Polymer-supported biopolymer synthesis: 3. Preparation of fully and partially protected βh-endorphin segments with a new phenolic poly [N-(2-methoxyethyl)-acrylamide]-based support

A new phenolic peptide resin, which is a bead-form derivative of crosslinked poly[N-(2-methoxyethyl)acrylamide], has been prepared by a suspension polymerization technique. The resin, which is highly expanded in all common peptide solvents, has been used for the solid (gel) phase synthesis of protected acylpeptide hydrazides. Fully protected acylpeptide hydrazide segments corresponding to β_h-endorphin (15–17), β_h-endorphin (6–14) and β_h-endorphin (6–17) have been prepared. Conversion, by selective catalytic transfer hydrogenation, of the fully protected acylpeptide hydrazide segment corresponding to β-_h-endorphin (15–17) to the partially protected form has been explored.     

Polymer-supported biopolymer synthesis: 5. Ultra-high load solid (gel) phase peptide synthesis—the stepwise elaboration of quasi-homogeneous peptide gel networks?

An approach to ultra-high load solid (gel) phase peptide synthesis is described in which a bead-form phenolic core polymer, crosslinked poly[N-{2-(4-hydroxyphenyl)ethyl}-acrylamide], is used as a support matrix at near theoretical maximum loading. Consecutive repeating units of the core polymer carry peptide chains undergling stepwise elongation. Synthesis proceeds through a series of solvated networks, which consist mainly of protected peptide. The solvated networks are deemed to be quasi-homogeneous, insofar as each has a regular covalent framework and each is believed to be uniformly distributed throughout the gel beads. Illustrative synthesis of two fully-protected acylpeptide hydrazide segments, corresponding to dynorphin(6–12) and to β_h-endorphin (18–26), are described.     

Essential arginyl residues in fructose-1,6-bisphosphatase.

Modification of pig kidney fructose-1,6-bisphosphatase with 2,3-butanedione in borate buffer (pH 7.8) leads to the loss of the activation of the enzyme by monovalent cations, as well as to the loss of allosteric adenosine 5'-monophosphate (AMP) inhibition. In agreement with the results obtained for the butanedione modification of arginyl residues in other enzymes, the effects of modification can be reversed upon removal of excess butanedione and borate. Significant protection to the loss of K+ activation was afforded by the presence of the substrate fructose 1,6-bisphosphate, whereas AMP preferentially protected against the loss of AMP inhibition. The combination of both fructose 1,6-bisphosphate and AMP fully protected against the changes in enzyme properties on butanedione treatment. Under the latter conditions, one arginyl residue per mole of enzyme subunit was modified, whereas three arginyl residues were modified by butanedione under conditions leading to the loss of both potassium activation and AMP inhibition. Thus, the modification of two arginyl residues per subunit would appear to be responsible for the change in enzyme properties. The present results, as well as those of a previous report on the subject (Marcus, F. (1975), Biochemistry 14, 3916-3921) support the conclusion that one arginyl residue per subunit is essential for monovalent cation activation, and another arginyl residue is essential for AMP inhibition. A likely role of the latter residue could be its involvement in the binding of the phosphate group of AMP.     

Phosphoglycerate mutase has essential arginyl residues

Phosphoglycerate mutase is inactivated by butanedione in borate buffer. Inactivation by 0.13 mM reagent correlates with the modification of one arginyl residue per subunit, and is prevented by either 2, 3-diphosphoglycerate or 3-phosphoglycerate. With 0.50 mM butanedione, inactivation is accompanied by the modification of three arginyl residues per subunit, two of which are protected by the combined presence of cofactor and substrate.     

Identification of essential arginyl residues in cytoplasmic malate dehydrogenase with butanedione.

