Myristic acid binding to human serum albumin investigated by dialytic exchange rate

Dialysis rate determinations of several fatty acids in the absence of albumin revealed that the myristate anion, like that of laurate, in aqueous solution, pH 7.5, is present as a monomer anion when the concentration is below 25 microM. Palmitate and oleate solutions, on the other hand, show a tendency to aggregation even at concentrations below 0.5 microM. Multiple binding of myristate to human serum albumin in phosphate buffer, at pH 7.5, 37 degrees C, was investigated by exchange of 14C-labeled myristate across a dialysis membrane under conditions of binding equilibrium. A binding isotherm was established by least squares fitting of the stoichiometric binding constants in the stepwise binding equation to the experimental data. The best-fit solution was supplemented with 30 acceptable solutions within a probability limit of 0.95. A concept of one or two distinct high-affinity sites for binding of fatty acids could not be verified; the observations allow a variety of binding mechanisms ranging from cooperativity of the first two myristates to a model with four equal and independent sites.     

Mitochondrial ATP synthase. Interaction of a synthetic 50-amino acid, beta-subunit peptide with ATP

A 50-amino acid peptide predicted by chemical modification studies of F1 and by comparison with adenylate kinase to comprise part of an ATP-binding domain within the beta-subunit of mitochondrial ATP synthase has been synthesized and purified. In the numbering system used for bovine heart beta, the peptide consists of amino acid residues from aspartate 141 at the N-terminal end to threonine 190 at the carboxyl end. In Tris-Cl buffer, pH 7.4, the peptide undergoes a dramatic reaction with ATP resulting in precipitate formation. Analysis of the precipitate shows it to contain both peptide and ATP. Similar to the ATPase activity of F1 and the binding of nucleotide to the enzyme, the capacity of ATP to induce precipitation of the peptide is decreased markedly by lowering pH. Interaction of the peptide with the fluorescent ATP analog, TNP-ATP (2'(3')-O-(2,4-6-trinitrophenyl)-adenosine 5'-triphosphate), can be demonstrated in solution at low concentrations. A 7-fold enhancement in fluorescence is observed when 2.5 microM TNP-ATP interacts with 2.5 microM peptide. Divalent cation is neither required for ATP-induced precipitation of the peptide nor for demonstrating interaction between TNP-ATP and peptide, just as Mg2+ is not required for nucleotide binding to F1. These results indicate that the beta-subunit peptide studied here comprises at least part of a nucleotide-binding domain within the mitochondrial ATP synthase complex.     

Relative spectral response as a function of sequential ligand binding

A unique method is presented for the determination of the critical number of ligands that must bind to a macromolecule to elicit a spectroscopic response. This method is based on analysis of ligand binding data. For example, four Ca2+ and two Mg2+ ions are necessary for mirroring the relative decrease in the intrinsic fluorescence of bovine prothrombin fragment 1. For application of the method, ligand loading and relative spectroscopic response data must be measured over a full range of concentrations.     

Identification and characterization of mitochondria autoantigens in progressive systemic sclerosis: identity with the 72,000 dalton autoantigen in primary biliary cirrhosis

Sera from eight out of 62 (14.5%) patients with progressive systemic sclerosis (PSS) reacted by immunoblotting with a 72,000 dalton antigen and one, a patient with concomitant primary biliary cirrhosis (PBC), reacted with the 72,000 dalton and a 47,000 dalton antigen. Reactivity with these antigens was not seen with any of 111 control sera. The antigens with minor variations in m.w. were present in a variety of cultured cells and tissue homogenates from different species. Subcellular fractionation studies localized the antigens to the mitochondria. Of 19 sera from patients with other diseases selected for immunofluorescence staining for anti-mitochondria autoantibody, nine reacted with the 72,000 dalton antigen, seven reacted with both the 72,000 and 47,000 dalton antigens, and three reacted with the 47,000 dalton antigen. These results show that serum reactivity with the 72,000 dalton and 47,000 dalton mitochondria autoantigens is found with some patients with PSS. Because mitochondria autoantibodies that are reactive with the 72,000 dalton and 47,000 dalton polypeptides are also found in patients with PBC, the present finding provides additional support for the association of PSS with PBC. Prior absorption of rat liver homogenate with PBC sera removed PSS serum reactivity with a 63,000 dalton antigen, the equivalent 72,000 dalton antigen in rodents, and vice versa, showing that both PBC and PSS sera recognize the same antigen.     

