Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Decavanadate is responsible for vanadate-induced two-dimensional crystals in sarcoplasmic reticulum

Two-dimensional protein crystals of the calcium pump protein of sarcoplasmic reticulum (SR) from fast skeletal muscle were induced using Na₃VO₃ as first described by Dux and Martonosi. These crystals exhibit repeat rows 11 nm apart which contain discrete units with 7 nm repeats. Four different methods of sample preparation for electron microscopy, i.e., negative staining, freezedrying, freeze-fracturing, and thin-sectioning electron microscopy, each give complimentary repeat units. The SR-membrane crystals exhibit surface structure by the freeze-drying technique and row-like structures on the normally smooth outer face of normal SR. The formation of the membrane crystals is dependent on the pH and concentration of the vanadate. Only conditions favoring the presence of decavanadate yield crystals. At low concentrations and neutral pH, decavanadate is unstable and with time converts to smaller oligomers and the monomer. The presence of membrane crystals was correlated with the life span of the decavanadate. Membrane crystals were obtained in the SR membrane from fast twitch muscle from light and heavy SR, referable to longitudinal and terminal cisternae as well as from reconstituted SR. Canine cardiac SR did not crystallize under these conditions.Abbreviations Tris (tris[hydroxymethyl])aminomethane - TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid), 2-(2-hydroxy-1-bis[hydroxymethyl]ethyl)aminoethanesulfonic acid - SR sarcoplasmic reticulum - CPP calcium pump protein Dedicated to the memory of Prof. David E. Green, friend, mentor, and colleague.     

Reversible modification of D-beta-hydroxybutyrate dehydrogenase by diamide

D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with a specific requirement of lecithin for function. The purified enzyme devoid of lipid (apodehydrogenase) is inactive but can be reactivated by forming a complex with phospholipid containing lecithin. We find that, of the six half cysteines present in D-beta-hydroxybutyrate dehydrogenase, only two are in the reduced form and available for modification with N-ethylmaleimide, even after denaturation in sodium dodecyl sulfate. Diamide treatment of either the inactive apodehydrogenase or the active enzyme-phospholipid complex resulted in complete loss of enzymic activity, the apodehydrogenase being assayed after addition of phospholipid. The inactivation by diamide can be reversed by the addition of dithiothreitol with full recovery of activity. Derivatization using N-[14C]ethylmaleimide showed that diamide modified only one sulfhydryl per enzyme monomer. The other sulfhydryl appears not to be essential for function since full activity can be restored after this sulfhydryl had been covalently derivatized with N-ethylmaleimide. Protein cross-linking was not observed after diamide modification of D-beta-hydroxybutyrate dehydrogenase, indicating that a disulfide bridge was not formed between enzyme subunits. The diamide-modified enzyme retains the ability to bind coenzyme, NAD(H), as detected by quenching of the intrinsic fluorescence of the protein. However, resonance energy transfer from protein to bound NADH and enhancement of NADH fluorescence were not observed, indicating that diamide modification of the protein alters the nucleotide binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane asymmetry and enhanced ultrastructural detail of sarcoplasmic reticulum revealed with use of tannic acid

Fixation of purified sarcoplasmic reticulum (SR) membrane vesicles, using glutaraldehyde supplemented with 1% tannic acid, reveals newly visualized ultrastructure in thin sections. The trilaminar appearance of the membrane is highly asymmetric; the outer electron-opaque layer is appreciably wider (70 A) than the inner layer (20 A). The asymmetry is not referable to lack of penetration of the tannic acid since: (a) SR vesicles made permeable with 1 mM EDTA, pH 8.5, show similar asymmetry; (b) treatment of SR with trypsin results in progressive loss in protein content and decrease in the thickness of the outer layer, until in the limit the trilayer has a symmetric appearance; (c) within the same muscle section, the SR membrane appears highly asymmetric whereas the sarcolemma has a more symmetric appearance; (d) reconstituted SR vesicles have a symmetric appearance with equally broad inner and outer layers (approximately 70 A); the symmetric structure is confirmed by freeze-fracture and negative staining electron microscopy. Heavy and light SR vesicles obtained by isopycnic density sedimentation of purified SR have the same asymmetric appearance of the membrane and seem to differ mainly in that the heavy vesicles contain internal contents consisting largely of Ca++-binding protein. The asymmetry of the SR membrane is referable mainly to the unidirectional alignment of the Ca++ pump protein, the major component (90% of the protein) of the membrane. The asymmetry of the SR membrane can be visualized now for the first time in situ in thin sections of muscle.     

