Purification and characterization of glutathione reductase from Rhodospirillum rubrum.

Glutathione reductase (NAD(P)H:GSSG oxidoreductase EC 1.6.4.2.) was purified 1160-fold to homogeneity from the nonsulfurous purple bacteria Rhodospirillum rubrum (wild type). Specific activity of the pure preparation was 102 U/mg. The enzyme displayed a typical flavoprotein absorption spectrum with maxima at 274,365, and 459 nm and an absorbance ratio A280/A459 of 7.6. The amino acid analysis revealed an unusually high content of glycine and arginine residues. Titration of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) showed a total of two free thiol groups per subunit, one of which is made accessible only under denaturing conditions. An isoelectric point of 5.2 was found for the native enzyme. Km values, determined at pH 7.5, were 6.1 and 90 microM for NADPH and GSSG, respectively. NADH was about 2% as active as NADPH as an electron donor. The enzyme's second choice in disulfide substrate was the mixed disulfide of coenzyme A and glutathione, for which the specific activity and Km values were 5.1 U/mg and 3.4 mM, respectively. A native molecular weight of 118,000 was found, while denaturing electrophoresis gave a value of 54,400 per subunit, thus suggesting that R. rubrum glutathione reductase exists as a dimeric protein. Other physicochemical constants of the enzyme, such as Stokes radius (4.2 nm) and sedimentation coefficient (5.71 S), were also consistent with a particle of 110,000.     

Characteristics of NADPH-dependent thymidylate synthetase purified from Streptomyces aureofaciens.

NADPH-dependent thymidylate synthetase from Streptomyces aureofaciens has been purified to homogenity by a two-step chromatographic procedure including anion-exchange chromatography and affinity chromatography on methotrexate-Sepharose 4B. The enzyme was purified 1025-fold with a 34% yield. Basic characteristics of the enzyme were determined: molecular weight of the enzyme subunit (28,000), pH and temperature optimum, effect of cations, dependency on reducing agents, Km values for dUMP, mTHF, and NADPH (3.78, 21.1, and 38.9 microM, respectively), and inhibition effect of 5-FdUMP. Binding studies revealed the enzyme mechanism to be ordered sequential: dUMP bound before mTHF. S. aureofaciens thymidylate synthetase exhibits an absolute requirement for NADPH for the enzyme activity--a unique feature not displayed by any of the thymidylate synthetases isolated so far. NADPH is not consumed during enzyme reaction, indicating its regulatory role. The properties of S. aureofaciens thymidylate synthetase show that it is a monofunctional bacterial enzyme.     

Two novel monoclonal antibodies to fibronectin that recognize the Hep II and CS-1 regions respectively: their differential effect on lymphocyte adhesion.

We have obtained two new mAbs to the carboxy-terminal region of fibronectin, namely P3D4 and P1F11, and have studied their binding sites and their ability to block lymphocyte adhesion to fibronectin. ELISA and Western blot analyses showed that P3D4 reacts with both fibronectin chains and both Hep II-containing fragments (58 kDa and 38 kDa). P1F11, raised against the synthetic peptide CS-1, reacted with the 38 kDa fragment and with a 190 kDa fragment derived from the A chain of fibronectin. P1F11 did not react with the 58 kDa fragment thus clearly establishing that 58 kDa comes from the B chain of fibronectin and lacks the CS-1 sequence. mAbs P3D4 and P1F11 were used to evaluate the contribution of the Hep II and CS-1 sites in cell attachment to fibronectin. P3D4 effectively inhibited B cell adhesion to 38 kDa, 58 kDa and fibronectin; P1F11 however produced only limited inhibition, suggesting that lymphocyte interaction with Hep II may modulate further binding to the CS-1 site.     

Structures of poly(dA-dT, ip5dU) containing various small amounts of the antiherpetic 5-isopropyl-2'-deoxyuridine.

Three different concentrations of the antiherpetic agent 5-isopropyl-2'-deoxyuridine (ip5dU) were introduced into the synthetic DNA poly(dA-dT) to analyze resulting copolymers by electron microscopy, UV absorption and CD spectroscopy. The poly(dA-dT, ip5dU) containing 1.3 and 4.3% ip5dU did not much differ from the parent poly(dA-dT) but poly (dA-dT, ip5dU) with 7.1% ip5dU behaved in an unusual way. Results are explained by the notion that if bulky isopropyls occur sufficiently close to each other then stable hairpins protruding from the double helix are formed, presumably to accommodate the ip5dU-s into the loops.     

Conformation of human fibrinogen in solution from polarized triplet spectroscopy.

The rotational motions of human fibrinogen in solution at 20 degrees C have been examined, in the 0.2-12-microseconds time range, by measuring the laser-induced dichroism of the triplet state of an erythrosin probe covalently bonded to the protein. The decay of the anisotropy was multiexponential, and up to three correlation times (phi 1 = 380 +/- 50 ns, phi 2 = 1.1 +/- 0.1 microseconds, and phi 3 = 3.3 +/- 0.6 microseconds) were needed to obtain a satisfactory analysis. The experimental data are consistent with the brownian motions of an elongated, rigid particle. If the correlation times are combined with previous data on the intrinsic viscosity of fibrinogen, the rotational and translational diffusive properties of the protein can be reproduced with high accuracy by idealizing it as an elongated ellipsoid of revolution with dimensions (2a x 2b) of (54 +/- 6) x (7.2 +/- 0.5) nm, having rotational diffusion constants of D parallel = (6.2 +/- 0.7) x 10(5) s-1 and D perpendicular = (5 +/- 1) x 10(4) s-1. The possibility of Ca(2+)-dependent changes in the rigidity or conformation of fibrinogen was excluded by examining the submicrosecond time-resolved fluorescence depolarization of 1-methylpyrene conjugates of the protein in the presence of different calcium concentrations. Although there are inherent difficulties to extrapolate the data on isolated fibrinogen molecules to the polymerizing species, this relatively stiff conformation meets the requirements of the classical half-staggered double-stranded model of fibrin polymerization rather better than those of the recently proposed interlocked single-stranded mechanism.     

