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Dinitrophenylation of a single cysteine side chain in phosphorylase b from rabbit muscle with concomitant blocking of AMP binding
Chapter 1.
M. Z. Atassi. Chemical modification and cleavage of proteins and chemical strategy in immunochemical studies of proteins. pages 1-161.
Chapter 2.
A. F. S. A. Habeeb. Influence of conformation on immunochemical properties of proteins. Pages 163-229.
Chapter 3.
Walter B. Dandliker. Investigation of immunochemical reactions by fluorescence polarization. Pages 231-261.
Chapter 4.
Abram B. Stavitsky. In Vitro immune responses of lymphoid cell populations to proteins and peptides. Pages 263-314.
Chapter 5.
Hakan Bergstrand. Immunochemistry of Encephalitogenic protein. Pages 315-370.
Chapter 6.
Dov Michaeli. Immunochemistry of Collagen. Pages 371-399.
Chapter 7.
Raymond N. Hiramoto and Vithal K. Ghanta. Histocompatibility antigens. Pages 401-439.
Author Index. Pages 441-468
Subject Index. Pages 469-485
Crystallographic binding studies of various metabolites to phosphorylase b in the presence of 2 mm-IMP have been carried out at low resolution (8.7 Å) with three-dimensional data and at high resolution (3 å) with two-dimensional data. From correlation of peaks observed in difference Fourier syntheses based on these two sets of data, the following binding sites have been identified: (1) the “active” site to which the substrate, glucose 1-phosphate, and the substrate analogues, maltotriose and arsenate, bind and which is close to the subunit-subunit interface of the phosphorylase dimer; (2) the allosteric adenine-nucleotide binding site to which the allosteric activator AMP and the allosteric inhibitor ATP bind and which is very close to the active site; (3) the inhibitor binding site for glucose 6-phosphate, which is also close to the active site. Glucose 6-phosphate causes extensive conformational changes in the protein, which are the largest observed for all the metabolites studied so far; (4) a glycogen binding site on the surface of the molecule to which maltotriose binds. The distance over the surface of the phosphorylase molecule from this site to the active site is 50 to 60 Å; (5) a second glucose 1-phosphate binding site situated in the interior of the molecule. The significance of this site is not yet understood but its position in the centre of the molecule suggests that it may have a key role in the control and catalysis of phosphorylase.
Reversed-phase systems have been developed that permit the detection and quantification of down to 0.1 nmole of non-UV-absorbing cations and anions using a UV detector. The samples give positive or negative peaks depending on their charge and retention relative to the UV-absorbing ionic component in the mobile phase. The relative detector response has a maximum, which can be considerably more than 100%, when the sample and the UV-absorbing mobile phase ion have about the same retention. Detection and separation studies on, e.g., sulphonates, sulphates, carboxylates, amino acids, dipeptides and alkylammonium compounds of different degrees of substitution are described.
This chapter discusses the molecular details of the regulation of the system of enzymes involved in the breakdown of glycogen to produce glucose 1-phosphate, one of the entry points into glycolysis in rabbit skeletal muscle. As phosphorylase is an enzyme of large molecular weight (2.0 × 105 for the more common active dimeric form), structural studies present considerable difficulties. This has been complicated by the fact that the most common crystal form of the enzyme contained the tetramer, which is the predominant form for phosphorylase a and the b form in the presence of AMP at high concentrations of enzyme. Proteolysis by trypsin of phosphorylase a results in a cleavage of the amino end of the molecule—the resulting hexapeptide contains the serine residue that is phosphorylated in the kinase-catalyzed phosphorylation of the enzyme and is rich in basic amino acids. Phosphorylase Kinase enzyme is responsible for phosphorylation of phosphorylase b.
1Phosphorylase b may be specifically labelled on one fast reacting sulphydryl group per subunit with 4-iodoacetamido-salicylic acid without loss of enzymic activity.2The fluorescence intensity of the covalently linked acetamido-salicylate is altered on the binding of substrate and effector ligands.3These fluorescence change were used to derive the “apparent” binding constants of the different ligands and to observe the interaction between them.4No change is observed in the fluorescence of the acetamido-salicylate label when phosphorylase b is converted into phosphorylase a. When the conversion is carried out in the presence of either AMP or glucose 6-phosphate there are fluoresence changes which reflect the differential ligand binding to the two forms of phosphorylase. The fluorescence method in the presence of ligands therefore provides a continuous assay for the b to a conversion.5Studies on the rate of interconversion revealed that the ratio of activities of non-phosphorylated phosphorylase b kinase at pH 6.8 and 8.4 is over 100 compared to a ratio of 3 for phosphorylated kinase.6Experiments carried out in the presence of glucose 6-phosphate directly demonstrated the presence of an intermediate active form of phosphorylase which binds glucose 6-phosphate tightly unlike fully phosphorylated phosphorylase a.7The association of phosphorylase a dimers to tetramers was detected in a mixture of acetamido-salicylate-phosphorylase and 4-nitrobenzo-2-oxa-1, 3-diazole phosphorylase. The quenching of the fluorescence of the former by the latter species in the mixed tetramer enabled us to confirm that tetramer formation was slower than the appearance of phosphorylase a activity.
