PubMed · 4676007
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M Wakashin, K Narita. 1972. [Urinary proteins].. https://pubmed.ncbi.nlm.nih.gov/4676007/
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An artificial operon that contains tandem holC-holD genes was used to overproduce a complex of the chi and psi subunits of the DNA polymerase III holoenzyme. Normally insoluble by itself, psi forms a tight soluble complex with chi. A purification procedure that yields pure, active chi psi complex in 100-mg quantities suitable for biophysical studies is reported. Sedimentation equilibrium studies demonstrate that chi psi is a 1:1 heterodimer. The presence of chi psi dramatically lowers the level of delta.delta' required to reconstitute holoenzyme to levels expected in vivo. That chi psi accomplishes this by binding to gamma or tau and increasing their affinity for delta.delta' was demonstrated by surface plasmon resonance using a Pharmacia BIA-core instrument. In the absence of delta.delta', chi psi binds to either the gamma or tau DnaX protein with Kd = 2 nM.
Eukaryotic initiation factor 5A (eIF-5A) requires posttranslational modification of lysine at position 50 to hypusine for its biological activity. We have expressed an unmodified variant of eIF-5A in Escherichia coli and show that it has structural properties different from those of the native protein in terms of its near- and far-UV circular dichroism spectra and its equilibrium unfolding transition with guanidinium chloride. In contrast to the hypusine-modified protein, which unfolds in a two-state process, the complex unfolding transition of unmodified eIF-5A suggests that this variant occurs in two differently folded conformations, F1 and F2. Both conformations are populated under near-physiological conditions at a ration of 60 to 40, respectively. Equilibrium unfolding consists of parallel events: unfolding of F1 to one or several intermediate states (I), and unfolding of F2 to the unfolded state (U). Although the establishment of each of these individual equilibria is fast, the interconversion is slow at guanidinium chloride concentrations between 0 M and 3 M. Kinetic analysis reveals activation energies of 24.3 kcal mol-1 for the reaction of F1 and F2 and 24.1 kcal mol-1 for the reaction of F2 to F1. Both F1 and F2 possess well-defined secondary and tertiary structure. However, the tertiary structures of the two conformations differ as indicated by their distinct near-UV circular dichroism spectra. These differences may be restricted to the C-terminal part of the protein as 2-dimensional 1H-NMR spectra of unmodified eIF-5A reveal no doubled set of proton resonances for aromatic amino acid and histidine residues, of which almost all are located in the N-terminal region.(ABSTRACT TRUNCATED AT 250 WORDS)
We purified a lipoxygenase to homogeneity from eggplant (Solanum melongena Linne) fruits employing the established visible spectrophotometric assay, in which the products of the lipoxygenase-catalyzed reaction, hydroperoxyfatty acids, oxidizes 2,2'-azino-di-[3-ethylbenzothiazoline-(6)-sulfonic acid] diammonium salt in the presence of a catalyst cytochrome c to yield chromophores in the visible region. This allowed for the rapid detection of the lipoxygenase activity and was helpful in accomplishing the efficient purification of the lipoxygenase. The purified lipoxygenase was a monomeric enzyme with a molecular weight of 95,000, s20,w of 5.3 S, and an isoelectric point of 4.4. The specific activity and Km value for the linoleate oxygenation were 7.8 mumol min-1 mg-1 and 0.8 mM, respectively, at 25 degrees C and pH 7.0. The enzyme contained 1.3 g-atom of nonheme iron per enzyme.