Complex behavior of marine animal tissue extracts in the competitive binding assay of brevetoxins with rat brain synaptosomes.

Brevetoxins are produced by the marine dinoflagellate Ptychodiscus brevis, an organism linked to red tide outbreaks, and the accompanying toxicity to marine animals and to neurotoxic shellfish poisoning in humans. Brevetoxins bind with high affinity to voltage-sensitive sodium channels and cause increased sodium ion conductance and nerve cell depolarization. The brevetoxin competitive binding assay with tritium-labeled brevetoxin 3 (3H-PbTx-3) and rat brain synaptosomes is a sensitive and specific assay for pure brevetoxins. Here we report that extracts of manatee, turtle, fish, and clam tissues contain components that interfere with the assay by cooperative, noncompetitive inhibition of 3H-PbTx-3 specific binding and increased nonspecific binding to synaptosomes. By determining the "apparent" toxin concentration ("[Toxin]") in the extract at several assay concentrations, a reasonable correction for the complex inhibition could be made using a semilog plot to extrapolate [Toxin] to zero extract concentration to obtain [Toxin]0. Spiking 4 extracts with 60 nM PbTx-3 caused [Toxin]0 to increase by 41 +/- 8 nM, indicating that the noncompetitive components did not prevent the assay of toxin but did reduce the accuracy of the result. Fourfold repetition of the assay of 4 samples gave standard deviations of 25 to 60% of the value of [Toxin]0, so the error can be fairly large, especially for samples with little toxin. Purification of an extract with a 1 g sample prep column of C-18 decreased the complex inhibition by about 3-fold but did not eliminate interference in the assay.

Possible mechanism for late gestational development of the antioxidant enzymes in the fetal rat lung.

We hypothesized that a possible mechanism to explain the significant increases that occur in the pulmonary antioxidant enzyme (AOE) system late in gestation might be an endogenous increase in the normal reactive O2 substrates for these enzymes. We found that lung O2 free radical formation increased approximately 175% between fetal day 18 and birth (p < 0.01). We also found that late fetal rat lung mitochondrial and microsomal rates of AOE substrate (H2O2) generation increased markedly, and there was also significantly increased lung lipid peroxidation products with increasing gestational age. These definite elevations in reactive O2 species production in parallel with the time course of maturational elevations in the pulmonary AOE system, suggest that increasing enzyme substrate concentrations could be a primary controlling mechanism for increasing lung AOE gene expression in preparation for birth of the newborn.

Complex association and dissociation kinetics of brevetoxin binding to voltage-sensitive rat brain sodium channels.

Brevetoxins bind with high affinity to the voltage-sensitive sodium channel and cause nerve cell depolarization and increased sodium ion conductance. Using 0.6 nM tritium-labeled brevetoxin-3 and freshly prepared synaptosomes from fresh or frozen rat brain, binding results at 6 degrees C fit well to a curve for 2-phase association with 65% of the binding in the rapid phase and t1/2 values of 11 and 74 min for the rapid and slow phases, respectively. Both phases were accelerated at higher toxin concentrations, binding of 9 nM brevetoxin-3 (PbTx-3) was close to equilibrium within 1 hr. The slow phase was not apparent when binding was done at 20 degrees C or when binding was done at 6 degrees C after the membrane sample had been preincubated at 4 degrees C for 1 day or at 22 degrees C for 1 hr. The 2-phase nature of association was not affected by substitution of KCl for choline chloride in the assay medium to produce sodium channel in the inactive state. Dissociation kinetics at 6 degrees C were also complex; the results fit well to a 2-phase curve with 55% of the dissociation in the rapid phase and t1/2 values of 13 and 64 min for the rapid and slow phases, respectively. The 2-phase nature did not change significantly after preincubation at 4 degrees C for 1 day. However, dissociation at 20 degrees C was rapid and fit a curve for 1-phase dissociation with a t1/2 of 2-6 min. At higher concentrations of PbTx-3, the binding is further complicated by the presence of 2-4 low-affinity binding sites with Kd values near 700 nM. In conclusion the association and dissociation of PbTx-3 with sodium channel from rat brain are complex processes that may involve changes in sodium channel conformation or interactions with other membrane (or membrane-associated) components.