Nuclear envelope membranes from rat liver cells contain ATPases, one of which can be inhibited and irreversibly labeled by (S-dinitrophenyl)-6-mercaptopurine riboside triphosphate. Inhibition and covalent substitution of the ATPase are achieved only after disruption of the nuclei, the ATP analogue is inactive on the ATPase activity of whole nuclei or on vesicles of the membrane prepared after a modified heparin method of Bornens and Courvalin. Electron micrographs and scanning micrographs helped to establish the characterization of closed vesicles and intact nuclei. With the aid of (alpha-32P)-labeled, and of the (beta, gamma-32P)-labeled analogue, it was possible to demonstrate the incorporation of the nucleotide into a few protein regions of the nuclear membrane disc electrophoresis pattern.
Phosphorylase b (1,4-alpha-D-glucan:1,6-alpha-D-glucan 6-alpha-glucosyltransferase, EC 2.4.1.1) can be specifically spin-labelled at a site essential for the catalytic action of the enzyme. A paramagnetic analogue of 1-fluoro-2,4-dinitrobenzene was synthesized and used as a dinitrophenylating agent. Reaction of phosphorylase b with the paramagnetic probe combined with the thiolysis method, leads to spin-labelling of a single -NH2 group (0.75 groups per subunit) with concomitant loss of 50% of the catalytic activity. Dinitrophenylation does not change the sedimentation profile of the enzyme. The ESR spectrum of modified phosphorylase b indicates that the attached label has rather limited segmental mobility and its environment is slightly hydrophobic. Small but subtle conformational changes induced by ligands in this critical site of the macromolecule can be directly detected by the spin-label. Also, sulfhydryl group modification of the spin-labelled enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) has a pronounced effect on the resonance spectrum.
This chapter describes the procedure and applicability of the sequential degradation plus dansylation method. This procedure is used extensively in several investigations of the amino acid sequences of proteins, notably that of α-chymotrypsin. All normal protein amino acids are encountered, and with few exceptions provided no difficulties. The yields of dansyl end groups decline slowly during successive stages of the degradation, and it is necessary to progressively increase the size of sample used for reaction. The method employs direct identification of the end groups with the very sensitive dansyl chloride technique. Approximately 1-5 millimicromoles are required for each step of the degradation, so that 20 millimicromoles is usually sufficient to establish the complete sequence of a penta- or hexapeptide. The limiting factor is the capacity of the electrophoresis equipment used in identifying end groups.
ENZYME regulation is apparently associated with ligand-induced conformational changes which have been detected in several instances1, but the nature and extent of these conformational changes or even the relationship between the regulatory and catalytic sites are not known. The purpose of this communication is to demonstrate that by double-labelling techniques detailed stereochemical information can be obtained about enzymes as complex as Phosphorylase b which could ultimately lead to a detailed understanding of allosteric regulation.
The synthesis of 6-(purine 5′-ribonucleotide)-5-(2-nitrobenzoic acid) thioether provided a reagent that will form stable thioether bonds between the 6 position of the purine moiety and aliphatic sulfhydryls. 2-Nitro-4-mercaptobenzoic acid is eliminated during this reaction. The nucleotide reagent, labeled with 32P, was used to activate phosphorylase b from rabbit muscle. The activation showed a stoichiometric relation to the amount of nucleotide incorporated into the enzyme. The nucleotide was covalently linked to the protein. We propose that the nucleotide became bound at or near the 5′-AMP binding site of the enzyme.
The isolation and sequence of active site peptides from glycogen phosphorylase is described. These cysteine peptides become exposed to preferential labeling upon removal of pyridoxal 5′-phosphate (PLP) from the enzyme and they have the sequence Ala-Cys, Asx-Ala-Cys-Asp and Asx-Glx-Lys-Cys-Gly-Gly. The first two of these peptides appear to be derived from the PLP binding site of phosphorylase.
