Effects of dehydroabietic acid on the physical state of cytoskeletal proteins and the lipid bilayer of erythrocyte membranes.

Dehydroabietic acid (DHAA) is a major aquatic toxic resin acid usually found in unbleached pulp mill effluents. This compound has been reported to accumulate in the red cells of rainbow trout and to cause hemolysis. To elucidate further understanding to the mechanism of action of this resin, the interaction of DHAA with human erythrocyte membranes has been monitored by electron paramagnetic resonance techniques of spin labeling. Results presented in this paper indicate that DHAA, in a concentration-dependent manner, significantly altered both the motion and order of the lipid bilayer and the physical state of cytoskeletal proteins, while DHAA had no effect on isolated lipids. It is proposed that the increase in the 'fluidity' of the lipid bilayer induced by DHAA is a secondary effect of primary changes in the physical state of the cytoskeletal proteins of the membrane, and that the latter effect is critically associated with the toxicity of DHAA.

Electron paramagnetic resonance studies of the effects of methoxyacetic acid, a teratologic toxin, on human erythrocyte membranes.

Methoxyacetic acid (MAA), a teratogenic toxin, is the major metabolite of ethylene glycol monomethyl ether (EGME, also referred to as 2-methoxyethanol, 2-ME). MAA causes a wide range of toxic effects in laboratory animals including reproductive and developmental toxicity, as well as hematotoxicity, by mechanisms that are not clear. In this study, we employed electron paramagnetic resonance (EPR) spin-labeling techniques in conjunction with spin labels specific for cytoskeletal proteins, bilayer lipids, cell-surface sialic acid, or cell-surface galactose and N-acetylgalactosamine residues of human erythrocyte membranes in order to gain insight into the mechanism of MAA toxicity. The major findings are: (1) MAA significantly increases the protein-protein interactions of skeletal proteins in a concentration-dependent manner (P < 0.001), while 2-ME has no effect (at even a 2.5-fold higher concentration). (2) Addition of MAA leads to significant increase in the rotational motion of spin-labeled terminal galactose and N-acetylgalactosamine residues (2.0 mM MAA, 38% decrease of the apparent rotational correlation time tau a, P < 0.01). (3) The rotational motion of spin-labeled sialic acid, 70% of which is on the major transmembrane sialoglycoprotein (glycophorin A or PAS 1), was not affected by MAA treatment. (4) MAA has no effect on the lipid bilayer fluidity, since no change in the motion of a lipid bilayer specific spin label (5-NS) in the erythrocyte membrane was observed. These results suggest that MAA may lead to teratologic toxicity by interacting not with lipid components but with certain, perhaps specific, protein components, i.e., transport proteins, cytoskeleton proteins or neurotransmitter receptors.

Menadione-induced cytotoxicity effects on human erythrocyte membranes studied by electron paramagnetic resonance.

Menadione (2-methyl-1,4-naphthoquinone) is cytotoxic to hepatocytes. In order to begin to investigate the changes in the physical state of membranes induced by this cytotoxic substance, electron paramagnetic resonance (EPR) spin-labeling techniques were used in conjunction with spin labels specific for cytoskeletal proteins, bilayer lipids, or cell-surface sialic acid or galactose to investigate erythrocyte membranes. We studied the molecular effects of oxidation of 200 microM menadione on the different membrane domains. The major findings are: (1) menadione increases protein-protein interactions (P < 0.001) of cytoskeletal proteins, (2) there is a slightly significant increase in the rotational motion of spin-labeled sialic acid (P < 0.05), while (3) the physical state of galactose residues was unaffected by menadione. Since glycophorin is coupled to the major cytoskeletal protein, spectrin, by protein 4.1, we suggest that menadione-induced oxidation could alter the conformation of protein 4.1. As a consequence, single or multiple sites of weakness could be induced leading to the alteration of the interactions of the cytoskeletal network and its anchoring domains in the membrane. These results are discussed with reference to possible mechanisms involved in the cytotoxic action of menadione.

Selective labeling of membrane protein sulfhydryl groups with methanethiosulfonate spin label.