Comparative responses of premature versus full-term newborn rats to prolonged hyperoxia.

Because fetal rat lungs have lower baseline levels of both surfactant and antioxidant enzymes than full-term newborn rats, we questioned whether prematurely delivered rats might be more susceptible to O2 toxicity than those born at term. In the present studies, prematurely delivered rats (gestational d 21 of 22) and full-term rat pups were simultaneously put in > 95% O2 after birth. Surprisingly, we found that the preterm rats were not more susceptible to O2-induced lung damage and lethality than full-term newborns, but, in fact, the composite percentage of survival was even greater in the preterm pups from 7 to 9 d in hyperoxia and were similar thereafter up to 14 d in high O2. In addition, the preterm rats showed significantly decreased lung wet/dry weight ratios and consistently less severe pathologic evidence of pulmonary edema compared with term rats at 6 and 8 d of O2 exposure. The premature pups demonstrated the capability of inducing pulmonary antioxidant enzyme responses to hyperoxia by 3 d, and had significantly elevated copper-zinc superoxide dismutase, catalase, and glutathione peroxidase activities (and lung surfactant contents) at 6 d of O2 exposure compared with the term rats in O2. The rates of lung total O2 consumption and cyanide-resistant O2 consumption at d 6 in hyperoxia were not different for preterm versus term pups.(ABSTRACT TRUNCATED AT 250 WORDS)

Does lung NAD(P)H:quinone reductase (DT-diaphorase) play an antioxidant enzyme role in protection from hyperoxia?

NAD(P)H:quinone reductase, or DT-diaphorase, has been studied primarily in the liver where it appears to function as an antioxidant-like enzyme in the 2-electron reduction of some quinones to less toxic hydroquinones. This property together with new molecular biology evidence that oxidants such as H2O2 can induce gene transcription of DT-diaphorase provide especially intriguing reasons to examine the possibility that lung DT-diaphorase could have an important antioxidant enzyme role versus pulmonary O2 toxicity during exposure to hyperoxia. We found that similar to the 'classical' lung antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) DT-diaphorase activity increased significantly in the late gestational fetal lung; also its activity was altered in the same way as the antioxidant enzymes by prenatal hormonal treatment. Another similarity is that DT-diaphorase activity was induced in the neonatal animal lung during hyperoxia, but not in the adult animal lung. However, using various drug treatments which markedly increased lung DT-diaphorase activity (e.g., 3-methylcholanthrene, butylated hydroxyanisole, methimazole) we found no improved hyperoxic survival in the treated adult rats. Also, dicumarol treatment, which markedly depressed DT-diaphorase activity, did not diminish the hyperoxic survival rate in an O2-tolerant adult rat model. Thus, we conclude that unlike the classical antioxidant enzymes, increased pulmonary DT-diaphorase activity is probably neither necessary nor sufficient to protect against pulmonary O2 toxicity during hyperoxia.

Negative regulation of antioxidant enzyme gene expression in the developing fetal rat lung by prenatal hormonal treatments.

Prenatal administration of thyrotropin-releasing hormone (TRH) or TRH plus dexamethasone (DEX) to pregnant rats accelerates lung surfactant system development in late gestation, but paradoxically depresses the normal late gestational elevation in fetal lung antioxidant enzyme (AOE) activities (Pediatr Res 30:522, 1991). In these present studies, we tested whether both prenatal hormonal treatments act to depress normal fetal lung AOE development by negative regulation of AOE gene expression. We used solution hybridization to quantitate the concentration of AOE mRNA. Results of the developmental studies revealed significantly decreased lung mRNA concentrations of copper-zinc superoxide dismutase, manganese superoxide dismutase, catalase, and glutathione peroxidase in late gestation as a result of prenatal TRH treatment. The addition of DEX administration did not reverse the lowered expression of lung AOE genes due to TRH treatment, but instead resulted in significant additional decreases in pulmonary AOE mRNA levels at both 21 and 22 d of gestation. The tested AOE mRNA half-lives (stabilities) revealed no significant differences between controls (8.0-10.5 h) and TRH-treated (8.2-9.5 h) and TRH-plus-DEX treatment (7.8-10.7 h) groups. These findings suggest that prenatal treatment with TRH and with TRH plus DEX acts to depress the normal late fetal lung AOE activity elevations by (direct) negative regulation of AOE gene expression, and the decreased AOE expression is likely regulated at the level of gene transcription rather than posttranscriptionally.