The inactivation of cytoplasmic malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) from porcine heart and the specific modification of arginyl residues have been found to occur when the enzyme is inhibited with the reagent butanedione in sodium borate buffer. The inactivation of the enzyme was found to follow pseudo-first order kinetics. This loss of enzymatic activity was concomitant with the modification of 4 arginyl residues per molecule of enzyme. All 4 residues could be made inaccessible to modification when a malate dehydrogenase-NADH-hydroxymalonate ternary complex was formed. Only 2 of the residues were protected by NADH alone and appear to be essential. Studies of the butanedione inactivation in sodium phosphate buffer and of reactivation of enzymatic activity, upon the removal of excess butanedione and borate, support the role of borate ion stabilization in the inactivation mechanism previously reported by Riordan (Riordan, J.F. (1970) Fed. Proc. 29, Abstr. 462; Riordan, J.F. (1973) Biochemistry 12, 3915-3923). Protection from inactivation was also provided by the competitive inhibitor AMP, while nicotinamide exhibited no effect. Such results suggest that the AMP moiety of the NADH molecule is of major importance in the ability of NADH to protect the enzyme. When fluorescence titrations were used to monitor the ability of cytoplasmic malate dehydrogenase to form a binary complex with NADH and to form a ternary complex with NADH and hydroxymalonate, only the formation of ternary complex seemed to be effected by arginine modification.     

Polymer-supported biopolymer synthesis: 1. High load solid (gel) phase strategy for peptide synthesis with a phenolic poly(acryloylmorpholine)-based support

An efficient, low-cost, reaction strategy for the solid (gel) phase synthesis of peptides and protected peptide segments has been developed. The strategy involves the use of a new poly(acryloylmorpholine)-based phenolic support matrix, Koch-Light Peptide Resin A. Illustrative syntheses of N-terminal Boc- and Z-protected[Leu]- enkephalin derivatives, including C-terminal acid hydrazides and esters, are described. The strategy, which is effective for the synthesis of peptides at high matrix loadings, is adopted readily for large-scale application.     

Essential arginyl residues in yeast phosphoglyceromutase.

Inactivation of yeast phosphoglyceromutase (tetramer) with 1,2-cyclohexanedione correlates with the modification of six arginyl residues per mole of the enzyme. Protection experiments using 3-phosphoglycerate suggest that four arginyl residues (one residue per subunit) are involved in the binding of the substrate to the enzyme. The modified enzyme reversibly regained its activity upon incubation with hydroxylamine. The reactivity of lysyl residues which have been shown to be involved in the active site is markedly reduced in the enzyme inactivated with 1,2-cyclohexanedione, indicating that the lysyl and arginyl residues are in close proximity in the active site.     

Yeast 3-phosphoglycerate kinase. Essential arginyl residues at the 3-phosphoglycerate binding site.

Yeast 3-phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phospho-transferase, EC 2.7.2.3) is inactivated by phenylglyoxal. Loss of activity correlates with the modification of two arginyl residues, both of which are protected by all of the substrates. The modification is not accompanied by any significant conformational change as determined by optical rotatory dispersion. Ultraviolet difference spectrophotometry indicates that the inactivated enzyme retains its capacity for binding the nucleotide substrates whereas the spectral perturbation characteristic of 3-phosphoglycerate binding is abolished in the modified enzyme. The data suggest that at least one of the two essential arginyl residues is located at or near the 3-phosphoglycerate binding site. A likely role of this residue could be its interaction with the negatively charged phosphate or carboxylate groups of 3-phosphoglycerate.     

Essential arginyl residues in thymidylate synthetase.

Thymidylate synthetase from amethopterin-resistant $\text{Lactobacillus}$$\text{casei}$ is rapidly and completely inactivated by 2,3-butanedione in borate buffer, a reagent that is highly selective for the modification of arginyl residues. The reversible inactivation follows pseudo-first order kinetics and is enhanced by borate buffer. dUMP and dTMP afford significant protection against inactivation while (±)-5,10-methylenetetrahydrofolate and 7,8-dihydrofolate provide little protection. Unlike native enzyme, butanedione-modified thymidylate synthetase is incapable of interacting with 5-fluoro-2′-deoxyuridylate and 5,10-(+)-methylenetetrahydrofolate to form stable ternary complex. The results suggest that arginyl residues participate in the functional binding of dUMP.     