Electron paramagnetic resonance was used to characterize the first use of a thio-specific spin label in membranes. Procedures for use of the spin-label, 1-oxyl-2,2,5,5-tetramethyl-delta 3-pyrroline-3-methyl (methanethiosulfonate MTS) covalently attached to membrane proteins in human erythrocyte membranes are reported. The major findings are: (1) MTS was found to be thiol-specific in membranes as it is for soluble proteins; (2) MTS labels ghost proteins in as few as 30 min at room temperature, providing a distinct advantage when sensitive or fragile membranes are to be used; (3) the distribution of the spin label suggests that the major cytoskeletal protein, spectrin, and the major transmembrane protein (Band 3) incorporate the highest percentage of spin label. This procedure expands the tools with which the researcher can investigate the physical state of membrane proteins and its alteration upon interaction of membrane perturbants or in pathological conditions.

Electrophoretic analysis of proteins from Phycomyces mutants with abnormal tropisms.

Certain phototropism mutants of Phycomyces blakesleeanus show defective bending responses (tropisms) to stimuli besides light, such as gravity, wind, and barriers. These so-called "stiff" mutants are affected in four genes (madD to madG). Using two-dimensional gel electrophoresis, we have analyzed polypeptides from microsomal and soluble fractions obtained from the wild type, four single mutants, and six double mutants affected in all pairwise combinations of the four genes. Consistent differences in spot patterns for madE and madF mutants were found in microsomal fractions but not in soluble fractions. In madE mutants, two spots designated E1 (52 kDa, pI 6.65) and E2 (50 kDa, pI 6.65) were altered. E1 appeared denser in the wild type than in the madE mutants, while the reverse was true for E2. The spots E1 and E2 are probably under regulatory control by madE, perhaps involving posttranslational modification. A protein spot, F1 (53 kDa, pI 6.1), was present on the wild-type gels but absent from all gels for madF mutants. The F1 polypeptide probably represents the madF gene product.

Modified Light-Induced Absorbance Changes in dim Y Photoresponse Mutants of Trichoderma.

A brief pulse of blue light induces the common soil fungus Trichoderma harzianum to sporulate. Photoresponse mutants with higher light requirements than the wild type are available, including one class, dim Y, with modified absorption spectra. We found blue-light-induced absorbance changes in the blue region of the spectrum, in wild-type and dim Y mutant strains. The light-minus-dark difference spectra of the wild type and of several other strains indicate photoreduction of flavins and cytochromes, as reported for other fungi and plants. The difference spectra in strains with normal photoinduced sporulation have a prominent peak at 440 nm. After actinic irradiation, this 440 nanometer difference peak decays rapidly in the dark. In two dim Y photoresponse mutants, the difference spectra were modified; in one of these, LS44, the 440 nanometer peak was undetectable in difference spectra. Detailed study of the dark-decay kinetics in LS44 and the corresponding control indicated that the 440 nanometer difference peak escaped detection in LS44 because it decays faster than in the control. The action spectrum of the 440 nm difference peak is quite different from that of photoinduced sporulation. The light-induced absorbance changes are thus unlikely to be identical to the primary photochemical reaction triggering sporulation. Nevertheless, these results constitute genetic evidence that physiologically relevant pigments participate in these light-induced absorbance changes in Trichoderma.

Identification and characterization of a nucleotide binding site of ovine prolactin with 2-azido-NAD.

Photoaffinity labeling of ovine prolactin with the NAD+ photoaffinity analog [alpha-32P]nicotinamide-2-azidoadenine dinucleotide has been used to identify an NADH/NADPH binding site. Specificity of nucleotide interaction was demonstrated by saturation and protection of labeling at physiologically relevant concentrations. Saturation of photoinsertion was observed at approximately 100 microM probe with an apparent Kd of approximately 25 microM. Protection of photoinsertion was observed with NAD+ and NADH. The photoinsertion was decreased by 75% and greater than 95%, respectively, upon addition of 200 microM of the above-mentioned compounds. The protection obtained with NADP+ and NADPH was of the same order, respectively. The adenine ring binding domain of NADH/NADPH binding site was identified by trypsin and chymotrypsin digestion of the photolabeled prolactin and purification of the photolabeled peptide by boronate affinity chromatography and immobilized Fe3+ affinity chromatography. The peptide was identified to be Ala22-Tyr28. These studies demonstrate that prolactin contains an NADH/NADPH binding site which may be significant in the mechanism of action of this hormone.