Double mutant cycle analysis of aspartate 69, 97, and 103 to asparagine mutants in the m2 muscarinic acetylcholine receptor.

Double mutant cycles provide a method for analyzing the effects of a mutation at a defined position in the protein structure on the properties of an amino acid at a second site. This approach was used to map potential interactions between aspartates 69, 97, and 103 in the m2 muscarinic acetylcholine receptor transmembrane helices 2 and 3. Receptors containing single and double aspartate to asparagine mutants were expressed in Chinese hamster ovary cells and their effects on ligand binding, signal transduction, and thermal stability determined. Analysis of the double mutant cycles showed that the mutations had approximately additive effects on ligand binding, signal transduction, and thermal stability. Ligand binding and thermal inactivation results support the conclusion that aspartate-103 is the ligand amine counterion. Effector coupling properties of the mutant receptors showed that aspartate-103 was also required for signal transduction activity. The mutation of aspartate-69 to asparagine completely eliminated signal transduction by the agonists acetylcholine, carbachol, and pilocarpine but not oxotremorine M, which caused reduced but significant inhibition of adenylyl cyclase and stimulation of phospholipase C. In contrast, adenylyl cyclase stimulation by the asparagine-69 mutant was elicited only by acetylcholine and carbachol but not by oxotremorine M. The variation in agonist-dependent effector coupling properties provides evidence that the asparagine-69 mutant can exist in activated receptor states that are different from the wild-type m2 muscarinic receptor.

Properties of glutathione peroxidase isolated from human plasma.

Human plasma glutathione peroxidase (GPx) was purified to homogeneity by ammonium sulfate fractionation, gel filtration on Sephadex G-150, chromatography on DEAE Sephacel, chromatofocusing with polybuffer, and gel filtration with Sephadex G-75. This isolation resulted in about 5,400-fold purification of the enzyme with a 32% yield in enzyme activity. The final preparation had a specific activity of about 28 units (mmoles NADPH oxidized) per milligram of protein. Determination of selenium on the purified enzyme revealed a content of 3.8 g atoms per mole GPx. Gel electrophoresis using SDS with standard proteins revealed a molecular weight of about 23,000 for the subunits, which would indicate a molecular weight of about 92,000 for the native enzyme. Amino acid analyses of the purified GPx indicated aspartate, glutamate, proline, glycine, alanine, and leucine as the predominant amino acids and cysteine, methionine, tryptophan, and histidine as the minor amino acids.

Physical properties of the purified cardiac muscarinic acetylcholine receptor.

The physical properties of the cardiac muscarinic acetylcholine receptor (mAcChR) purified from porcine atria as recently described [Peterson, G.L., Herron, G.S., Yamaki, M., Fullerton, D.S., & Schimerlik, M.I. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4993-4997] have been examined by D2O/H2O sucrose gradient sedimentation and Sephacryl S-300 gel filtration in Triton X-405 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). From the sedimentation experiments the partial specific volume and sedimentation constant for the mAcChR-Triton X-405 complex were determined to be 0.813 cm3/g and 5.30 S, respectively, which lead to an estimate of the molecular weight of the complex of 143 000. Gel filtration in Triton X-405 gave an estimate of the Stokes radius (4.29 nm) and an apparent molecular weight of 116 000. Combination of sedimentation and gel filtration gave an apparent molecular weight of 137 000 and a frictional ratio (f/f0) of 1.21 for the complex. The partial specific volume of the receptor calculated from composition was 0.717 cm3/g assuming 26.5% by weight carbohydrate. The amount of bound Triton X-405 was estimated at 1.011 g/g of mAcChR, which gave an apparent molecular weight of 70 900 (sedimentation) or 68 200 (sedimentation plus gel filtration) for the uncomplexed receptor. SDS-PAGE experiments at acrylamide concentrations ranging from 6% T [monomer plus bis(acrylamide)] to 17% T gave a linear range of apparent molecular weight from 67 600 (6% T) to 98 600 (17% T), and calibration against the retardation coefficient, Kr, determined from Ferguson plots gave an apparent molecular weight of 89 100 +/- 6700. From a newly developed, novel evaluation scheme the anomalous migration of the mAcChR in SDS-PAGE was found to be due to both an excess charge density and an abnormally large shape parameter (Kr), and the true molecular weight of the protein portion of the mAcChR ligand binding polypeptide was estimated to be between 50 000 and 60 000.

The efficacy of direct, 24-hour culture, and mitotic synchronization methods for cytogenetic analysis of bone marrow in neoplastic hematologic disorders.

Bone marrow aspirates from 90 patients suspected of having a hematologic disorder were processed by using different cytogenetic methods to determine if any procedure was more likely to reveal a chromosomally abnormal clone or produce better-quality metaphases. All specimens were processed by a direct technique and 24-hr culture without mitogens; 50 specimens were also processed by an amethopterin mitotic synchronization method. In each case, the microscope slides were coded by the processing technologist and analyzed by two other experienced cytogenetic technologists. The results were not known to any of the investigators until all 90 specimens were analyzed. With the exception of one specimen, in which a chromosomally abnormal clone was identified only in the direct preparation, no apparent differences were found in the karyotypes among the three methods. Also, the differences in the quality or number of metaphases found among the three methods were not statistically significant; however, 24-hr unstimulated cultures produced more metaphases than the mitotic synchronization procedure. The greatest source of discordance was caused by one test yielding either no metaphases or an uncertain result when the other tests produced a successful study. We suggest that in routine practice at least two different methods should be used, and it may be best if at least one of these methods is a direct technique.

Localization of minutes to specific polytene chromosome bands by means of overlapping duplications.

Overlapping duplications recovered as suppressors of Minute loci have been used to localize M(2)z and M(3)w(124) to specific polytene bands 25A1(2) and 95A1(2). The surprising efficiency of M localization by duplication may result from the tendency of M suppressors to be at least a visible fraction of a polytene band in length.

Properties and evolutionary potential of newly induced tandem duplications in Drosophila melanogaster.

Most of some 33 X-ray-induced duplications recovered as Suppressors of Minute loci proved to be direct tandem duplications. When heterozygous, most duplications were crossover suppressors, and duplications of short to moderate size did not reduce the fitness of their bearers. Crossover suppression by tandem duplication may be attributed to intrastrand foldbacks of the type regularly seen in somatic polytene chromosomes. As a consequence, linkage disequilibrium between duplicated elements and normal chromosomes should be more profound than has been supposed. Tandem duplications appear to be predisposed by reason of frequency of generation, crossover suppression and fitness effects to serve as the primary source of new genes.

Metabolic fate of hydrazine.

Studies of the disposition of hydrazine administered to mammals have not succeeded in accounting for more than a modest fraction of the dose, nor have the excretory products been completely identified. We have utilized 15N-labeled hydrazine and conventional methods to account for about 75% of single doses of about 0.5 LD50 (1 mmol/kg). In 48 h, about 30% appeared in urine as hydrazine and about 20% emerged as a derivative that is acid-hydrolyzable to hydrazine. About 25% was converted to N2 gas, most of which appeared less than 30 min after administration. The percentage converted to N2 at 4 h increased only slightly with dose between 0.5 and 2.0 mmol/kg. Disappearance of hydrazine from blood was biphasic with half-times of 0.74 and 26.9 h.