The role of arginyl residues in porphyrin binding to ferrochelatase

The role of cationic amino acid residues in the binding of porphyrin substrates by purified bovine ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) have been examined via chemical modification with camphorquinone-10-sulfonic acid, phenylglyoxal, butanedione, and trinitrobenzene sulfonate. The data obtained show that modification of arginyl, but not lysyl, residues results in the rapid inactivation of ferrochelatase. The 2,4-disulfonate deuteroporphyrin, which is a competitive inhibitor of mammalian ferrochelatase, protects the enzyme against inactivation. Ferrous iron has no protective effect. Reaction with radiolabeled phenylglyoxal shows that modification of 1 arginyl residue causes maximum inhibition of enzyme activity. The inactivation does not follow simple pseudo-first order reaction kinetics, but is distinctly biphasic in nature. Comparison of the enzyme kinetics for modified versus unmodified enzyme show that modification with camphorquinone-10-sulfonic acid has no effect on the Km for iron but does alter the Km for porphyrin.     

Implication of arginyl residues in mRNA binding to ribosomes

Modification of Escherichia coli robosomes with phenylglyoxal and butanedione, protein reagents specific for arginyl residues, inactivates polypeptide polymerization, assayed as poly(U)-dependent polyphenylalanine synthesis, and the binding of poly(U). Inactivation is produced by modification of the 30-S subunit. Both the RNA and the protein moieties of 30-S subunits are modified by phenylglyoxal, and modification of either of them is accompanied by inactivation of polypeptide synthesis. Modification of only the split proteins released from 30-S subunits by prolonged dialysis against a low-ionic-strength buffer, which contain mainly protein S1, produces inhibition of poly(U) binding and inactivation of polypeptide synthesis. Amino acid analysis of the modified split proteins showed a significant modifications of arginyl residues. These results indicate that the arginyl residues of a few 30-S proteins might be important in the interaction between mRNA and the 30-S subunit, which agrees with the general role assigned to the arginyl residues of proteins as the positively charged recognition site for anionic ligands.     

Role of arginyl residues in yeast hexokinase PII.

Yeast hexokinase PII is rapidly inactivated (assayed at pH 8.0) by either butanedione in borate buffer or phenylglyoxal, reagents which are highly selective for the modification of arginyl residues. MgATP alone offers no protection against inactivation, consistent with low affinity of hexokinase for this nucleotide in the absence of sugar. Glucose provides slight protection against inactivation, while the combined presence of glucose and MgATP gives significant protection, suggesting that modified arginyl residues may lie at the active site, possibly serving to bind the anionic polyphosphate of the nucleotide in the ternary enzyme:sugar:nucleotide complex. Extrapolation to complete inactivation suggests that inactivation by butanedione correlates with the modification of 4.2 arginyl residues per subunit, and complete protection against inactivation by the combined presence of glucose and MgATP correlates with the protection of 2 to 3 arginyl residues per subunit. When the modified enzyme is assayed at pH 6.5, significant activity remains. However, modification by butanedione in borate buffer abolishes the burst-type slow transient process, observed when the enzyme is assayed at pH 6.5, to such an extent that after extensive modification the kinetic assays are characterized by a lag-type slow transient process. But even after extensive modification, hexokinase PII still demonstrates negative cooperativity with MgATP and is still strongly activated by citrate when assayed at pH 6.5.     

Essential arginyl residues in yeast enolase.

Yeast enolase is rapidly inactivated by butanedione in borate buffer, complete inactivation correlating with the modification of 1. 8 arginyl residues per subunit. Protection against inactivation is provided by either an equilibrium mixture of substrates or inorganic phosphate, a competitive inhibitor of the enzyme. Complete protection by substrates correlates with the shielding of 1. 3 arginyl residues per subunit, while phosphate protects 1. 0 arginyl residue per subunit from modification.     

Efrapeptin prevents modification by phenylglyoxal of an essential arginyl residue in mitochondrial adenosine triphosphatase.

Studies of phenylglyoxal incorporation by beef-heart mitochondrial ATPase reveal one fast-reacting arginyl residue/enzyme molecule. Modification of this group proceeds at a rate which parallels the loss of enzymatic activity. Efrapeptin protects the arginyl residue from reaction with phenylglyoxal. The data suggest that efrapeptin binds at the catalytic site and blocks accessibility of an essential arginine at the adenine nucleotide binding site. The detection of a single, fast-reacting, essential arginine on an enzyme with multiple copies of the catalytic subunit, provides further evidence in support of the alternating site mechanism for ATP synthesis proposed by Kayalar et al. (Kayalar, C., Rosing, J., and Boyer, P.D. (1977) J.Biol. Chem. 252, 2486--2491).     

