The DNA polymerase from the bacteriophage T4 is part of a multienzyme complex required for the synthesis of DNA. As a first step in understanding the contributions of individual proteins to the dynamic properties of the complex, e.g., turnover, processivity, and fidelity of replication, the minimal kinetic schemes for the polymerase and exonuclease activities of the gene 43 protein have been determined by pre-steady-state kinetic methods and fit by computer simulation. A DNA primer/template (13/20-mer) was used as substrate; duplexes that contained more single-strand DNA resulted in nonproductive binding of the polymerase. The reaction sequence features an ordered addition of 13/20-mer followed by dATP to the T4 enzyme (dissociation constants of 70 nM and 20 microM) followed by rapid conversion (400 s-1) of the T4.13/20-mer.dATP complex to the T4.14/20-mer.PPi product species. A slow step (2 s-1) following PPi release limits a single turnover, although this step is bypassed in multiple incorporations (13/20-mer-->17/20-mer) which occur at rates > 400 s-1. Competition between correct versus incorrect nucleotides relative to the template strand indicates that the dissociation constants for the incorrect nucleotides are at millimolar values, thus providing evidence that the T4 polymerase, like the T7 but unlike the Klenow fragment polymerases, discriminates by factors > 10(3) against misincorporation in the nucleotide binding step. The exonuclease activity of the T4 enzyme requires an activation step, i.e., T4.DNA-->T4.(DNA)*, whose rate constants reflect whether the 3'-terminus of the primer is matched or mismatched; for matched 13/20-mer the constant is 1 s-1, and for mismatched 13T/20-mer, 5 s-1. Evidence is presented from crossover experiments that this step may represent a melting of the terminus of the duplex, which is followed by rapid exonucleolytic cleavage (100s-1). In the presence of the correct dNTP, primer extension is the rate-limiting step rather than a step involving travel of the duplex between separated exonuclease and polymerase sites. Since the rate constant for 13/20-mer or 13T/20-mer dissociation from the enzyme is 6 or 8 s-1 and competes with that for activation, the exonucleolytic editing by the enzyme alone in a single pass is somewhat inefficient (5 s-1/(8 s-1+5 s-1)), ca. 40%. Consequently, a major role for the accessory proteins may be to slow the rate of enzyme.substrate dissociation, thereby increasing overall fidelity and processivity.

are observed. If the vI were associated with a residual ground-state population, it is obvious that modes associated with the bpzo of this residual ground-state complex should also be observed and that their intensities (relative to the features labeled u, in Figure 4B) would closely approximate those observed in Figure 2D. We have confirmed the fact that the relative enhancements of bpyo and bpzo at 350.7 nm are very similar to those at 356.4 nm. Thus, it is not reasonable to argue that relative enhancements at 354.7 nm would be significantly different than at 356.4 nm. Therefore, the absence of wi features in Figure 4B is inconsistent with assignment of the v, features to a residual ground-state population.The TR3 spectrum of the heteroleptic partner complex, Ru-is also consistent with the Dallinger-Woodruff assignment of the u, modes. As can be seen in Figure 2H, the RR spectra of the ground-state species, excited at 356.4 nm, exhibits strong bands associated with both fragments, bpyo and bpzo. The TR3 spectrum ( Figure 4C) excited at 354.7 nm exhibits both u, and w, features, as is expected, given the interpretation that the 3MLCT state is properly formulated as [Ru3(bpy)-(bp~)(bpz-)]~+. To summarize this section, while it is obviously true that a residual population of ground-state species can contribute intensity to the features labeled v, and wI in the TR3 spectrum, these modes are'also enhanced in the spectrum of the 3MLCT state species, Le., even in the absence of a residual ground-state population. We have also confirmed the fact that the enhancements (relative to Sod2-internal standard) of the bpyo "neutral ligand" modes in the TR3 spectrum of R~( b p y ) ,~+ and Ru(bpy),(bp~)~+ (Figure 4, traces A and B, respectively) are identical within experimental error. Such behavior obviously supports the view that the origin of the "neutral ligand" modes in both cases is identical; Le., in both cases the fragment responsible for scattering is formulated as R~~+ ( b p y )~.Thus, the original (Dallinger-Woodruff) assignment of these features to modes of the neutral bipyridine fragments of the 3MLCT state is supported bq the present work.For a number of years we have studied the resonance Raman spectra of flavins and flavoproteins; these studies included investigation of the solvent effect on the 13 resonance Raman (RR) bands between 1700 and 1100 cm-'. These investigations led us to the conclusion that two bands, I1 and X, in the Raman spectrum of flavins are strongly influenced by the degree of hydrogen bonding. Other investigators have made similar suggestions based on shifts in these two bands and in others. We report the QCFF-?r calculations of 87 normal modes of lumiflavin (7,8,1O-trimethylisoalloxazine). We have also calculated resonance Raman intensities and isotope shifts of each resonance Raman active mode. The generally good agreement between calculated and observed values supports our assignment of the experimental bands. The QCFF-a assignments are in general agreement with earlier calculations leadin...

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Cytotoxic T lymphocytes (CTL) play a vital role in host defense against viral and intracellular bacterial infections. However, nonreplicating vaccines administered by intramuscular injection using a syringe and needle elicit predominantly humoral responses and not CTL responses. Here we report that epidermal powder immunization (EPI), a technology that delivers antigens on 1.5-to 2.5-m gold particles to the epidermis using a needle-free powder delivery system, elicits CTL responses to nonreplicating antigens. Following EPI, a majority of the antigen-coated gold particles were found in the viable epidermis in the histological sections of the target skin. Further studies using transmission electron microscopy revealed the intracellular localization of the gold particles. Many Langerhans cells (LCs) at the vaccination site contained antigen-coated particles, as revealed by two-color immunofluorescence microscopy, and these cells were found in the draining lymph nodes 20 h later. Immune responses to several viral protein antigens after EPI were studied in mice.

The rate of association of equine liver alcohol dehydrogenase and its coenzymes exhibits a large pH dependence with slower rates at basic pH and an observed kinetic pKa value of approximately 9-9.5. This pH dependence has been explained by invoking local active site electrostatic effects which result in repulsion of the negatively charged coenzyme and the ionized hydroxyl anion form of the zinc-bound water molecule. We have examined a simpler hypothesis, namely, that the pH dependence results from the electrostatic interaction of the coenzyme and the enzyme which changes from an attractive interaction of the negatively charged coenzyme and the positively charged enzyme to a repulsive interaction between the two negatively charged species at the isoelectric point for the enzyme (pH 8.7). We have tested this proposal by examining the ionic strength dependence of the association rate constant at various pH values. These data have been interpreted by using the Wherland-Gray equation, which we have shown can be applied to the kinetics of enzyme-coenzyme association. Our results indicate that the shielding of the buffer electrolyte changes from a negative to a positive value as the charge on the protein changes at the isoelectric point. This result is exactly that which is predicted for electrostatic effects that depend on the charge of the protein molecule and is not consistent with predictions based upon the local active site effects. At low ionic strength values of 10 mM or less, approximately 75% of the observed pH dependence results from the enzyme electrostatic effects; the remaining pH dependence may result from active site effects.(ABSTRACT TRUNCATED AT 250 WORDS)