Dinitrophenylation of phosphorylase b with an eightfold molar excess of 2,4-dinitrofluorobenzene (DNFB) results in inactivation of the enzyme, and subsequent analyses of the protein showed modification of four to five groups. ε-Amino groups of lysine and SH groups of cysteine were modified. In the presence of either α-D-glucose 1-phosphate (glucose-1-P) or adenosine 5′-monophosphate (AMP), inactivation is retarded and in the presence of both, 75% of the activity could be retained with modification of 3-3.5 groups. When glucose-1-P or AMP was present during dinitrophenylation, their respective binding sites were preserved as indicated by kinetic studies DNP-phosphorylase b, prepared in the presence of glucose-1-P or AMP or both, could be converted into crystalline DNP-phosphorylase a derivatives. DNP-phosphorylase a prepared from phosphorylase b dinitrophenylated in the presence of glucose-1-P and AMP was found to be electrophoretically homogeneous and sedimented essentially as a single component in the ultracentrifuge. No change in KM for glucose-1-P and AMP was observed with this derivative in comparison with the unmodified enzyme but a clear difference existed in the KM for glycogen.
A method for the removal of pyridoxal 5′-phosphate (PLP) from rabbit muscle phosphorylase b is described. The procedure involves two simultaneous operations, namely, distortion of the protein by an appropriate deforming agent which exposes the covalently bound PLP, and removal of the cofactor by interaction with a PLP reagent. A number of deforming buffers were tested, of which imidazolium citrate proved to be the most effective. Using this buffer and L-cysteine as the carbonyl reagent, resolution of phosphorylase occurred with a half-life of ca. 5 min at pH 6.2, 0°, <1 min at pH 7.0 and 37°; the energy of activation at pH 7.0 was 11.7 kcal/mole. Resolution was specific with respect to the PLP reagent. Of a number of cysteine analogs tested, L-cysteine was found to be the most effective ; penicillamine was barely active and cysteamine totally inactive. Under these same condi-tions neither imidazolium citrate nor L-cysteine alone brought about resolution. The deforming buffer probably facilitates removal of PLP by causing gross conformational changes in the enzyme: even in the absence of L-cysteine, it caused dissociation of phosphorylase b into monomers (S20,w = 5.5 S) and a rapid exchange of the protein-bound PLP with free [32P]PLP. Resolution of phosphorylase b was blocked by addition of adenosine 5′-phosphate or by phosphorylation of the protein as it occurs during the conversion of phosphorylase b to a. Apophosphorylase b prepared by this procedure has a residual activity of <1% and a correspondingly low PLP content. There is no indication that the protein has undergone any irreversible denaturation during resolution since the apoenzyme can be fully reactivated by incubation with PLP.
FDNB∗ has been widely used in structural and functional studies of peptides and proteins (Sanger, 1945; Hirs , 1961; Sokolovsky , 1964; Di Prisco, 1967). Being a reactive aryl halide, this reagent may react with several of the functional groups of proteins such as α- and ϵ- amino groups, imidazoles, sulfhydryls and aliphatic or phenolic hydroxyls. This paper describes a method for quantitative removal of dinitrophenyl groups from histidine, tyrosine and cysteine side chains. The reaction is referred to as “thiolysis” since cleavage is brought about by thiols (e. g. 2-mercaptoethanol). In view of the mild conditions under which the reaction proceeds (aqueous medium, pH = 8.0 and 22°), it may find a variety of applications in peptide and protein chemistry.
A method for the purification of the enzyme catalyzing the conversion of phosphorylase b to a is described. After a 65-fold increase in specific activity, the enzyme obtained is free of PR enzyme activity.The course of the reaction at several concentrations of converting enzyme is illustrated, and converting enzyme units are defined. The optimum pH for the enzyme is approximately 9.0; the reaction requires Mn++ or Mg++ ions and ATP. It is shown that a mono-manganous-ATP complex is probably acting in the reaction.Conversion of phosphorylase b to a is carried out in the presence of 32P-ATP, and an incorporation of at least 2 moles of 32P per mole of phosphorylase a is found to occur.
Dialyzed and Norit-treated muscle phosphorylase a, after recrystallization from versene-glycerophosphate, contains 8 organic phosphate groups per mole or 2 phosphate groups per subunit of molecular weight of 125,000. Four of these phosphate groups are extracted by precipitation of the enzyme with trichloroacetic or perchloric acid. The extracted phosphate compound was isolated as the barium salt and identified as pyridoxal-5-phosphate by its spectrum and by specific enzymic tests. Column chromatography of the trichloroacetic acid extract and paper electrophoresis did not reveal the presence of other phosphorylated compounds. In particular, pyridoxamine-5-phosphate, adenylic acid and other nucleotides could not be detected. Free pyridoxal, pyridoxamine or pyridoxine were also absent.
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