but under excess energization the local H+ ion concentration may build up high enough to displace the bound Ca++, resulting in delocalization of the H+ gradient. The latter situation seems, in chloroplasts at least, to function as a signal for over-energization; i.e., excess light absorption, a potential stress situation for plants. Lumenal acidification appears to be a trigger for initiating stress alleviation responses.

Subunit III of the chloroplast ATP-synthase can form a Ca 2+-binding site on the lumenal side of the thylakoid membrane

Subunit III, the 8 kDa component of the chloroplast CF 0 H + channel, was isolated and purified from pea thylakoids for the purpose of studying its Ca 2+-binding properties. After n-butanol extraction and ether precipitation, HPLC purification was accomplished using a poly(styrenedivinylbenzene) column which removes lipid and protein contaminations. The main components of protein contamination were two hydrophobic proteins of near 4 kDa molecular mass, psaI and psbK gene products associated with PSI and PSII reaction centers, respectively. Purified subunit III as well as the unfractionated organic-solvent soluble preparation were used in a 45Ca 2+-ligand blot assay known to detect high affinity Ca 2+-binding sites in proteins. Polypeptides were separated with SDS-PAGE and were transferred onto PVDF membranes. Treatment of the membrane with 45CaCl 2 in the presence of 10-fold excess of MgCl 2 and 200-fold excess KCl led to the labeling of only the 8 kDa polypeptide. The Ca 2+ binding was inhibited after derivatizing aqueously exposed carboxyl groups with a water soluble carbodiimide plus a nucleophile, after de-formylation of the N-terminal methionine, or with a subsequent treatment with La 3+. Ca 2+ binding was maximum at pH 7.5–8.5 and was greatly decreased at acidic pH. Dicyclohexylcarbodiimide treatment (no nucleophile was added) of thylakoid membranes, which derivativizes the hydrophobically located Glu-61, decreased the electrophoretical mobility of isolated subunit III but did not inhibit the Ca 2+ binding. The data indicate that the carbonyl group of the formylated N-terminal Met-1 and probably the carboxyl group of the subunit III C-terminal Val-81 provide some of seven essential oxygen ligands normally required for defining a Ca 2+-binding site in proteins. It is probable, but not yet established that an oligomeric form of subunit III polypeptides is essential for forming the Ca 2+-binding site. Based on the accepted models for the hairpin conformation of the subunit III, it does seem clear that the Ca 2+-binding site can form on the lumenal side of the membrane in the functional CF 0 structure.

Photoexcitation of rhodopsin: conformation changes in the chromophore, protein and associated lipids as determined by FTIR difference spectroscopy.

Abstract— The visual pigment rhodopsin is the major membrane protein in the rod photoreceptor membrane. Rhodopsin's function is to transduce the light induced isomerization (ll‐cis to all‐trans) of its internally located retinylidene chromophore into transient expression of signal sites at the surface of the protein. Fourier transform infrared (FTIR) difference spectroscopy has been used to study all of the steps in the photobleaching sequence of rhodopsin. Early protein alterations involving the peptide backbone and aspartic and/or glutamic carboxyl groups were detected which increase upon lumirhodopsin formation and spread to water exposed carboxyl groups by metarhodopsin II. The intensified and frequency shifted hydrogen‐out‐of‐plane vibrations of the chromophore that are present in bathorhodopsin are absent in lumirhodopsin. This indicates that by lumirhodopsin, the chromophore has relaxed relative to its more strained all‐frans form in bathorhodopsin. Finally, the transition to metarhodopsin II is found to involve perturbation of the acyl tail region of unsaturated phospholipid molecules possibly in response to small changes in the shape of the rhodopsin.

Some Properties of the Chloroplast Envelope as Revealed by Electrophoretic Mobility Studies of Intact Chloroplasts

The electrophoretic mobility of mature spinach (Spinacia oleracea L. var. Americana) chloroplasts sampled over a 7-month period was between -2.03 and -2.45 micrometers per second per volt per centimeter when suspended in a solution containing 1 millimolar CaCl(2). The surface charge density of EDTA-treated chloroplasts was calculated to be -7,400 electrostatic units per square centimeter representing, on the average, one electronic charge per 645 square Angstroms. Electrophoretic mobility increases during plastid maturation. Calcium, but not magnesium, generally stabilized the envelope of isolated plastids against small increases in surface charge that occur with time in the absence of calcium. Pronase caused a sharp, but temporary, decrease in the electrophoretic mobility of chloroplasts. This was interpreted as representing a transient binding of pronase to the envelope surface during proteolysis. No -SH groups were detected on the surface of the plastid envelope. Inasmuch as the isoelectric point of intact chloroplasts was found to be at pH 4.5, it is likely that the major part of the total surface charge results from the presence of exposed carboxyl groups of intrinsic envelope proteins that are not readily hydrolyzed by mild pronase treatment.