The molecular weight of Ehrlich tumor cell ribonucleotide reductase and its subunits: Effector-induced changes

The molecular weights of Ehrlich tumor cell ribonucleotide reductase and its individual components were determined by sedimentation equilibrium in the Beckman Airfuge. The distribution of enzyme after sedimentation equilibrium was determined by measurement of the CDP reductase and ADP reductase activities associated with ribonucleotide reductase. The apparent molecular weight of the intact enzyme was 304,000 when assayed for CDP reductase and 254,000 when assayed for ADP reductase. This difference in apparent molecular weights was statistically significant with a P value of 0.0002. The molecular weights of the individual components of ribonucleotide reductase were determined in a similar fashion by assaying in the presence of an excess of the complementary component. The non-heme iron component had a molecular weight of 81,000 when assayed for either CDP or ADP reductase activity. The effector-binding component had an apparent molecular weight of 127,000 when assayed for CDP reductase and 95,000 when assayed for ADP reductase. This difference in apparent molecular weights was statistically significant with a P value of 0.004. The effectors ATP and dGTP altered the apparent molecular weights of the intact enzyme and individual components. In the presence of ATP the molecular weight of intact CDP reductase was 481,000 while the apparent molecular weight of the effector-binding component of CDP reductase alone was 418,000. In the presence of dGTP, the molecular weight of intact ADP reductase was 293,000 while the apparent molecular weight of the effector-binding component of ADP reductase alone was 154,000. These results indicate that the proportion of the non-heme iron component and the effector-binding component is not equimolar and that the composition of the enzyme is not constant but is altered by the presence of effectors. Our data also suggest that CDP reduction and ADP reduction are catalyzed by different molecular species of the enzyme which apparently have different effector-binding components.     

An evolutionary conserved mechanism of T cell activation by microbial toxins. Evidence for different affinities of T cell receptor-toxin interaction.

The enterotoxins produced by Staphylococcus aureus are the most potent mitogens known. They belong to a group of distantly related mitogenic toxins that differ in other biologic activities. In this study we have compared the molecular mechanisms by which these mitogens activate human T lymphocytes. We used the staphylococcal enterotoxins A to E, the staphylococcal toxic shock syndrome toxin, the streptococcal erythrogenic toxins A and C (scarlet fever toxins, erythrogenic toxins (ET)A, ETC), and the soluble mitogen produced by Mycoplasma arthritidis. We found that all these toxins can activate both CD4+ and CD8+ T cells and require MHC class II expression on accessory and target cells. However, T cells could be activated in the absence of class II molecules if the toxins ETA or SEB were co-cross-linked on beads together with anti-CD8 or anti-CD2 antibodies. Enterotoxins, toxic shock syndrome toxin and scarlet toxins stimulate a major fraction of human T cells, and show preferential, but not exclusive, stimulation of T cells carrying certain TCR V beta. In contrast, the mitogen of M. arthritidis, a pathogen for rodents stimulates only a minority of human T cells but activates a major fraction of murine T cells. Analysis of human T cell clones expressing V beta 5 or V beta 8 TCR showed that these clones responded also to those toxins that did not stimulate V beta 5+ and V beta 8+ T cells in bulk cultures. These results indicate that different TCR bind to these toxins with different affinities and that the specificity of the TCR-V beta-toxin interaction is quantitative rather than qualitative in nature. Taken together our findings suggest that these toxins use a common mechanism of T cell activation. They are functionally bivalent proteins crosslinking MHC class II molecules with variable parts of the TCR. Besides V beta, other parts of the TCR must be involved in this binding. The finding that murine T cells responded more weakly to the toxins produced by the human-pathogenic bacteria than to the Mycoplasma mitogen could indicate that the toxins have been adapted to the host's immune system in evolution.     

