Pressure inactivation of tetrameric lactate dehydrogenase homologues of confamilial deep-living fishes

Summary The susceptibility to inactivation by hydrostatic pressure of the tetrameric (Fig. 1) muscletype (M₄) lactate dehydrogenase homologues (LDH, EC 1.1.1.27;l-lactate: NAD⁺ oxidoreductase) from six confamilial macrourid fishes was compared at 4 °C. These marine teleost fishes occur over depths of 260 to 4815 m. The pressures necessary to half-inactivate the LDH homologues are related to the pressures which the enzymes are exposed to in vivo (Table 1); higher hydrostatic pressures are required to inactivate the LDH homologues of the deeper-occurring macrourids. The resistance of the LDH homologues to inactivation by pressure is affected by protein concentration (Fig. 3). After an hour of incubation at pressure, the percent remaining activity approaches an asymptotic value (Fig. 2). The inactivation of the macrourid LDH homologues by pressure was not fully reversible. Assuming that inactivation by pressure was due to dissociation of the native tetramer to monomers, apparent equilibrium constants (K _eq) were calculated. Volume changes (V) were calculated over the range of pressures for which plots inK _eq versus pressure were linear (Fig. 4). The V of dissociation values of the macrourid homologues range from –219 to –439 ml mol^–1 (Table 1). Although the hydrostatic pressures required to inactivate the LDH homologues of the macrourid fishes are greater than those which the enzymes are exposed to in vivo, the pressure-stability of these enzymes may reflect the resistance of these enzymes to pressure-enhanced proteolysis in vivo.     

Identification of novel rhodopsin mutations responsible for retinitis pigmentosa: Implications for the structure and function of rhodopsin

Ten rhodopsin mutations have been found in a screen of 282 subjects with retinitis pigmentosa (RP), 76 subjects with Leber congenital amaurosis, and 3 subjects with congenital stationary night blindness. Eight of these mutations (gly⁵¹-to-ala, val¹⁰⁴-to-ile, gly¹⁰⁶-to-arg, arg¹³⁵-to-gly, cys¹⁴⁰-to-ser, gly¹⁸⁸-to-glu, val²⁰⁹-to-met, and his²¹¹-to-arg) produce amino acid substitutions, one (gln⁶⁴-to-ter) introduces a stop codon, and one changes a guanosine in the intron 4 consensus splice donor sequence to thymidine. Cosegregation of RP with gln⁶⁴-to-ter, gly¹⁰⁶-to-arg, arg¹³⁵-to-gly, cys¹⁴⁰-to-ser, gly¹⁸⁸-to-glu, his²¹¹-to-arg, and the splice site guanosine-to-thymidine indicates that these mutations are likely to cause retinal disease. Val¹⁰⁴-to-ile does not cosegregate and is therefore unlikely to be related to retinal disease. The relevance of gly⁵¹-to-ala and val²⁰⁹-to-met remains to be determined. The finding of gln⁶⁴-to-ter in a family with autosomal dominant RP is in contrast to a recent report of a recessive disease phenotype associated with the rhodopsin mutation glu²⁴⁹-to-ter. In the present screen, all of the mutations that cosegregate with retinal disease were found among patients with RP. The mutations described here bring to 35 the total number of amino acid substitutions identified thus far in rhodopsin that are associated with RP. The distribution of the substitutions along the polypeptide chain is significantly nonrandom: 63% of the substitutions involve those 19% of amino acids that are identical among vertebrate visual pigments sequenced to date.     

Molecular evolution of the ZFY and ZNF6 gene families

Members of the ZFY and ZNF6 gene families have been cloned from species representing different taxa and different modes of sex determination. Comparisons of these genes show the ZFY-like and ZNF6 sequences to be strongly conserved across marsupials, birds, and lepidosaurians. Sequence analyzed by neighbor-joining indicated that both gene families are monophyletic with a high bootstrap value. Pairing of sequences from males and females of nonmammalian species showed there to be no significant difference between male and female sequences from a single species, consistent with autosomal locations. The molecular distances between murine Zfy-1, Zfy-2, and other ZFY-like sequences suggested that Zfy genes have undergone a period of rapid evolutionary change not seen in human ZFY.      