Soluble beta-galactoside specific lectin is developmentally regulated in lungs of neonatal black mice and beige mice.

The beige mouse, a mutant of the C57 black mouse, is best known as a model of the Chediak-Higashi syndrome. Recently, it was found that alveolar maturation in neonatal beige mice is impaired, resulting in abnormally large alveoli. In guinea pigs, hamsters, and rats there is an elevated activity of a soluble, beta-galactoside-binding lectin in lungs at the age when alveolar maturation is in progress. Our present studies were done to find out if the temporal relationship between elevated lectin activity and alveolar maturation also occurs in mice and, further, if the impaired alveolar maturation in beige mice might be linked to the lectin. We found that the temporal relationship between lectin activity and alveolar maturation is also present in black and beige mice, with a peak in specific lectin activity occurring at about 8 days after birth. We also found that the major lectin purified from black or beige mice has essentially the same subunit molecular weight, isoelectric point, and amino acid composition. In conclusion, we found nothing abnormal about the lectin or its developmental regulation that can explain the impaired alveolar maturation in neonatal beige mice. The results do not rule out the possibility of an important role for the lectin in normal lung development or the possibility that some aspect of function or localization of the lectin or its ligands, not related to total lung lectin hemagglutinating activity, may be altered in the beige mouse.

Rat lung lectin synthesis, degradation and activation. Developmental regulation and modulation by dexamethasone.

Soluble lectins are widely distributed cell-agglutinating proteins. Their activity is developmentally regulated in several tissues, including the lung, but virtually nothing is known about the mechanisms of the developmental regulation or the turnover of these proteins. We studied mechanisms that might be responsible for the developmentally regulated changes in the activity of a lectin (beta-galactoside-binding protein) found in the lung, and determined if its activity or turnover could be modulated by treatment of rat pups with a glucocorticosteroid hormone (dexamethasone). Our studies on the activity and turnover of the lectin indicated that the peak of lectin activity (units/mg of protein) that occurred at age 12 days appeared to be brought about by two means: an increase in the activity of the lectin molecule itself (units/micrograms of lectin) that occurred at age 8 days, and 1.5-fold increase in the absolute rate of lectin synthesis at age 11 days. The decline in lectin activity was associated with a decrease in its rate of synthesis, return to the baseline extent of activation, and an increased rate of degradation. Treatment of rat pups with dexamethasone diminished the peak of lectin activity (units/mg of protein) by about 25%. This effect of dexamethasone was due, at least in part, to the complete prevention of activation of the lectin molecule (units/micrograms of lectin) and a premature increase in the rate of lectin degradation. Perhaps the normal fall in lectin activity after age 11 days is caused by mechanisms induced by the increase in serum corticosteroid that occurs at that age.

Pulmonary interstitial fibrosis induced in hapten-immune hamsters.

A model for pulmonary interstitial fibrosis (PIF) based on cell-mediated immune response is described. Animals were primed for contact hypersensitivity responses by skin painting with trinitrophenol (TNP), but instead of challenging with the antigen on the skin, animals were challenged with a single intratracheally administered dose of the immunizing hapten. Primed animals developed inflammation followed by pulmonary fibrosis, as determined by histologic examination. Furthermore, immunized animals developed an increase in hydroxyproline (as an indirect measure of collagen synthesis) that could be recovered from the lung by 7 days after an intratracheal challenge with TNP. The increase in hydroxyproline within the lung persisted through 30 days. The response was specific because little or no fibrosis or increase in collagen deposition was observed in immune animals that were challenged with an unrelated hapten (dinitrophenol). Unimmunized animals demonstrated a slight increase in hydroxyproline in the lung 7 days after challenge, but with time the collagen content of these control animals approached normal levels. These studies demonstrate that a specific cell-mediated immune response to a hapten within the lung can induce pulmonary interstitial fibrosis.

Importance of the mitochondrial amino acid pool in the sensitivity of erythroid cells to chloramphenicol: role of glycine and serine.