Variations in concentration of oxytocin and vasopressin in the paraventricular nucleus of the hypothalamus during the estrous cycle in rats

Oxytocin (OT) and arginine-8-vasopressin (AVP) were measured by radioimmunoassay in micropunched hypothalamic neurosecretory nuclei of estrous cycling female Sprague-Dawley rats. In the paraventricular nucleus (PVN): the concentration (pg/microgram protein) of OT was significantly higher in rats in diestrus than during proestrus, estrus, or metestrus, while the concentration during metestrus was significantly greater than in proestrus and estrus; the concentration of AVP was significantly lower in animals in estrus than during the other three stages; because the paraventricular OT levels dropped before proestrus, the AVP/OT ratio was significantly greater in animals in proestrus than in diestrus, metestrus, and estrus. In the supraoptic nucleus (SON) a similar trend was noted: the concentration of OT was highest during diestrus, and AVP was lowest during estrus, though neither was significantly different from other stages. Because the OT and AVP cycles in the SON were asynchronous, the ratio of AVP to OT was significantly higher in proestrus than in metestrus or diestrus and significantly greater in estrus than during diestrus. In contrast to these two areas, peptide concentrations did not vary significantly across the estrous cycle in other sites of nonapeptide synthesis, i.e. the anterior commissural nucleus (ACN) and the suprachiasmatic nuclei (SCN).     

Location of ligand-binding sites on the nicotinic acetylcholine receptor alpha-subunit

The portions of the Torpedo californica nicotinic acetylcholine receptor (AChR) alpha-subunit that contribute to the allosteric antagonist-binding site and to the agonist-binding site have been localized by affinity labeling and proteolytic mapping. [3H]Meproadifen mustard was employed as an affinity label for the allosteric antagonist-binding site and [3H]tubocurare as a photoaffinity label for the agonist-binding site. Both labels were found in a 20-kDa proteolytic fragment generated from the AChR alpha-subunit by Staphylococcus aureus V8 protease. This 20-kDa peptide also contains the 3H-labeled 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide-reactive site and binds 125I-alpha-bungarotoxin. N-terminal sequencing established that the 20-kDa fragment began at Ser-173 of the alpha-subunit. Fluorescein isothiocyanate-conjugated concanavalin A could be bound to the second of the two major V8 cleavage products, an 18-kDa peptide. This peptide was also sensitive to treatment with endo-beta-N-acetyl-glucosaminidase H, consistent with the presence of N-linked carbohydrate on this fragment. The N terminus of this peptide was found to be Val-46 of the alpha-subunit sequence. Experiments designed to map disulfide bonds within the AChR alpha-subunit indicate that no bonds exist between the 18-kDa fragment (containing Cys-128 and Cys-142) and the 20-kDa fragment (containing Cys-192, Cys-193, and Cys-222). These results establish that the 20-kDa fragment contributes to both the acetylcholine and the allosteric antagonist-binding sites, whereas there is no evidence that the 18-kDa fragment is part of either site.     

