Origin of pathogenic determinants of recombinant murine leukemia viruses: analysis of Bxv-1-related xenotropic viruses from CWD mice.

The acquisition of U3 region sequences derived from the endogenous xenotropic provirus Bxv-1 appears to be an important step in the generation of leukemogenic recombinant viruses in AKR, HRS, C58, and some CWD mice. We report here that each of three CWD lymphomas produced infectious xenotropic murine leukemia virus related to Bxv-1. In Southern blot experiments, these proviruses hybridized to probes that were specific for the xenotropic envelope and Bxv-1 U3 region sequences. Nucleotide sequence analysis of a cloned CWD xenotropic provirus, CWM-S-5X, revealed that the envelope gene was closely related to but distinct from those of other known xenotropic viruses. In addition, the U3 region of CWM-S-5X contained a viral enhancer sequence that was identical to that found in MCF 247, a recombinant AKR virus that is thought to contain the Bxv-1 enhancer. Finally, restriction enzyme sites in the CWM-S-5X provirus were analogous to those reported within Bxv-1. These results establish that the virus progeny of Bxv-1 have the potential to donate pathogenic enhancer sequences to recombinant polytropic murine leukemia viruses. Interestingly, the three CWD polytropic viruses that were isolated from the same tumor cells that produced the Bxv-1-like viruses had not incorporated Bxv-1 sequences into the U3 region.     

Measurement and prediction of the rates of spontaneous transfer of phospholipids between plasma lipoproteins

The purpose of this report is to develop a correlation between the hydrophobicity of a phospholipid as measured by reversed-phase high-performance liquid chromatography and its rate of spontaneous transfer and to use this correlation to predict the rate of transfer of any homologous lipid from any lipoprotein. We have studied the mechanism of transfer of a series of fluorescent or radiolabeled phospholipids among natural and reassembled serum lipoproteins. Fluorescent phosphatidylcholines included those with 9-(1-pyrenyl)nonanoic acid in the sn-2 position and lauric, myristic, palmitic, stearic, oleic or linoleic acid at sn-1. The radioactive phosphatidylcholines contained [3H]oleic acid in the sn-2 position and lauric, myristic, or palmitic acid at sn-1. The kinetics of transfer of the pyrene-labeled lipid were followed by changes in the excimer fluorescence, and that of the radioactive lipids by separation of the donor (lipid-apolipoprotein recombinant) from the acceptor (single bilayer vesicles) on a column of Sephacryl S-200. The retention time of each lipid was measured by high-performance hydrophobic chromatography through a Waters radially compressed C18 column eluted with 75% isopropanol and 25% triethylammonium phosphate (0.15 M). A linear relationship was observed between the rate-constant of transfer and the retention time which suggest that the rate of desorption of phosphatidylcholines from lipoproteins and vesicles is controlled predominately by the hydrophobic effect. For a homologous series of lipids, the rate of transfer can be predicted from retention times obtained from hydrophobic chromatography. The kinetics of transfer of 1-lauroyl-2-[9-(1-pyrenyl)nonanoyl] phosphatidylcholine between isolated human serum lipoproteins exhibits a linear correlation between the transfer half-time and the size of the donor lipoproteins. As a consequence, transfer from very-low-density lipoprotein is 10-times slower than that observed from high-density lipoproteins. The observed correlations between phospholipid transfer rates and both the Stokes radius of the donor and the retention time of the phospholipid on a hydrophobic column permit one to calculate the rate of transfer of homologous molecules between lipid-protein complexes. The results predict that the spontaneous transfer of phospholipids between plasma lipoproteins would be too slow to be a physiologically important phenomena.     

On the structure of flavin-oxygen intermediates involved in enzymatic reactions.