The amino acid pool of mitochondria has been recently implicated in the sensitivity of erythroid cells to chloramphenicol (CAP) [8]. In the present study, we have analyzed the composition of that pool in the sensitive erythroleukemia mitochondria (EM) and compared it with that of the resistant chloroma mitochondria (CM). We have also tested the effect of every major component in each pool on the sensitivity to CAP. The study of endogenous amino acid composition of EM and CM was performed with a JEOL 5AH amino acid analyzer, and the sensitivity to the drug was assayed by determining its interaction with the mitochondrial protein synthesis activity. Analysis of the total endogenous pool showed about 20% more amino acids in CM compared to EM. However, some amino acids were present in significantly higher (e.g. glycine, serine and histidine) or lower (e.g. proline, leucine, arginine, glutamic acid and threonine) quantity within EM. When compensating for each low amino acid in either CM or EM by addition of that particular acid to the incubation medium, only glycine and serine had a significant effect. Thus, the addition of glycine or serine enhanced the sensitivity to CAP from 14 to 49-52% in CM, but were without effect in EM. Addition of the other acids to either mitochondria gave little or no effect. Since serine could be interconvertible intramitochondrially to glycine, and because the latter with succinate are the first reactants in heme biosynthesis which is initiated inside the mitochondria, it would appear that erythroid cell sensitivity to CAP is predetermined by the mitochondrial glycine-serine pool and might be somehow related to the pathway of heme biosynthesis in these cells.

Oxidation and chemical modification of lung beta-galactoside-specific lectin.

Galaptins are small, soluble, lectins with a specificity for beta-galactose residues. Many galaptins are inactivated by atmospheric oxygen and are protected by disulphide-reducing reagents. We find that each subunit of rat lung galaptin contains one residue of tryptophan and six of cysteine. Oxygen inactivates rat lung galaptin by oxidation of the cysteine residues. During oxidation, the normal dimeric structure is maintained and all disulphide bonds are formed within individual subunits. Exogenous thiols protect against inactivation, but oxidized thiols accelerate inactivation. Human lung fibroblast galaptin is almost completely inactivated within 1 h in tissue culture medium at 37 degrees C. Alkylation of native rat lung galaptin with iodoacetate or ethyleneimine causes substantial loss of activity. The dimeric galaptin structure is maintained. In contrast, alkylation with iodoacetamide yields carboxamidomethyl-galaptin, which is fully active and stable to atmospheric oxygen in the absence of disulphide-reducing reagents. This derivative is very useful for studies of galaptin properties and function.

Effect of glycerol on protein acetylation by acetic anhydride.

We investigated the basis for the previously unexplained stabilization of proteins by glycerol during reaction with acetic anhydride [S. Siegel and W. M. Award, Jr. (1973) J. Biol. Chem. 248, 3233-3240]. Model studies showed that glycerol competes successfully for acetylation against protein hydroxyl groups. In contrast, amino groups are much more potent nucleophiles and their acetylation is not apparently affected. Since alpha-amino and phenolic pKa's did not change significantly in increasing glycerol concentrations, these findings are ascribed to glycerol's lower pKa value as compared to water, leading to the decreased acetylation of tyrosine, threonine, and serine hydroxyl groups in Pronase guanidine-stable chymoelastase. An additional mechanism is important and predominates in the protection against inactivation of bovine delta-chymotrypsin during acetylation and is explained by the recently described basis for protein stabilization in glycerol [K. Gekko and S. N. Timasheff (1981) Biochemistry 20, 4667-4676; 4677-4686]. Those studies demonstrated that glycerol increased the hydrophobicity of nonpolar residues, augmenting their tendency to be removed from protein surfaces. Therefore, the stabilization afforded by glycerol for chymotrypsin is attributed in part to a favoring of the native folded state which forces the side chains of isoleucine-16 and valine-17 to be buried, increasing the apparent pKa of the alpha-amino group of isoleucine-16 as it forms the charge pair with the beta-carboxyl group of aspartate-194. This conclusion was supported by stopped-flow analyses of the interaction of delta-chymotrypsin with proflavin in increasing concentrations of glycerol.