Isolation and reconstitution of the n-butylmalonate-sensitive dicarboxylate transporter from rat liver mitochondria

The mitochondrial dicarboxylate carrier has been substantially purified from rat liver mitoplasts by extraction with Triton X-114 in the presence of cardiolipin followed by chromatography on hydroxylapatite. Upon incorporation of the hydroxylapatite eluate into phospholipid vesicles, an n-butylmalonate-sensitive malonate/malate exchange has been demonstrated. This exchange activity is enhanced 226-fold relative to the starting material (i.e. detergent-extracted mitoplasts). Silver-stained sodium dodecyl sulfate-polyacrylamide gradient gels verify the high purity of this fraction relative to the starting material. Nonetheless, the banding pattern indicates that several protein species are still present. As isolated, the dicarboxylate transporter is rather unstable but can be stabilized either by the addition of 10% ethylene glycol and subsequent storage at -20 degrees C or by incorporation into phospholipid vesicles in the presence of malate followed by freezing in liquid nitrogen. Such proteoliposomes catalyze a [14C]malonate uptake which is characterized by a first order rate constant of 1.02 min-1 and a t 1/2 of 41 s. This uptake can be inhibited by dicarboxylates (e.g. succinate, malate, unlabeled malonate) but not by either alpha-ketoglutarate or by tricarboxylates (e.g. citrate, threo-Ds-isocitrate). Furthermore, the reconstituted malonate transport is dependent on internal malate and can be inhibited by n-butylmalonate, mersalyl, p-chloromercuribenzoate, and Pi, but not by N-ethylmaleimide. It is concluded that this highly purified fraction contains a reconstitutively active dicarboxylate transporter which, based on its substrate specificity and inhibitor sensitivity, appears to be identical to the native dicarboxylate transport system found in intact rat liver mitochondria.     

The value of provoked expectoration in obtaining sputum samples for cytologic investigation. A prospective, consecutive and controlled investigation of 134 patients

A prospective controlled investigation in 134 consecutive outpatients compared the cytologic adequacy of sputum samples obtained by spontaneous and provoked expectoration. Inhalation of nebulized 10% sodium chloride was used for provoked expectoration. A significantly higher number of adequate samples was produced after provocation, as judged by the presence of alveolar macrophages (X2 = 5.63; p less than 0.02). The improvement in sample adequacy was limited to the nonsmokers and ex-smokers in the study. This result, together with the relatively high cost of cytologic sputum examinations, indicates that provoked expectoration should at least be applied to the collection of sputum samples from nonsmokers and ex-smokers.     

Inhibition of DNA replication in preimplantation mouse embryos by aphidicolin

The regulation of trophectoderm differentiation in mouse embryos was studied by inhibiting DNA synthesis with aphidicolin, a specific inhibitor of DNA polymerase alpha. Embryos were exposed to aphidicolin (0.5 micrograms/ml) for 16 h at various preimplantation stages and scored for their ability to form a blastocyst and develop beyond the blastocyst stage. Embryos were most sensitive to aphidicolin at the late 4-cell stage and became progressively less sensitive as they developed. Aphidicolin inhibited blastocyst formation by 70%, 100%, 77%, and 24% after treatment at the 2-cell, 4-cell, noncompacted 8-cell, and compacted 8-cell stages, respectively. Although the inhibitory effect of aphidicolin on blastocyst formation decreased markedly as 8-cell embryos underwent compaction, developmental capacity beyond the blastocyst stage was poor after treatment of either noncompacted or compacted 8-cell embryos. Treatment at the morula and early blastocyst stages was less harmful to embryos than treatment at earlier stages but reduced the number of trophoblast outgrowths by interfering with hatching. Autoradiographic analysis showed that during aphidicolin treatment, incorporation of 3H-thymidine was inhibited over 90% at all stages examined, indicating an inhibition of DNA synthesis. Because inhibition of blastocyst formation by aphidicolin decreased at the compacted 8-cell stage, we suggest that approximately the first half of the fourth DNA replication cycle is critical for subsequent blastocyst formation. Furthermore, the poor further development of blastocysts formed after aphidicolin treatment of compacted 8-cell embryos suggests that the DNA replication requirements for initial trophectoderm differentiation are distinct from requirements for further development of blastocysts in vitro.