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The steady-state kinetics of a full-length and truncated form of the type 2 human methionine aminopeptidase (hMetAP2) were analyzed by continuous monitoring of the amide bond cleavage of various peptide substrates and methionyl analogues of 7-amido-4-methylcoumarin (AMC) and p-nitroaniline (pNA), utilizing new fluorescence-based and absorbance-based assay substrates and a novel coupled-enzyme assay method. The most efficient substrates for hMetAP2 appeared to be peptides of three or more amino acids for which the values of k(cat)/K(m) were approximately 5 x 10(5) M(-1) min(-1). It was found that while the nature of the P1' residue of peptide substrates dictates the substrate specificity in the active site of hMetAP2, the P2' residue appears to play a key role in the kinetics of peptidolysis. The catalytic efficiency of dipeptide substrates was found to be at least 250-fold lower than those of the tripeptides. This substantially diminished catalytic efficiency of hMetAP2 observed with the alternative substrates MetAMC and MetpNA is almost entirely due to the reduction in the turnover rate (k(cat)), suggesting that cleavage of the amide bond is at least partially rate-limiting. The 107 N-terminal residues of hMetAP2 were not required for either the peptidolytic activity of the enzyme or its stability. Steady-state kinetic comparison and thermodynamic analyses of an N-terminally truncated form and full-length enzyme yielded essentially identical kinetic behavior and physical properties. Addition of exogenous Co(II) cation was found to significantly activate the full-length hMetAP2, while Zn(II) cation, on the other hand, was unable to activate hMetAP2 under any concentration that was tested.

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The three-dimensional structure of a novel four amino acid truncated form of the CXC chemokine GRObeta [5-73] isolated from bone marrow stromal cells with potent hematopoietic and anti-infective activities has been determined by two-dimensional (1)H nuclear magnetic resonance (NMR) spectroscopy in solution. On the basis of 1878 upper distance constraints derived from nuclear Overhauser effects (NOE) and 314 dihedral angle constraints, a group of 20 conformers representing the solution structure of the human GRObeta [5-73] was computed with the program DYANA. At the concentrations used for NMR study, GRObeta [5-73] forms a dimer in solution that is architectured by a six-stranded antiparallel beta-sheet (residues 25 to 29, 39 to 44, 49 to 52) and a pair of helices (residues 58 to 68) with 2-fold symmetry, while the C terminus of the protein is disordered. The average of the pairwise root-mean-square deviations of individual NMR conformers relative to the mean coordinates for the backbone atoms N, C(alpha) and C' of residues 5 to 68 is 0.47 A. Overall, the global fold of GRObeta [5-73] is similar to that of the previously reported NMR structure of GROalpha and the NMR and X-ray structures of interleukin-8. Among these three CXC chemokines, GRObeta [5-73] is most similar in structure to GROalpha. Significant differences between GRObeta [5-73], GROalpha and interleukin-8 are in the N-terminal loop comprising residues 12 to 19. The N-terminal arm containing the conserved ELR motif and the loop of residues 30 to 38 containing the GPH motif are different among these three CXC chemokines. The structural differences in these two regions may be responsible for the specificity of the receptor binding and biological activity of different chemokines.

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A surface plasmon resonance (SPR) biosensor was used to study the interaction of human interleukin-5 (hIL5) with its receptor. IL5 is a major growth factor in the production and activation of eosinophils. The receptor for IL5 is composed of two subunits, alpha and beta. The alpha subunit provides the specificity for IL5 and consists of an extracellular soluble domain, a single transmembrane region and a cytoplasmic tail. We expressed the soluble domain of the human IL5 receptor alpha subunit (shIL5R alpha) and human IL5 (hIL5) in Drosophila. Both hIL5 and shIL5R alpha were immobilized separately through amine groups onto the carboxylated dextran layer of sensor chips of the BIAcore (Pharmacia) SPR biosensor after N-hydroxysuccinimide/carbodiimide activation of the chip surface. Interactions were measured for the complementary macromolecule, either shIL5R alpha or hIL5, in solution. Kinetics of binding of soluble analyte to immobilized ligand were measured and from this the association rate constant, dissociation rate constant and equilibrium dissociation constant (Kd) were derived. With immobilized shIL5R alpha and soluble hIL5, the measured Kd was 2 nM. A similar value was obtained by titration calorimetry. The Kd for Drosophila expressed receptor and IL5 is higher than the values reported for proteins expressed in different systems, likely due to differences in the methods of interaction analysis used or differences in protein glycosylation. Receptor-IL5 binding was relatively pH independent between pH 6.5 and 9.5. Outside this range, the dissociation rate increased with comparatively little increase in association rate.(ABSTRACT TRUNCATED AT 250 WORDS)