Purification and Characterization of a Novel Chemorepellent Receptor from Tetrahymena thermophila

Chemosensory transduction and adaptation are important aspects of signal transduction mechanisms in many cell types, ranging from prokaryotes to differentiated tissues such as neurons. The eukaryotic ciliated protozoan, Tetrahymena thermophila, is capable of responding to both chemoattractants (O'Neill et al., 1985; Leick, 1992; Kohidai, Karsa & Csaba, 1994, 1995) and chemorepellents (Francis & Hennessey, 1995; Kuruvilla, Kim & Hennessey, 1997). An example of a nontoxic, depolarizing chemorepellent in Tetrahymena is extracellular lysozyme (Francis & Hennessey, 1995; Hennessey, Kim & Satir, 1995). Lysozyme is an effective chemorepellent at micromolar concentrations, binds to a single class of externally facing membrane receptors and prolonged exposure (10 min) produces specific chemosensory adaptation (Kuruvilla et al., 1997). We now show that this lysozyme response is initiated by a depolarizing chemoreceptor potential in Tetrahymena and we have purified the membrane lysozyme receptor by affinity chromatography of solubilized Tetrahymena membrane proteins. The solubilized, purified protein is 42 kD and it exhibits saturable, high affinity lysozyme binding. Polyclonal antibodies raised against this 42 kD receptor block the in vivo lysozyme chemoresponse. This is not only the first time that a chemoreceptor potential has been recorded from Tetrahymena but also the first time that a chemorepellent receptor has been purified from any unicellular eukaryote. Received: 28 July 1997/Revised: 14 November 1997     

Organization and nucleotide sequence of ten ribosomal protein genes from the region equivalent to the spectinomycin operon in the archaebacterium Halobacterium marismortui.

Summary We have created missense mutations in the indirect flight muscle (IFM)-specific Act88F actin gene of Drosophila melanogaster by random in vitro mutagenesis. Following P element-mediated transformation into wild-type flies and subsequent transfer of the inserts into Act88F null strains, the effects of the actin mutants on the structure and function of the IFMs were examined. All of the mutants were antimorphic for flight ability. E316K and G368E formed muscle with only relatively small defects in structure whilst the others produced IFMs with large amounts of disruption. E334K formed filaments but lacked Z discs. V339I formed no muscle structure in null flies and did not accumulate actin. E364K and G366D both had relatively stable actin but did not form myofibrils. Using an in vitro polymerisation assay we found no significant effects on the ability of the mutant actins to polymerise. E364K and G366D also caused a strong induction of heat shock protein (hsp) synthesis at normal temperatures and accumulated large amounts of hsp22 which, together with the mutant actin, was resistant to detergent extraction. Both E316K and E334K caused a weak induction of hsp synthesis. We discuss how the stability, structure and function of the different mutant actins affects myofibril assembly and function, and the induction of hsps.     

Circadian Rhythms in Photosynthesis : Oscillations in Carbon Assimilation and Stomatal Conductance under Constant Conditions

Net carbon assimilation and stomatal conductance to water vapor oscillated repeatedly in red kidney bean, Phaseolus vulgaris L., plants transferred from a natural photoperiod to constant light. In a gas exchange system with automatic regulation of selected environmental and physiological variables, assimilation and conductance oscillated with a free-running period of approximately 24.5 hours. The rhythms in carbon assimilation and stomatal conductance were closely coupled and persisted for more than a week under constant conditions. A rhythm in assimilation occurred when either ambient or intercellular CO₂ partial pressure was held constant, demonstrating that the rhythm in assimilation was not entirely the result of stomatal effects on CO₂ diffusion. Rhythms in assimilation and conductance were not expressed in plants grown under constant light at a constant temperature, demonstrating that the rhythms did not occur spontaneously but were induced by an external stimulus. In plants grown under constant light with a temperature cycle, a rhythm was entrained in stomatal conductance but not in carbon assimilation, indicating that the oscillators driving the rhythms differed in their sensitivity to environmental stimuli.     