Elicitation of Casbene Synthetase Activity in Castor Bean

Endopolygalacturonase isolated from culture filtrates of the fungus Rhizopus stolonifer was shown previously to act as an elicitor of biosynthetic capacity for the antifungal agent, casbene, in castor bean (Ricinus communis L.) seedlings (S.-C. Lee, C.A. West 1981 Plant Physiology 67:633-639). Selective amidation of exposed carboxyl groups of the pure fungal endopolygalacturonase using intermediate activation with a water-soluble carbodiimide under mild conditions leads to inactivation of its enzymic activity. Tests of active and partially inactivated preparations of the enzyme reveal a close correlation between the levels of catalytic and elicitor activities. This suggests that the catalytic activity of the enzyme is necessary for its function as an elicitor. Treatment of the cell-free particulate fraction of homogenates of castor bean seedlings with the active fungal endopolygalacturonase results in the production of a heat-stable, water-soluble component which is highly active as an elicitor of casbene synthetase activity. Several additional lines of evidence, including the susceptibility of the heat-stable elicitor fraction to partial inactivation following prolonged treatment with endopolygalacturonase, indicate that the heat-stable elicitor is most likely a pectic fragment of the plant cell wall and that it is a required intermediate in the process of elicitation of casbene synthetase activity by the fungal endopolygalacturonase.

Specificity of alpha-chymotrypsin with exposed carboxyl groups blocked.

The 15 exposed carboxyl groups of alpha-chymotrypsin were modified with glycine ethyl ester at low pH using barbodiimide reagent. The specificity of the modified enzyme (Chy-15) was studied over the pH range of 4 to 9 with both N-acylated and non-N-acylated amino acid esters. The modified enzyme had lower reactivity toward N-acylated esters than non-N-acylated esters compared to the native enzyme. Typical substances such as acetyl- and benzoyl-L-tyrosine ethyl esters retained 4 and 9% activity, whereas phenylalanine ethyl ester was slightly more reactive with the modified than with the native enzyme. The pH-rate profiles of acetyl-L-phenylalanine ethyl ester and tryptophan ethyl and benzyl esters were investigated in detail. Analysis of these profiles revealed three pKa values of approximately 5, 7, and 9 related to a functional carboxyl, imidazoyl, and an amino group, respectively. Since similar pKa values occur for the native enzyme, modification did not block the carboxyl corresponding to pKa 5. A mechanism is proposed for catalysis which includes both the protonated and unprotonated form of the imidazoyl (His-57) and utilizes water rather than a carboxyl (Asp-102) as the proton sink.

Conversion of exposed aspartyl and glutamyl residues in proteins to asparaginyl and glutaminyl residues

1. 1. The feasibility of converting exposed carboxyl groups in proteins to carboxamido groups using 1-ethyl-3-dimethylaminopropylcarbodiimide (EDC) as a condensing agent was demonstrated. 2. 2. The effectiveness of ammonia as a nucleophile in the EDC-mediated condensation with carboxyl groups in proteins was determined. In 5.5 M NH 4Cl at pH 4.75, 11.5, 14.7, 9.0, 5.0 and 5.1 carboxyl groups are converted to carboxamido groups in chymotrypsinogen A, α-chymotrypsin (EC 3.4.4.5), lysozyme (EC 3.2.1.17), ribonuclease (EC 2.7.7.16) and trypsin (EC 3.4.4.4), respectively. 3. 3. The number of carboxamido groups found was determined from the amount of extra ammonia appearing in acid-hydrolyzed protein which was reacted with EDC and NH 4Cl. In one case, the number of β- and γ-carboxamido groups formed was also determined enzymically by digesting treated protein with proleolytic enzymes and then enzymically assaying the digest for free asparagine and glutamine. Both acid hydrolysis and enzymatic assays gave essentially the same value for the yield of carboxamido groups. 4. 4. 5.5 M NH 4Cl, 1 M glycine methylester, and 1 M glycinamide were all equally effective in modifying carboxyl groups. Since previous studies showed that the glycine derivatives quantitatively modify exposed carboxyl groups in proteins, it was concluded that NH 4Cl may be used to quantitatively convert exposed carboxyl groups in proteins to carboxamido groups. Now that it is possible to convert aspartyl and glutamyl residues in proteins to asparaginyl and glutaminyl residues, it should be possible to establish with greater certainty the role of carboxyl groups in the function and structure of proteins.