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Human interleukin-1 alpha, cloned and expressed in E. coli, has been purified and structurally characterized by various physiochemical methods, including mass spectrometry. The recombinant protein has been crystallized by the hanging drop vapor diffusion method using dimethyl sulfoxide as the precipitating agent. The space group is P2(1)2(1)2(1). Unit cell dimensions are a = 44.1, b = 57.1, and c = 61.7 A and alpha = beta = gamma = 90 degrees. The crystals diffract to beyond 1.7 A and are suitable for high resolution data collection. Native diffraction data were collected. Screens for heavy atom derivatives have been initiated.

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A human interleukin-1 (IL-1) beta cDNA was cloned, and the region coding for the mature protein was expressed in Escherichia coli. The 17-kDa biologically active product was purified in 40% yield to apparent homogeneity, without chaotropes, from the soluble fraction of sonicated cell lysates. The recombinant IL-1 beta was characterized by amino acid analysis, NH2- and COOH-terminal sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, and biological assay. Specific biological activity was 4.6 X 10(8) units/mg in a co-mitogenic IL-2 induction assay using cultured EL-4 T-lymphocytes. The molar extinction coefficient was determined to be 10,300 cm-1 M-1 at 280 nm. NH2-terminal sequence analysis revealed that 70% of the product begins with the Ala corresponding to the NH2 terminus of the natural protein, while 30% begins with the following Pro. No initiator Met was observed. Both of the sulfhydryl groups are reactive to Ellman's reagent and to iodoacetamide under nonreducing conditions, indicating that the Cys residues do not form disulfide bonds. S-Carboxamidomethyl-Cys-rIL-1 beta retained biological activity in the IL-2 induction assay. Circular dichroism suggested an extensive beta sheet structure for rIL-1 beta.

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We have purified the beta subunit of the DNA polymerase III holoenzyme to homogeneity from an overproducing strain (Blanar, M., Sandler, S., Armengod, M., Ream, L., and Clark, A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4622-4626). From this procedure we can obtain 100 mg quantities of protein. The beta isolated from the overproducer is indistinguishable from that isolated from wild-type cells in terms of its activity and molecular weight. Partial amino acid sequence analysis has confirmed the DNA sequence of the dnaN gene (Ohmori, H., Kimura, M., Nagata, T., and Sakakibara, Y. (1984) Gene (Amst.) 28, 159-170) and established the sites for initiation and termination of translation. No processing that removes amino acid residues from beta occurs since the active protein begins with the initiating methionine and terminates at the position predicted from the DNA sequence. Our knowledge of the precise amino acid composition has been used to determine the extinction coefficient of beta to be 17,900 and 18,700 cm-1 M-1 at 280 and 277 nm, respectively. The extinction coefficient at 280 nm is reduced to 14,700 cm-1 M-1 under denaturing conditions in guanidine HCl. Conditions have been optimized so that 1 N-ethylmaleimide residue can be incorporated per beta monomer with full preservation of activity.

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Adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) will substitute for ATP in the formation of an initiation complex between the DNA polymerase III holoenzyme of Escherichia coli and primed DNA. The initiation complex formed in the presence of ATP gamma S between the DNA polymerase III holoenzyme and single-stranded DNA-binding protein-encoated primed M13 Gori DNA is stabile and isolable by gel filtration at room temperature. Upon addition of the four required deoxynucleoside triphosphates, this complex is rapidly converted to the duplex replicative form without dissociation of the polymerase. Initiation complexes formed in the presence of either ATP gamma S or ATP are indistinguishable by their resistance to antibody directed against the beta subunit of the holoenzyme and by their ability to elongate without further activation. A 2-fold difference was observed, however, in both the extent of initiation complex formation and in the dissociation of initiation complexes once formed. This difference is discussed in the light of previous proposals regarding a dimeric polymerase capable of replicating both strands at a replication fork concurrently.