Identification of the membrane-embedded regions of the Neurospora crassa plasma membrane H(+)-ATPase.

Reconstituted proteoliposomes containing functional Neurospora crassa plasma membrane H(+)-ATPase molecules oriented predominantly with their cytoplasmic surface exposed were treated with trypsin and then subjected to Sepharose CL-6B column chromatography to remove the liberated peptides. The peptides remaining associated with the liposomes were then separated from the phospholipid by Sephadex LH-60 column chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six H(+)-ATPase peptides with approximate molecular masses of 7, 7.5, 8, 10, 14, and 21 kDa were found to be tightly associated with the liposomal membrane. Amino acid sequencing of the 7-, 7.5-, and 21-kDa peptides in the LH-60 eluate identified them as H(+)-ATPase fragments beginning at residues 99 or 100, 272, and 660, respectively. After further purification, the approximately 10- and 14-kDa peptides were also similarly identified as beginning at residues 272 and 660. The approximately 8-kDa fragment was purified further but could not be sequenced, presumably indicating NH2-terminal blockage. To identify which of the liposome-associated peptides are embedded in the membrane, H(+)-ATPase molecules in the proteoliposomes were labeled from the hydrophobic membrane interior with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine and cleaved with trypsin, after which the membrane-associated peptides were purified and assessed for the presence of label. The results indicate that the approximately 7-, 7.5-, and 21-kDa peptides are in contact with the lipid bilayer whereas the approximately 8-kDa peptide is not. Taken together with the results of our recent analyses of the peptides released from the proteoliposomes, this information establishes the transmembrane topography of nearly all of the 919 residues in the H(+)-ATPase molecule.     

<Emphasis Type="Italic">Hilara</Emphasis> sp. (Diptera: Empididae; Empidinae): Mating System,Swarm Movements,and Inbreeding Avoidance

In a study spanning parts of nine years, an undescribed species of Hilara Meigen was observed to form mating swarms displaying complex behaviors. Typically, swarms were shaped like a flattened torus rotating rapidly about a horizontal axis. Many swarms also moved up and down and turned slowly back-and-forth about a vertical axis. Both up-and-down and turning movements were random in extent and direction, suggesting that they might arise as random, asymmetric density fluctuations within the swarms themselves. A rotating secondary swarm appeared intermittently inside one end of some primary swarms. Swarm membership changed continually as flies left one swarm to join another and as entire swarms coalesced. At one site the set of all swarms displayed properties not found in the swarms individually: spatial extension, daily dissipation and reconstitution over a period of weeks or months, reproductive potential, and gene flow. Such emergent properties qualify the set as a multicomponent swarm, an object heretofore known only in computer models. Hilara sp. appears to be protandrous, univoltine, and promiscuous. Generally, males paired preferentially with somewhat smaller females, but some small and medium-sized males paired with much larger females. Although males of nearly all known Hilara species present nuptial gifts of prey or other items to females, nuptial gifts were not observed at any time during the present study. Many characteristics of swarms of Hilara sp. can be understood as adaptations that reduce inbreeding.     

Experimental errors and their effect on analyzing circular dichroism spectra of proteins

Seven types of error that may interfere with the analysis of protein circular dichroism (CD) spectra for secondary structure are examined. Three of these errors are operational encompassing wavelength synchronization, and proper choice of spectral bandwidth and scan speed. Three are experimental involving intensity adjustments and two sources of baseline shift. The skew baseline shift is analogous to error in CD intensity at short wavelengths due to high sample absorption and low source intensity. The final source of error deals with constrained analyses. We have investigated these types of error to determine how they may be affecting our analysis of protein CD spectra and the role they may play in causing our analyses to fail for some proteins. We find that small errors in the baseline (which are independent of the protein spectrum) will rationalize our poor analyses. Spectroscopists must adopt new standards of precision if sophisticated analyses are to succeed.