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A comparative study of recombinant 51- and 66-kDa subunits comprising equine infectious anemia virus reverse transcriptase (EIAV RT) is reported. Both polypeptides sedimented as stable homodimers (molecular mass, 102 and 132 kDa, respectively) when analyzed by rate sedimentation through glycerol gradients. Consistent with their dimer composition, each preparation displayed considerable levels of both RNA- and DNA-dependent DNA polymerase activity on different homopolymeric template/primer combinations. However, a detailed analysis of the polymerization products indicated qualitative differences. Whereas p66 EIAV RT proceeded essentially unimpaired along both RNA and DNA templates, p51-catalyzed DNA synthesis was interrupted close to or in the immediate vicinity of the primer. A series of "programmed" 2-step polymerization reactions suggests that p51 EIAV RT enters an abortive mode of polymerization. Duplication of this observation with p51 human immunodeficiency virus-1 RT, together with recent observations from murine RT, suggests that lack of a ribonuclease H domain and loss of contact with the nascent product from the polymerase active center have profound consequences on the mode of polymerization.

A contribution of the 51-kDa subunit of human immunodeficiency virus type-1 reverse transcriptase to activities of the parental heterodimer (p66/p51) was assessed in "selectively deleted" heterodimers whose p51 component contained C-terminal truncations of 13, 19, or 25 residues. Analyses included (i) efficiency of reconstitution into heterodimer, (ii) retention of polymerase and ribonuclease H (RNase H) function, and (iii) interaction with the HIV replication primer, tRNA(Lys,3). Our data suggest that these features of heterodimer reverse transcriptase can be modulated by the extent of the C-terminal p51 deletion. Severely impaired tRNA binding in a selectively deleted heterodimer whose 51-kDa subunit lacks 13 residues, despite retention of enzymatic functions, strengthens arguments for p51 involvement in tRNA binding.

The interaction of several forms (p51, p66, and p66/p51) of recombinant human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) with a synthetic derivative of its cognate replication primer, tRNA(Lys-3), has been determined by gel-mobility shift analysis. While p66/p51 RT is proficient in tRNA binding, preparations of p66 and p51 display only weak binding at elevated protein:tRNA ratios, despite the former containing both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activity. Gel permeation analysis of purified p66 RT indicate this to be predominantly monomeric, suggesting that dimerization may be a prerequisite for efficient tRNA binding. Prolonged incubation of a mixture of the 66- and 51-kDa polypeptides results in heterodimer reconstitution, restoration of tRNA binding, and recovery of appreciable levels of RNA-dependent DNA polymerase activity. Under the same conditions, both the tRNA binding and RNA-dependent DNA polymerase activities of the 66- and 51-kDa polypeptides are unaffected, suggesting that they remain in the monomeric conformation.

Metal chelate affinity chromatography has been used to follow reconstitution of the 66- and 51-kDa human immunodeficiency (HIV)-1 and HIV-2 reverse transcriptase (RT) subunits into heterodimer, as well as chimeric enzymes comprised of heterologous subunits. By adding a small N-terminal polyhistidine extension to the 51-kDa subunit of either enzyme, reconstituted RT could be recovered from a cell lysate by chromatography on Ni(2+)-nitrilotriacetic acid-Sepharose. Homologous RT subunits rapidly associated to form the respective heterodimers (1-p66.1-p51 and 2-p66.2-p51) when bacterial lysates containing the individual components were mixed. Under the same conditions, association of p66 HIV-2 and p51 HIV-1 RT was inefficient and could be improved slightly by prolonged incubation of the respective p66 and p51 subunits. In contrast, HIV-1 p66 RT rapidly associated with the 51-kDa subunit of the HIV-2 enzyme. RNA-dependent DNA polymerase activity was associated with all reconstituted enzymes, and the response of each chimeric RT to an inhibitor selective for the HIV-1 enzyme indicated that sensitivity to inhibition was determined by the source of its 66-kDa subunit.

In animal cells, unliganded steroid receptors are complexed with a 90-kDa heat shock protein, HSP90; hormone binding by the receptor leads to the release of HSP90. We found that the 795-amino acid rat glucocorticoid receptor protein formed oligomeric complexes in vitro upon synthesis in rabbit reticulocyte lysates; these oligomers also dissociated in the presence of hormone. Similar complexes formed when X795, a receptor derivative containing only the C-terminal half (amino acids 407-795) of the protein, was translated in vitro. Moreover, X795 was co-immunoadsorbed from the reticulocyte lysates together with HSP90 by three different anti-HSP90 monoclonal antibodies, indicating that the in vitro translated receptor binds HSP90 and that the interaction occurs within the C-terminal half of the receptor. To localize the HSP90 binding region in greater detail, various deletion mutants of X795 were translated in vitro and assayed for oligomer formation and for co-immunoadsorption with HSP90. The results indicated that HSP90 interacted with the receptor within a subregion of the hormone binding domain, between amino acids 568 and 616. These findings are consistent with the proposal that HSP90 may participate in the mechanism of signal transduction by steroid receptors.

