Wendy Parris

Late stages in bacteriophage λ head morphogenesis: In vitro studies on the action of the bacteriophage λ D-gene and W-gene products

Abstract The in vitro maturation of bacteriophage λ can be divided into discrete steps. Concatemers of λ DNA bind terminase to form complex I. This DNA-terminase complex then binds a prohead to form a ternary complex (II). Complex II in turn can be converted to infectious phage by the addition of extracts containing the products of the phage genes D, W, FII , as well as phage tails. By using in vitro complementation assays gpD and gpW have been partially purified and their interactions with complex II studied. gpD can bind to complex II in vitro to form a new complex (III) which can be isolated by sedimentation on neutral sucrose gradients. This complex requires only the addition of gpW, gpFll, and phage tails to form mature phage particles. The sedimentation of complex III is virtually identical to that of complex II; however, the resistance of the former to inactivation by DNase is higher, likely due to the partial packaging of the DNA. In similar experiments it was shown that gpW cannot bind to complex II but can effectively interact with complex III. This latter reaction converts complex Ill to a DNase-resistant form which sediments in a manner identical to that of full phage heads (complex IV). After isolation of the complex IV only gpFll and tails are required for mature phage formation in vitro . gpW is a heat-stable protein of molecular weight approximately 10,000.

THE IN VITRO TRANSLOCASE ACTIVITY OF LAMBDA TERMINASE AND ITS SUBUNITS : KINETIC AND BIOCHEMICAL ANALYSIS

The terminase holoenzyme of bacteriophage λ is a multifunctional protein composed of two subunits, gpNu1 and gpA. In vitro, under certain conditions, terminase can render DNAs from various sources, of varying lengths and termini, resistant to degradation by high concentrations of DNase I. This reaction is completely dependent on the presence of terminase, proheads, a hydrolyzable triphosphate, and a divalent metal ion, and we propose that it is the result of translocation of DNA into proheads by terminase. This reaction is stoichiometric with respect to terminase, DNA, and proheads and can be supported by all deoxyribo- and ribonucleoside triphosphates, but not by the corresponding diphosphates or nonhydrolyzable ATP analogs. Mg2+ and Ca2+ promote the reaction, but Mn2+ and Zn2+ do not. In the absence of spermidine, translocase activity is low, but addition of the Escherichia coli protein integration host factor (IHF) promotes specific translocation of only those DNA fragments containing the terminase-binding site, cosB. When spermidine is present, nonspecific translocation of DNA from any source is stimulated. Under these conditions IHF no longer promotes specificity, but translocation of only cosB-containing DNA fragments can be restored by addition of small amounts of a dialyzed and RNase-treated E. coli extract, suggesting that additional host factor(s) may be involved in determination of packaging specificity. To a limited extent, gpA alone can promote translocation, but gpNu1, which has no translocase activity on its own, must be added to approach the holoenzyme-like activity levels. Formation of viable phage cannot be accomplished by gpA in the absence of gpNu1.

Site-specific immobilization of an L-lactate dehydrogenase via an engineered surface cysteine residue

SummaryIn order to facilitate immobilization of the L-lactate dehydrogenase from Bacillus stearothermophilus, a single cysteine residue has been introduced by site-directed mutagenesis whose freely accessible thiol group is located on the protein surface without interfering with enzyme catalysis. The active lactate dehydrogenase mutant Arg331Cys could be coupled covalently to thiopropyl- or organomercurial-functionalized agarose beads with at least 56% recovery of enzymatic activity. The immobilized catalyst showed saturation kinetics similar to the free enzyme, but had an increased thermal stability.

The substrate specificity of the muscle l-lactate dehydrogenase of spiny dogfish (Squalus acanthis)

Abstract The substrate specificity of the l -lactate dehydrogenase ( l -LDH) from the muscle of the spiny dogfish (DMLDH) was evaluated for 13 structurally varied α-ketoacid substrates and the results were compared with the specificity data for the l -LDH from Bacillus stearothermophilus (BSLDH), the latter being the most valuable l -LDH for preparative-scale production of chiral α-hydroxy acid synthons of asymmetric synthetic value. DMLDH has a significantly narrower specificity than BSLDH, and also than heart LDHs. Reasons for its narrower specificity, based on X-ray data analyses that provide new insights into the factors determining the specificities of l -LDHs are advanced.

THR246 mutations decrease substrate inhibition in lactate dehydrogenase

Threonine 246 in Bacillus stearothermophilus L-lactate dehydrogenase has been changed to valine, serine, and alanine by site-directed mutagenesis. Kinetic analyses show a decrease in substrate inhibition for pyruvate reduction with the T246S mutant and virtual elimination of substrate inhibition for the T246A and T246V mutants. The results indicate that the absence of substrate inhibition in the 246A/V-catalyzed reactions is due to the elimination of a key hydrogen bond between the hydroxyl group of threonine and pyruvate in the wild-type complex that is an important contributor in the formation of the abortive enzyme-NAD(+)-pyruvate complex responsible for substrate inhibition.