Endogenous ligands of rat lung beta-galactoside-binding protein (galaptin) isolated by affinity chromatography on carboxyamidomethylated-galaptin-Sepharose.

Rat lung beta-galactoside-binding protein (galaptin) is developmentally regulated during postnatal lung development. In common with other vertebrate galaptins, it is very labile when purified and dependent on the presence of exogenous thiol reagents. Reaction of rat lung galaptin with iodoacetamide resulted in a stable active carboxyamidomethylated galaptin that could be coupled to Sepharose. The resultant affinity matrix bound asialoglycoproteins, and these could be quantitatively eluted with disaccharide haptens. The carboxyamidomethylated-galaptin-Sepharose affinity matrix was used to search for endogenous ligands in 13-day-rat lung. Cytosolic fractions of developing rat lung contained no moieties that could be specifically eluted with disaccharide hapten. Only when membranous fractions were extracted with 1% Triton were glycoproteins solubilized that bound to the affinity matrix and could be specifically eluted with disaccharide hapten. The eluted glycoproteins were potent inhibitors of galaptin binding to asialo-orosomucoid. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis identified these glycoproteins as being of high Mr, with three components of Mr 160000-200000 and a smaller component of Mr 75000. This is the first evidence for specific membrane-associated glycoproteins being the ligands of rat lung galaptin.

Evidence for direct methyl transfer in betaine: homocysteine S-methyl-transferase.

We described recently the purification and preliminary characterization of human hepatic betaine: homocysteins S-methyltransferase (Skiba, W. E., Taylor, M. P., Wells, M. S., Mangum, J. H., and Awad, W. M., Jr. (1982) J. Biol. Chem. 258, 14944-14948) where it was shown that isovalerate and 3,3-dimethylbutyrate, analogs of dimethylglycine and betaine, respectively, were good inhibitors. The present study demonstrates that butyrate is a modest competitive inhibitor, binding at the betaine site. This led to the consideration and synthesis of a putative dual-substrate analog, S(delta-carboxybutyl)-DL-homocysteine, which bound with high affinity to the active site of the methyltransferase; presumably this effect is due to the L-isomer only. Homologs, S(gamma-carboxypropyl)-DL-homocysteine and S(beta-carboxyethyl)-DL-homocysteine, do not inhibit at concentrations 100-fold higher than where inhibition is noted with the dual-substrate analog, indicating the latter's specificity. These findings support the hypothesis that methyl transfer in this enzyme occurs directly from one substrate to the other.

Effects of acetylation and guanidination on alkaline conformations of chymotrypsin.

Guanidination leads to stabilization of several globular proteins, including bovine chymotrypsinogen, as determined by hydrogen isotope exchange (P. Cupo, W. El-Deiry, P.L. Whitney, and W.M. Awad, Jr. (1980) J. Biol. Chem. 255, 10828-10833). The present study examined the binding of proflavin to guanidinated, acetylated, and native chymotrypsins in order to compare conformational flexibilities. The order of decreasing alkaline stabilities of the catalytically active conformations of the different delta-chymotrypsin forms was guanidinated, native, and acetylated proteins; delta-chymotrypsin showed greater stability than alpha-chymotrypsin. In each case removal of calcium reduced the amount of the catalytically active conformation. The alkaline pH dependence for the decrease of the catalytically active conformation of guanidinated alpha-chymotrypsin could not be attributed to the titration of a single group, indicating that the alpha-amino group of isoleucine-16 is not the sole feature regulating the conformational transition for this derivative. At neutral pH values delta-chymotrypsin exists completely in an active conformation and the percentage of alpha-chymotrypsin in this form is only slightly lower. These differences from earlier results are possibly due to differences in buffers, calcium ion concentrations, and ionic strength. The rate of inactivation of guanidinated delta-chymotrypsin with methyl acetimidate was much lower than the corresponding rate for the native enzyme. This suggests that guanidination increases enzyme stability which in turn leads to a reduced accessibility of the alpha-amino group of isoleucine-16.

Membrane-associated carbonic anhydrase purified from bovine lung.