Solid (gel) phase synthesis of fully-protected acylpeptide hydrazides with hydrazine-labile sidechains

A simple, efficient strategy for the preparation of fully-protected acylpeptide hydrazides with hydrazine-labile sidechains is described. The method involves controlled hydrazinolytic cleavage of target peptides from assemblies elaborated by solid (gel) phase synthesis on phenolic peptide resins.     

An arginyl in the N-terminus of the V1a vasopressin receptor is part of the conformational switch controlling activation by agonist.

Defining how the agonist-receptor interaction differs from that of the antagonist-receptor and understanding the mechanisms of receptor activation are fundamental issues in cell signalling. The V1a vasopressin receptor (V1aR) is a member of a family of related G-protein coupled receptors that are activated by neurohypophysial peptide hormones, including vasopressin (AVP). It has recently been reported that an arginyl in the distal N-terminus of the V1aR is critical for binding agonists but not antagonists. To determine specific features required at this locus to support high affinity agonist binding and second messenger generation, Arg46 was substituted by all other 19 encoded amino acids. Our data establish that there is an absolute requirement for arginyl, as none of the [R46X]V1aR mutant constructs supported high affinity agonist binding and all 19 had defective signalling. In contrast, all of the mutant receptors possessed wildtype binding for both peptide and nonpeptide antagonists. The ratio of Ki to EC50, an indicator of efficacy, was increased for all substitutions. Consequently, although [R46X]V1aR constructs have a lower affinity for agonist, once AVP has bound all 19 are more likely than the wildtype V1aR to become activated. Therefore, in the wildtype V1aR, Arg46 constrains the inactive conformation of the receptor. On binding AVP this constraint is alleviated, promoting the transition to active V1aR. Our findings explain why arginyl is conserved at this locus throughout the evolutionary lineage of the neurohypophysial peptide hormone receptor family of G-protein coupled receptors.     

Affinity labeling of arginyl residues at the catalytic region of estradiol 17 beta-dehydrogenase from human placenta by 16-oxoestrone

16-Oxoestrone inhibited competitively the activity of estradiol 17 beta-dehydrogenase from human placenta against estradiol in phosphate buffer (pH 7.2), suggesting reversible binding of 16-oxoestrone to the substrate-binding site. 16-Oxoestrone irreversible inactivated the estradiol 17 beta-dehydrogenase in borate buffer (pH 8.5) in a time-dependent manner, following pseudo-first-order kinetics. The rate constant (k3) obtained for the inactivation by 16-oxoestrone was 8.3 x 10(-4) s-1. The rate of inactivation was significantly decreased by addition of estrone, estradiol, estriol, NAD(H) and NADP+. Also, the rate was reduced markedly by 2'AMP, 5'ATP and 2',5' ADP, but not by NMN(H) and 3-pyridinealdehyde adeninediphospho nucleotide. The inactivation by 16-oxoestrone was neither prevented by sodium azide nor influenced by light. From these data, 16-oxoestrone, an alpha-dicarbonyl steroid, was suggested to inactive estradiol 17 beta-dehydrogenase by modification of arginyl residues located around the substrate-binding site of the enzyme. Biphasic inactivation of the enzyme by 16-oxoestrone was observed with an increase of modified arginyl residues. The first phase of the inactivation was regarded as an affinity labeling of the arginyl residues at or near the substrate-binding site of the enzyme. Stoichiometry of the inactivation indicated that two arginyl residues were essential for maintenance of the enzyme activity. The second phase was considered as chemical modification of the arginyl residues outside of the catalytic region of the enzyme.     