During the catalytic reactions of flavoprotein hydroxylases and bacterial luciferase, flavin peroxides are formed as intermediates [see Massey, V. and Hemmerich, P. (1976) in The Enzymes, 3rd edn (P. Boyer, ed.) pp. 421--505, Academic Press, New York]. These intermediates have been postulated to be C(4a) derivatives of the flavin coenzyme. To test this hypothesis, modified flavin coenzymes carrying an oxygen substituent at position C(4a) of the isoalloxazine ring were synthesized. They are tightly bound by the apoenzymes of D-amino acid oxidase, p-hydroxybenzoate hydroxylase and lactate oxidase; the resulting complexes show spectral properties closely similar to those of the transient oxygen adducts of the hydroxylases. The optical spectra of the lumiflavin model compounds were found to be highly dependent on the solvent environment and nature of the subsituents. Under appropriate conditions they simulate satisfactorily the spectra of the transient enzymatic oxygen adducts. The results support the proposal that the primary oxygen adducts formed with these flavoproteins on reaction of the reduced enzymes with oxygen are flavin C(4a) peroxides.     

Effect of pH on the interaction of benzoate and D-amino acid oxidase.

The kinetic and equilibrium dissociation constants of the reversible binding of benzoate to hog kidney D-amino acid oxidase (DAAO) were studied at 19 degrees C over the pH range 5.3-10.5 by means of a stopped-flow apparatus and spectrophotometric titrations. A simple bimolecular reaction of the form second order-first order was observed; a two-step reaction was seen. Analysis of the pH dependence of the bimolecular rate constants and equilibrium dissociation constants is consistent with three ionizable groups which are important for benzoate binding. The pK values of the enzyme-related ionization are 6.3, 9.2, and 9.6. Analysis of the change in extinction coefficient at 360 nm indicates the pK of 9.6 can be assigned to the 3-imino group of the enzyme-bound flavin. The effect of benzoate on the apparent pK for the ionization of the 3-imino group of the enzyme-bound Fad has been reexamined. The presence of benzoate causes an apparent shift of this ionization from a pK value of 9.6 to 10.7.     

Mice with Spontaneous Mammary Tumors Develop Type-Specific Neutralizing and Cytotoxic Antibodies Against the Mouse Mammary Tumor Virus Envelope Protein gp52

Sera from C3H mammary tumor-bearing mice contain cytotoxic antibodies for mouse mammary tumor virus (MMTV)-producing cells, based on (51)Cr release in a complement-dependent serum cytotoxicity assay. The cytotoxic antibodies could be absorbed by purified C3H MMTV gp52 and C3H MMTV-infected cat cells (C3H [MMTV] CrFK) containing cell surface MMTV gp52. However, purified MMTV p27 and uninfected CrFK cat cells were negative. Absorption of the sera with GR (MMTV) CrFK cells also removed all of the cytotoxicity, whereas absorption with RIII (MMTV) CrFK cells was negative, even though all three infected cat cells contained equivalent amounts of gp52. The same C3H cytotoxic sera also neutralized the focus-forming capacity of a C3H MMTV pseudotype of Kirsten sarcoma virus containing MMTV gp52. In contrast, sera from mammary tumor-bearing GR and RIII mice did not neutralize the pseudotype. Furthermore, neutralization could be achieved only by anti-gp52 but not by anti-gp36, -p27, -p14, or -p10 C3H MMTV sera. The gp52's of C3H, GR, and RIII MMTV could also be distinguished by using a type-specific competition radioimmunoassay employing (125)I-gp52 of C3H MMTV and a hyperimmune rabbit anti-C3H MMTV serum. To demonstrate these differences directly, we studied the primary structure of gp52 on the surface of the C3H, GR, and RIII (MMTV) CrFK cells. Two-dimensional tryptic peptide maps of the cell surface lactoper-oxidase-catalyzed iodinated gp52's revealed a greater number of peptides common to the gp52's of C3H and GR MMTVs than to RIII MMTV gp52. These results demonstrate that gp52 is a major target antigen for both cytotoxic and neutralizing antibodies, that the cell surface and virion-associated gp52's of C3H, GR, and RIII MMTV contain both group- and type-specific determinants, and that C3H and GR MMTV gp52's are antigenically more related to each other than to RIII MMTV gp52. Furthermore, C3H mammary tumor-bearing mice develop type-specific antibodies capable of recognizing unique gp52 determinants and, therefore, are able to distinguish the gp52 of C3H MMTV from the gp52's of GR and RIII MMTV.     