We found carbonic anhydrase activity associated with particulate fractions of homogenates of rat, rabbit, human, and bovine lungs. These membrane-associated carbonic anhydrases were remarkably stable in solutions containing sodium dodecyl sulfate (SDS). The bovine enzyme was dissolved with SDS and purified by affinity chromatography and gel filtration. The purified enzyme contains glucosamine, galactose, and sialic acid; it is at least 20% carbohydrate. The apparent molecular weight by SDS-polyacrylamide gel electrophoresis (52,000) may be higher than the actual molecular weight due to the presence of carbohydrate. The enzyme contains cystine, an amino acid that is absent in bovine erythrocyte carbonic anhydrase. Dithiothreitol greatly accelerated the rate of inactivation of the membrane-associated enzyme in SDS, so disulfide bonds appear to stabilize this enzyme. The specific CO2-hydrating activity was about half that of the erythrocyte enzyme. Acetazolamide inhibits the membrane-associated enzyme (Ki = 10 nM) nearly as well as the erythrocyte enzyme (Ki = 3 nM). Antibody to bovine erythrocyte carbonic anhydrase did not inhibit the membrane-associated enzyme. Other investigators have accumulated a good deal of evidence for carbonic anhydrase on the luminal surface of pulmonary capillaries. The enzyme described here appears to be a new isozyme whose properties are consistent with such a localization.

Localization of carbonic anhydrase on pulmonary artery endothelial cells in culture.

Bovine pulmonary artery endothelial cells in culture possess carbonic anhydrase activity and immunoreactivity. The intact cells and cell homogenates lower the pH of 25 mM triethanolamine sulfate buffer saturated with CO2 (starting pH 8.1). The intact cells are more reactive than the cell homogenates, and the enzymic activity is enriched in association with the plasma membrane fraction. Specific immunofluorescence is obtained when the cells are incubated with rabbit antibovine erythrocyte carbonic anhydrase B and then with goat antirabbit immunoglobulin G coupled to fluorescein. At the level of electron microscopy, antibodies to carbonic anhydrase B are reactive with sites along the plasma membrane and associated caveolae. Multivesicular bodies are the only intracellular sites labeled and appear to correspond to the globular sites of intracellular immunofluorescence. Cells maintained and propagated in culture in the absence of an exogenous source of carbonic anhydrase nonetheless possess carbonic anhydrase activity, suggesting that the cells are capable of synthesizing the enzyme. Taken together, our results indicate that pulmonary artery endothelial cells possess carbonic anhydrase situated so that the enzyme could readily catalyze the dehydration of plasma HCO-3 to facilitate CO2 excretion and participate in the regulation of blood pH as central venous blood is converted into systemic arterial blood.

Immunochemical studies of infectious mononucleosis--X. Characterization of a glycoprotein from horse erythrocytes which reacts with Paul-Bunnell antibody.

A highly purified preparation of horse erythrocyte glycoprotein was prepared from an aqueous ethanolic extract of hemoglobin-free membranes. The subunit apparent mol. wt was 30,000. In aqueous solution the glycoprotein formed globular aggregates of 93 +/- 16 A diameter. The glycoprotein had a receptor for the Paul-Bunnell antibody of infectious mononucleosis which was associated with an O-glycosidically linked oligosaccharide and dependent on the presence of N-glycolylneuraminic acid. In addition the glycoprotein had a neuraminidase-sensitive receptor for human peripheral blood lymphocytes. Fifty per cent inhibition of the rosetting of sheep red cells by 4 x 10(5) lymphocytes was caused by 30 microgram of glycoprotein.

Comparison of receptor properties of erythrocyte membrane glycoproteins.

Membrane glycoproteins from horse, sheep, goat and bovine erythrocytes were solubilized and purified. These glycoproteins could be placed in three groups based on their degrees of glycosylation: The major bovine erythrocyte glycoprotein (BGII) had 77% sugar, the minor bovine glycoprotein (BGI) had 27% sugar and the others had approximately 50% sugar. Four of the glycoproteins aggregated in a uniform way in aqueous solution--one, BGII, did not. Four had similar subunit sizes of 25-34,000 daltons, but BGII was larger--55,000 daltons. Receptor functions (for plant and invertebrate lectins, antibodies and cell surfaces) of these glycoproteins were primarily those dependent upon the presence of terminal sialic acid residues.