FK506 binding protein associated with the calcium release channel (ryanodine receptor).

The calcium release channel (CRC)/ryanodine receptor (RyRec) has been identified as the foot structure of the sarcoplasmic reticulum (SR) and provides the pathway for calcium efflux required for excitation-contraction coupling in skeletal muscle. The CRC has previously been reported to consist of four identical 565-kDa protomers. We now report the identification of a 12-kDa protein which is tightly associated with highly purified RyRec from rabbit skeletal muscle SR. N-terminal amino acid sequencing and cDNA cloning demonstrates that the 12-kDa protein from fast twitch skeletal muscle is the binding protein for the immunosuppressant drug FK506. In humans, FK506 binds to the 12-kDa FK506-binding protein (FKBP12) and blocks calcium-dependent T cell activation. We find that FKBP12 and the RyRec are tightly associated in skeletal muscle SR on the basis of: 1) co-purification through sequential heparin-agarose, hydroxylapatite, and size exclusion chromatography columns; 2) coimmunoprecipitation of the RyRec and FKBP12 with anti-FKBP12 antibodies; and 3) subcellular localization of both proteins to the terminal cisternae of the SR, and not in the longitudinal tubules of SR, in fast twitch skeletal muscle. The molar ratio of FKBP12 to RyRec in highly purified RyRec preparations is approximately 1:4, indicating that one FKBP12 molecule is associated with each calcium release channel/foot structure.     

Molecular cloning and characterization of (R)-3-hydroxybutyrate dehydrogenase from human heart.

The complete amino acid sequence of human heart (R)-3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) has been deduced from the nucleotide sequence of cDNA clones. This mitochondrial enzyme has an absolute and specific requirement of phosphatidylcholine for enzymic activity (allosteric activator) and is an important prototype of lipid-requiring enzymes. Despite extensive studies, the primary sequence has not been available and is now reported. The mature form of the enzyme consists of 297 amino acids (predicted M(r) of 33,117), does not appear to contain any transmembrane helices, and is homologous with the family of short-chain alcohol dehydrogenases (SC-ADH) (Persson, B., Krook, M., and J?rnvall, H. (1991) Eur. J. Biochem. 200, 537-543) (30% residue identity with human 17 beta-hydroxysteroid dehydrogenase). The first two-thirds of the enzyme includes both putative coenzyme binding and active site conserved residues and exhibits a predicted secondary structure motif (alternating alpha-helices and beta-sheet) characteristic of SC-ADH. Bovine heart peptide sequences (174 residues in nine sequences determined by microsequencing) have extensive homology (89% identical residues) with the deduced human heart sequence. The C-terminal third (Asn-194 to Arg-297) shows little sequence homology with the SC-ADH and likely contains elements that determine the substrate specificity for the enzyme including the phospholipid (phosphatidylcholine) binding site(s). Northern blot analysis identifies a 1.3-kilobase mRNA encoding the enzyme in heart tissue.     

Retention of endocrine function by an insulin-secreting pancreatic islet cell tumour from Syrain hamster through serial transplantation in nude mice.

An insulin-secreting islet cell tumour of the Syrian hamster has been transplanted serially in the congenitally immune-deficient nude mouse, in order to test the potential usefulness of this mouse mutant as a graft carrier of heterologous tumours with stable differentiated phenotypes. The incidence of tumour growth was very high, and the hamster tumour retained its functional and histologic characteristics during consecutive passages in nude mice. These results show that nude mice may be useful carriers of differentiated tumours from non-inbred species including man, and for the isolation of cell lines from such tumours.