Kinetic and isotopic studies of the oxidative half-reaction of phenol hydroxylase

Phenol hydroxylase, an FAD-containing monooxygenase, catalyzes the conversion of substituted phenols to the corresponding catechol. Use of metapyrocatechase, capable of dioxygenation of several catechols to give highly absorbing products, permitted determination of the time course of product release from phenol hydroxylase. Product dissociated prior to complete reoxidation of the enzyme, most likely concomitant with formation of the 4a-hydroxyflavin species (intermediate III). Deuterated phenol and thiophenol exhibited no kinetic isotope effect during the oxidative half-reaction. Isotope effects of 1.7 to 3.7 were found with resorcinol for the conversion of the second intermediate to intermediate III. These effects limited the possible models for phenol hydroxylation. An attempt was made to distinguish whether the spectrum of intermediate II is due entirely to that of the flavin moiety of phenol hydroxylase or whether some radical intermediate form involved in the formation of catechol makes a significant visible contribution. Reduced native and 6-hydroxy-FAD phenol hydroxylase were reacted with oxygen and resorcinol in order to provide evidence for the identity of intermediate II.     

Purification and properties of milk xanthine dehydrogenase.

Milk xanthine oxidase (XO) has been prepared in a dehydrogenase form (XDH) by purifying the enzyme in the presence of 2.5 mM dithiothreitol. Unlike XO, which reacts rapidly only with oxygen and not with NAD, the XDH form of the enzyme reacts rapidly with NAD. XDH has a turnover number for the NAD-dependent conversion of xanthine to urate of 380 mol/min/mol at pH 7.5, 25 degrees C, with a Km = < or = 1 microM for xanthine and a Km = 7 microM for NAD, but has very little O2-dependent activity. There is evidence that the two forms of the enzyme have different flavin environments: XDH stabilizes the neutral form of the flavin semiquinone and XO does not. Further, XDH binds the artificial flavin 8-mercapto-FAD in its neutral form, shifting the pK of this flavin by 5 pH units, while XO binds 8-mercapto-FAD in its benzoquinoid anionic form. XDH can be converted back to the XO form by the addition of three to four equivalents of the disulfide-forming reagent 4,4'-dithiodipyridine, suggesting that, in the XDH form of the enzyme, disulfide bonds are broken; this may cause a conformational change which creates a binding site for NAD and changes the protein structure near the flavin.     

19F NMR studies on 8-fluoroflavins and 8-fluoro flavoproteins

The 19F NMR spectra of the oxidized and reduced forms of 8-fluororiboflavin, 8-fluoro-FAD, and the 8-fluoroflavin-reconstituted flavoproteins flavodoxin, riboflavin binding protein, D-amino acid oxidase, p-hydroxybenzoate hydroxylase, Old Yellow Enzyme, anthranilate hydroxylase, general acyl-CoA dehydrogenase, glucose oxidase, and L-lactate oxidase were measured. For the proteins studied the oxidized resonances appeared over a 10.1-ppm range, while the reduced resonances were spread over 10.3 ppm. Reduction caused an upfield shift of about 27 ppm for the free 8-fluoroflavins and most of the 8-fluoro flavoproteins. The notable exception was 8-fluoro-FMN flavodoxin, which was shifted 37.6 ppm, indicating an unusually high electron density in the benzene ring. Ligand binding to the oxidized 8-fluoro flavoproteins caused either upfield or downfield shifts of 1.5-5 ppm, depending on the protein/ligand combination. The 8-fluoro-FAD anthranilate hydroxylase resonance was shifted downfield and split into two peaks in the presence of anthranilate. The 8-fluoro-FMN Old Yellow Enzyme resonance was shifted upfield upon complexation with charge-transfer-forming, para-substituted phenolates. The upfield shift increased from less than 1 to 5 ppm as the electron-donating capacity of the phenolate increased. Complexation of native Old Yellow Enzyme with 2,4-difluorophenol caused the fluorine resonances of the ligand to shift and split into two pairs of signals. Each pair of signals was associated with a different isozyme of Old Yellow Enzyme.