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We have previously shown that the processive synthesis of long DNA products on a poly(dA) X oligo(dT)10 primer-template is facilitated by formation of an isolable initiation complex between the Escherichia coli DNA polymerase III holoenzyme and DNA in the presence of ATP (Fay, P. J., Johanson, K. O., McHenry, C. S., and Bambara, R. A. (1982) J. Biol. Chem. 257, 5692-5699). Here we have demonstrated that the ATP requirement for processive synthesis can be obviated by a large excess of the beta subunit of the DNA polymerase III holoenzyme. The reaction which occurs in the presence of excess beta can be distinguished from the ATP-mediated reaction by its salt sensitivity and the lack of stabile initiation complex formation between polymerase and primed DNA. A model is presented which suggest that one of the functions of ATP in the DNA polymerase III holoenzyme reaction is to lock beta into the replicative complex such that it does not readily equilibrate with solution.

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The initiation of the DNA polymerase III holoenzyme-catalyzed reaction is blocked by antibody directed against the beta subunit; elongation is unaffected (Johanson, K., and McHenry, C. (1980) J. Biol. Chem. 255, 10984-10990). We have developed an immunological method for quantitating nanogram quantities of beta in reaction complexes. Using this method, we have demonstrated that beta is present in all stages of the DNA polymerase III holoenzyme reaction. Upon initiation complex formation, the antigenic determinants of beta become inaccessible to anti-beta immunoglobulin G. The methods described herein should be generally applicable to the study of a variety of multienzyme complexes. Even after conversion of a primed G4 single strand to the duplex replicative form, beta does not readily dissociate. This creates a kinetic barrier to the overall holoenzyme replicative reaction.

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Two forms of Escherichia coli DNA polymerase III, DNA polymerase III', and DNA polymerase III have been shown to synthesize DNA products via a processive mechanism with product sizes distinctive for each enzyme form. These forms of DNA polymerase III are intermediate in complexity between the core DNA polymerase III and the DNA polymerase III holoenzyme. In a previous publication (Fay, P. J., Johanson K. O., McHenry, C. S., and Bambara, R. A. (1981) J. Biol. Chem. 256, 976-983), we demonstrated that on a randomly primed fd DNA template or on an oligo(dT)10 . poly(dA) template, the DNA polymerase III holoenzyme adds more than 100 nucleotides before dissociation, whereas the core enzyme adds 10 to 15 nucleotides. Now we show that DNA polymerase III' adds 30 to 40 nucleotides before dissociation. This number can be increased to approximately 60 if spermidine is present, but it is insensitive to the presence of E. coli single-stranded DNA-binding protein. DNA polymerase III adds about 50 nucleotides before dissociation, but this value can be increased to 200 nucleotides in the presence of the binding protein. Using measurement of product sizes made on an oligo(dT)10 . poly(dA) template, reconstitution of holoenzyme activity from DNA polymerase III and the beta subunit was monitored. Finally, it is shown that the products obtained from a purified initiation complex of holoenzyme and oligo(dT)10 . poly(dA) derive solely from the holoenzyme.

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The beta subunit of the DNA polymerase III holoenzyme has been purified 10,000-fold to homogeneity from Escherichia coli HMS-83. The native and denatured molecular weights of beta are 72,000 and 37,000, as determined by equilibrium sedimentation. Thus, beta appears to exist as a dimer when in a state free of other holoenzyme components. This conclusion is supported by the native molecular weight calculated from the sedimentation coefficient (5.0 S) and the Stokes radius (32.5 A) and subunit molecular weights determined from denaturing polyacrylamide gel electrophoresis. An antibody directed specifically against the beta subunit has been prepared and found to block the DNA polymerase III holoenzyme-catalyzed reaction, but not reactions which require only the core DNA polymerase III. Furthermore, this antibody blocks the initiation, but not the elongation reaction catalyzed by the DNA polymerase III holoenzyme. Thus, beta may only be required for formation of the initiation complex between polymerase components and a primed template. Immunoprecipitation studies demonstrate that the beta subunit forms a complex with other holoenzyme components in solution, in the absence of DNA.

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