The role of arginyl residues in estrogen receptor activation and transformation

Receptor-estradiol complexes (RE2) formed at 0 degree C in hypotonic buffers bind poorly to nuclei (nonactivated state); their sedimentation coefficient in low or high salt sucrose density gradients (SDG) is 8 S or 4 S, respectively (untransformed state); estradiol dissociates from untransformed RE2 at a high rate (k-1 = 0.44 min-1). Brief heating (28 degrees C, 30 min) induces activation (increased binding of RE2 to nuclei and polyanions), transformation (formation of receptor dimers which sediment at 6 S in 0.4 M KCl/borate SDG) and RE2 transition into a state from which E2 dissociates at a lower rate (k-2 = 8 X 10(-3) min-1). We have examined the role of arginyl residues in the above changes in receptor properties. It is well established (Patthy, L., and Smith, E. L. (1975) J. Biol. Chem. 250, 557-564; 565-569) that 1,2-cyclohexanedione (1,2-CHD) is a highly specific arginine-modifying agent; in borate buffer at 28 degrees C, but not at 0 degrees C, peptide arginyls are covalently modified. RE2 complexes heated in the presence of 1,2-CHD (50 mM) bind poorly to nuclei; 1,4-cyclohexamedione and 1,2-cyclohexanediol had no effect. This reagent also prevents the temperature-induced transition of RE2 into a state with slow E2 dissociation rates although it does not interfere with heat transformation (formation of 6 S dimer). Modification of heat-activated and transformed RE2 by 1,2-CHD causes a loss in receptor binding to nuclei and alters RE2 from a state with slow into a state with fast E2 dissociation rates, although the receptor remains unaltered in the transformed 6 S state. At 0 degree C, i.e. in the absence of covalent arginyl modification, 1,2-CHD promotes dissociation of the 8 S aggregate into 4.6 S subunits which bind to nuclei to the same extent as heat-transformed control RE2. Heating of the molybdate-stabilized 8 S receptor in the presence of 1,2-CHD yields a nonactivated 8 S receptor (4.6 S on high salt SDG); removal of molybdate and unreacted 1,2-CHD by gel filtration at 0 degree C followed by exposure to high ionic strength causes 8 S to 4 S dissociation; these 4 S subunits, however, do not bind to nuclei, suggesting that their nucleotropic domain was accessible to 1,2-CHD modification while the receptor was in the aggregated 8 S state. It is proposed that the nuclear binding site of the estrogen receptor contains arginyl residues. Furthermore, a distinct set of arginyl residues appears to be related to the estrogen-binding domain; its integrity is required for the heat-induced formation and maintenance of the RE2 state with slow E2 dissociation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sea urchin egg-cortical granule protease has arginyl proteolytic specificity

Sea urchin eggs secrete esteroproteolytic activity at fertilization. This enzyme has been shown to be proteolytic toward embryo protein and casein, but a systematic study of its substrate specificity has not been done. In this communication we present data that demonstrates for the first time that the cortical granule protease from Strongylocentrotus purpuratus eggs cleaves arginyl residues in a protein substrate, lysozyme. We have developed a sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) assay that detects femtomole levels of trypsin and chymotrypsin protease activity [Green, 1986: Anal Biochem 152:83–88]. In the sea urchin system, we have detected protease activity from as few as 50 eggs. Correlating the RP-HPLC analysis with a spectrophotometric Nα-benzoyl-L-arginine ethyl ester assay, we have found that each egg secretes approximately 40 attomoles of trypsin-like activity. This general method should be quite useful in investigations into the natural substrate of the egg protease.     

Functional consequences of modifying highly reactive arginyl residues of fructose 1,6-bisphosphatase. Loss of monovalent cation activation.

Modification of pig kidney fructose 1,6-bisphosphatase with 2,3-butanedione (in the presence of AMP) results in the loss of activation of the enzyme by monovalent cations. Under these conditions about 8 arginyl residues per mole of enzyme were modified. No other residues were modified. No loss of monovalent cation activation occurs when modification with 2,3-butanedione is carried out in the presence of AMP plus the substrate fructose 1,6-bisphosphate and 3.2 less arginyl residues were modified. Since fructose 1,6-bisphosphatase contains 4 subunits, it is suggested that one arginyl residue per subunit plays an essential role in monovalent cation activation of the enzyme. Studies on sulfhydryl group reactivity toward 5,5'-dithiobis(2-nitrobenzoic acid) explain the protection exerted by fructose 1,6-bisphosphate against the loss of monovalent cation activation in terms of an enzyme conformational change induced by substrate, which makes unreactive the essential arginyl residue. The results of the present paper, as well as previous evidence, are discussed in terms of the mechanism of monovalent cation activation of fructose 1,6-biphosphatase.