A kinetic pulse-chase labeling technique was used to measure the intracellular half-life of the glucocorticoid receptor in S49 mouse lymphoma cells. Cells were pulse-labeled with [35S]methionine for 30 min and then cultured in the presence of unlabeled methionine (chase). Labeled receptors were quantitated at periodic time points during the chase by immunoadsorption to protein A-Sepharose using the BuGR2 monoclonal antireceptor antibody. The decay of labeled receptors during the chase was linear on a semilog plot, consistent with first order kinetics. Receptor half-life was 9 h when cells were cultured in either phenol red-containing medium supplemented with fetal calf serum or in phenol red free-medium supplemented with charcoal extracted serum, indicating that endogenous steroids do not affect receptor half-life. Receptor half-life was also unchanged when cells were cultured in the presence of 0.1 microM dexamethasone, a glucocorticoid hormone, or 0.1 microM RU486 (11 beta-(4-dimethylamino-phenyl)-17 beta-hydroxy-17 alpha-(propynylestra-4,9- diene-3-one), an antiglucocorticoid hormone. We conclude that the intracellular half-life of the glucocorticoid receptor in S49 mouse lymphoma cells is not regulated by either glucocorticoid or antiglucocorticoid hormones.

We have investigated the association of the glucocorticoid receptor in S49 mouse lymphoma cells with the 90-kDa heat shock protein, HSP 90. Proteins were synthetically labeled by culturing cells with [35S]methionine and immunoadsorbed to protein A-Sepharose using monoclonal antireceptor antibody (BuGR-2). HSP 90 coimmunoadsorbed with the receptor when cytosol was incubated with antireceptor antibody for 1 h in the absence of molybdate, indicating that molybdate is not required for either formation or recovery of HSP 90-receptor complexes. Molybdate stabilized HSP 90-receptor complexes during prolonged incubation in vitro and rendered the complexes resistant to dissociation by 500 mM NaCl. In pulse-chase experiments, cells were incubated with [35S]methionine for 30 min (pulse label) and then cultured with excess unlabeled methionine (chase). At different times in the chase, cytosol was prepared from cells, and labeled proteins were immunoadsorbed by antireceptor antibody. The amount of labeled receptor recovered from cytosol decreased during the chase with a half-life of about 4 h. Labeled HSP 90 coimmunoadsorbed with the receptor at 4 h into the chase but not at the beginning of the chase or at 2 h into the chase, indicating that newly synthesized HSP 90 associated with the receptor in a time-dependent manner during the chase. Labeled HSP 90 did not associate with the receptor when dexamethasone was added to cells at the beginning of the chase. These findings support the hypothesis that HSP 90 associates with the glucocorticoid receptor in intact cells rather than in cytosol and indicates that dexamethasone promotes dissociation of HSP 90-receptor complexes in vivo.

A series of plasmids encoding various Klebsiella pneumoniae nif (nitrogen fixation) genes were constructed to determine which were required to produce active iron (Fe) protein in Escherichia coli, a species which does not normally fix nitrogen. The greatest success was achieved with binary plasmid systems that produced nifA regulatory protein under the control of a tac promoter on one plasmid, which then induced synthesis of nifH and nifM proteins from their native promoter sites on a second plasmid. nifH protein, the monomeric subunit of Fe protein, produced in the presence of nifM constituted nearly 10% of the whole cell protein and exhibited the corresponding amount of C2H2-reducing activity in nitrogenase assays conducted in vitro. nifH protein formed in the absence of nifM constituted 4.7% of the whole cell protein and exhibited no detectable activity in assays of whole cell extracts. The plasmid-encoded Fe protein was purified to homogeneity and was found to be indistinguishable from that isolated from derepressed wild type K. pneumoniae, having a similar specific activity, approximately 4 Fe/dimer of 68 kDa, and similar epr features. Although these experiments do not exclude the participation of other E. coli gene products in the maturation of nifH protein, they limit the nif-specific genes required for active Fe protein production to nifA, nifH, and nifM. Since nifA is thought to be the required activator protein involved in nif operon transcription, the simplest explanation for these observations is that nifH codes for the peptide of the Fe protein, while nifM acts to convert this nifH peptide to the functioning Fe protein of nitrogenase. In the absence of nifM, only an inactive nifH polypeptide is produced.