The T cell recognition of globular protein antigens requires the processing and presentation of the antigen by Ia-expressing APCs. Processing is believed to involve the uptake of antigen into an acidic compartment where proteolysis occurs. The resulting peptides containing the T cell antigenic determinant are associated with Ia and presented at the cell surface to the specific T cells. The mechanisms by which antigenic peptides become associated with Ia is not known. We previously described a peptide binding protein of 72/74 x 10(3) Mr (PBP72/74) that plays a role in antigen presentation as shown by the ability of an antiserum raised in rabbits to affinity-purified PBP72/74 to block presentation of cytochrome c to a cytochrome c-specific T cell hybrid. Here we show that PBP72/74 is recognized by mAbs specific for members of the HSP70 family of proteins. In Western blots PBP72/74 is bound by mAb 7.10, specific for an evolutionarily conserved epitope of HSP proteins and by mAb N27, specific for both the constitutively expressed and inducible 72/73 x 10(3) Mr HSP70 proteins. In addition, PBP72/74 shares a second common feature of the HSP proteins, that of binding to ATP. Indeed, ATP causes the release of PBP72/74 from binding to a peptide fragment of cytochrome c (Pc 81-104) and PBP72/74 can be eluted from ATP columns by Pc 81-104. Finally, a portion of PBP72/74 is shown to be present on B cell surfaces by immunofluorescence staining. Thus, it appears that characteristics of the heat shock proteins are shared by a protein playing a role in antigen presentation, suggesting some commonality in function.
A single catalytic site model is proposed to account for the multiphasic kinetics of oxidation of ferrocytochrome c by cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1). This model involves nonproductive binding of substrate .to sites near the catalytic site on cytochrome c oxidase for cytochrome c, decreasing the binding constant for cytochrome c at the catalytic site. This substrate inhibition results in an increase.in the first-order rate constant for the dissociation of the ferricytochrome c-cytochrome c oxidase complex, the rate-limiting step in the steady-state turnover of electrons between cytochrome c and cytochrome c oxidase in the spectrophotometric assay, yielding increases in the initial rate as well as the Michaelis constant-namely, multiple kinetic phases. (2) and Borei (3) proposed that the reaction involved the formation of an enzyme-substrate complex. In 1949, Slater (4) confirmed and extended the observations of these authors, demonstrating that at any single concentration of enzyme [provided in the form of a KeilinHartree heart muscle particle preparation (5)], the kinetics indeed fit the Michaelis-Menten relation. However, the oxidation of ferrocytochrome c monitored spectrophotometrically (namely, in the absence of any added reducing agent) was generally observed to follow a first-order time course (6-13). Because the observed first-order rate constants decrease with increasing cytochrome c concentration rather than remaining unchanged, as one would expect for a simple bimolecular collisional mechanism with no precursor-pair formation, Smith and Conrad (13) suggested that the hyperbolic dependence of the initial velocity on cytochrome c concentration was due to inhibition of the reaction by cytochrome c itself.That this was not the only possible explanation of these kinetics followed from Minnaert's (14) elegant analysis, in which it was shown that a first-order time course could arise from equal binding of ferro-and ferricytochromes c to cytochrome oxidase, with ferrocytochrome c forming a productive complex while ferricytochrome c acted as a competitive inhibitor. This hypothesis was supported by Yonetani and Ray (15), who demonstrated that (i) under conditions in which first-order kinetics are observed, the K1 for ferricytochrome c is the same as the apparent Michaelis constant for ferrocytochrome c and (ii) at more alkaline pH values, where the kinetics deviate from a first-order time course, the K1 for ferricytochrome c is no longer equal to the apparent Km for the reaction.Indeed, inspection of the Michaelis-Menten equation, which takes into account binding of product to the enzyme, leads to the expression: dp _ Vmax 15= kobS, dt LKm + (s + P)when Km is a good approximation of Ks, the equilibrium substrate binding constant, and the binding of substrate and product are equivalent, namely K, = Kp (16). Thus, kobs is a first-order rate constant that is distinguishable from an ordinary first-order rate constant because it is a function of s + p, the tot...
oligosaccharide portions of these molecules in Me2S0 and Me2NCH0. In these studies, the amide side chain (in GalNAcj3( 1-.3)Gal) was found to exist in two conformations basically involving torsions about the trans arrangement (T = 160° J. Am. Chem. SOC. 1991, 113, 6822-6831 and 60'). In this report we have shown by comparing experimental and simulated NOES from the NH proton that for the oligosaccharides LND-I and LNF-I only one of these two conformations agrees with experiment.Abstract: Conformational dynamics within the complex between Zn-substituted cytochrome c peroxidase (ZnCcP) and cytochrome c (Cc) has been studied by examining the quenching of the 'ZnP excited state by the ferriheme of Cc. The temperature and solvent dependence of the triplet-state quenching rate constants (k,) show that complexes of ZnCcP with a large set of Fe3+ cytochromes c undergo a transition between a low-temperature state that does not exhibit triplet quenching and a high-temperature state that does. Within the narrow transition range (220 K C T C 250 K), the decay traces for the [ZnCcP, Fe3+Cc] complexes are nonexponential, and outside of this range they are exponential. This behavior is displayed by complexes with Cc(Drosophi1a melanogaster), Cc(Candida krusei) and a suite of site-directed mutants of Cc(yeast iso-I) where position 82 contains either an aliphatic (Met, Ser, Leu, or Ile) or an aromatic (Phe) residue. Above 250 K, k, varies strongly among these complexes and decreases sharply with the concentration of cosolvent (EG = ethylene glycol), apparently because of increasing viscosity, while both the breadth of the transition range and its midpoint vary little within this class. MCD and optical spectra between ambient and 4 K rule out the trivial explanation that the transition might reflect changes in the coordination state, and the invariance of k, with a IO-fold increase in [Cc] shows that the proteins remain bound as a complex upon cooling. As the midpoint and breadth of the transition are unaffected by changes in percent EG, the transition does not appear to arise from a solventdriven process. Instead, we propose that, at ambient temperatures, the binding interface of the [CcP, Cc] complex undergoes rapid dynamic rearrangements between the subset of conformers that exhibit 3ZnP quenching and the subset that does not. Below the transition range, the complex exists in the latter form, and it is suggested that, upon heating, there is a cooperative loosening of the binding interactions within the interface. We present an heuristic description of the complex based on the statistical mechanical description of the cooperative helix-coil transition in poly(amino acids). In contrast, members of a second class of complexes, those with Cc(horse), Cc(tuna), and Cc(rat), have low quenching rate constants ( k , = 40 s-I at ambient) that decrease smoothly to k, = 0 s-] by 250 K. Furthermore, k, for these complexes shows little dependence upon either solvent or cytochrome, and the triplet decay traces remain exponential at all t...
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