Nonoxidative cadmium-dependent dimerization of Cd7-metallothionein from rabbit liver.

The effect of free Cd(II) ions on monomeric Cd7-metallothionein-2 (MT) from rabbit liver has been studied. Slow, concentration-dependent dimerization of this protein was observed by gel filtration chromatographic studies. The dimeric MT form, isolated by gel filtration, contains approximately two additional and more weakly bound Cd(II) ions per monomer. The incubation of MT dimers with complexing agents EDTA and 2-mercaptoethanol leads to the dissociation of dimers to monomers. The results of circular dichroism (CD) and electronic absorption studies indicate that the slow dimerization process is preceded by an initial rapid Cd-induced rearrangement of the monomeric Cd7-MT structure. The 113Cd NMR spectrum of the MT dimer revealed only four 113Cd resonances at chemical shift positions similar to those observed for the Cd4 cluster of the well-characterized monomeric 113Cd7-MT. This result suggests that on dimer formation major structural changes occur in the original three-metal cluster domain of Cd7-MT.     

Heparin binding to the urokinase kringle domain.

The binding of urokinase to immobilized heparin and dextran sulfate was studied using activity assays of the bound urokinase. The markedly higher binding observed with high M(r) urokinase compared to low M(r) urokinase indicated a role for the amino-terminal fragment (ATF). This was confirmed by the use of inactive truncated urokinase and monoclonal antibodies specific for the ATF in competition assays of urokinase binding. Antibody competition assays suggested a site in the kringle domain, and a synthetic decapeptide Arg-52-Trp-62 from the kringle sequence (kringle numbering convention) was competitive in assays of urokinase binding to dextran sulfate and heparin. Heparin binding to the urokinase kringle was unambiguously demonstrated via 1H NMR spectroscopy at 500 MHz. Effective equilibrium association constants (K(a)*) were determined for the interaction of isolated kringle fragment and low M(r) heparin at pH 7.2. The binding was strong in salt-free 2H2O (K(a)* approximately 57 mM-1) and remained significant in 0.15 M NaCl (K(a)* approximately 12 mM-1), supporting a potential physiological role for the interaction. This is the first demonstration of a function for the kringle domain of urokinase, and it suggests that while the classical kringle structure has specificity for lysine binding, there may also exist a class of kringles with affinity for polyanion binding.     

Quantitation of molybdopterin oxidation product in wild-type and molybdenum cofactor deficient mutants of Chlamydomonas reinhardtii.

A simple and reliable procedure of oxidation of molybdenum cofactor (MoCo) from molybdoenzymes by autoclaving samples at 120 degrees C for 20 min yielded a single predominant fluorescent species that could be quantitatively determined by reverse phase high performance liquid chromatography. This method allowed detection and quantitation of molybdopterin in cell-free extracts of the green alga Chlamydomonas reinhardtii. The MoCo oxidation product from C. reinhardtii has the same chromatographic and spectral properties as that of milk xanthine oxidase and chicken liver sulfite oxidase. The oxidized species was also detected in molybdenum cofactor mutants of Chlamydomonas reinhardtii defective at the nit-3, nit-4, nit-5/nit-6 and nit-7 loci, which strongly suggests that active molybdenum cofactor itself is not directly involved in the control of its own biosynthetic pathway.     

Interaction of retinol and retinoic acid with phospholipid membranes. A differential scanning calorimetry study.

The influence of retinol and retinoic acid, two retinoids of major interest, on the main gel to liquid-crystalline phase transition of different phospholipid membranes has been studied by means of differential scanning calorimetry. Both compounds exerted perturbing effects on the phase transition of membranes composed of dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylethanolamine. At concentrations up to 42.5 mol% of retinoid in the membrane, the delta H was not much affected with respect to the pure phospholipid, indicating a rather slight interaction. As the concentration of retinol was increased the Tc transition temperature decreased. A fluid-phase immiscibility was observed for the system DPPC/retinol at concentrations between 0 and 33 mol%. Almost ideal phase diagrams were obtained for the mixture DPPE/retinol. At concentrations of 33 mol% and higher retinol was able to induce phase separations in DPPC membranes, but not in DPPE. The effect of retinoic acid was much weaker, the Tc and delta H remaining almost unaltered and equal to that of the pure phospholipid up to concentrations of 30 mol%, at neutral pH. Retinoic acid exerted a pH-dependent effect. As the pH decreased, and therefore increased the extent of protonation of retinoic acid, the pertubation of the membrane induced by this compound was less. A strong effect, both on Tc and delta H, was observed at pH 10, where the retinoic acid moiety will be mainly unprotonated and the negative charge will generate repulsive forces thus destabilizing the membrane. The mixture DPPC/retinoic acid presents a region of fluid-phase immiscibility. At low pH, when the retinoic acid moiety was fully protonated, this fluid-immiscibility region extended from 0 to 36 mol% of retinoic acid, but its size decreased with increasing pH, and at pH 10 it was only found from 0 to 3 mol%. These results are discussed in terms of the possible retinoid/phospholipid interactions and the disposition of the retinoid moiety in the bilayer.