The NADH-dehydrogenase subunit 5 gene in Oenothera mitochondria contains two introns and is co-transcribed with the 5 S rRNA gene

Summary The gene encoding subunit 5 of the NADH dehydrogenase complex (ND 5) has been identified in Oenothera mitochondria from a cDNA clone. The coding region is interrupted by a type II intron of 850 nucleotides and a second intervening sequence of 357 nucleotides. Genomic sequence rearrangement within the first intron creates a nontranscribed partial copy of the gene. The intact ND 5 gene is transcribed in a complex pattern with mRNAs including the 5 S rRNA sequence. Excision of the two introns appears to proceed slowly in vivo since the steady state mitochondrial RNA contains significant proportions of unprocessed precursor molecules.     

Determination of reaction volumes and polymer distribution characteristics of tobacco mosaic virus coat protein

A method that allows the quantitative determination of reaction volumes from sedimentation velocity experiments in an analytical ultracentrifuge is presented. Combined with a second method for detecting pressure-induced depolymerization, general characteristics of polymer distributions may be probed. We show that it is possible to determine if a sample is in an equilibrium or metastable state of subunit association. Our approach to probe macromolecular aggregation systems by small pressure perturbations is not restricted to the use of centrifuges. This method has been applied to characterize certain aspects of the polymerization of tobacco mosaic virus coat protein (TMVP). There are at least two helical polymer conformations in RNA-free coat protein rods. The smaller, helix I, polymers are limited to sizes below about 70 subunits (four to five helical turns) and undergo some kind of cooperative conformational change before further subunits may be added indefinitely. In contrast to helix I, the larger helix II polymers occur as broader and skewed size distributions. Under moderately strong polymerization conditions, the equilibrium state can contain both types of helical rods. The reaction volume for the addition of trimers is -220 ml/mol for both types of helical polymers. These results are compared with the results of previous thermodynamic analyses of TMVP polymerization.     

Structure and thermodynamics of the dihexadecylphosphatidylcholine-water system

X-ray small- and wide-angle diffraction, differential scanning calorimetry (DSC), temperature scanning densitometry (TSD) and electron microscopy were used to study the lyotropic and thermotropic properties of the system 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine-water over a wide range of compositions from the dry lipids to a large excess of water, and in the temperature range between 0 degrees C and 150 degrees C. The results were used to construct a temperature-composition phase diagram. The phases have been characterized with respect to their molecular arrangements and hydrocarbon chain packing subcells. In the fully hydrated state (greater than 45 wt% H2O) four thermotropic phases were found, with readily reversible transitions at 5 degrees C, 32.5 degrees C and 43.6 degrees C, respectively. The two lower temperature phases deviate from all others in consisting of bilayers with fully interdigitated hydrocarbon chains, while above 32.5 degrees C the structures resemble closely those of the analog diester lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). At hydrations between 30 and 45 wt% H2O, and below 32 degrees C, interdigitated and non-interdigitated multilayers coexist in one coherent phase. A bilayer tilting mechanism is proposed for the formation of this coexistence of two regular structures. Below 30 wt% H2O, hydrated 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) exists in lamellar, non-interdigitated bilayers, showing very weak interbilayer swelling. There, the water molecules appear to occupy voids between the polar headgroups.     

Plastid DNA in the mitochondrial genome of Oenothera: Intra- and interorganellar rearrangements involving part of the plastid ribosomal cistron

Summary Part of the plastid rRNA cistron is present in the mitochondrial genome of Oenothera. This sequence of 2081 nucleotides contains the 3 half of the plastid 23 S rRNA, the adjacent intergenic region and the 4.5 S rRNA. Secondary intramitochondrial sequence rearrangements involve this region of plastid origin and the gene encoding the putative mitochondrial small ribosomal protein S13. Sequence comparison suggests that the interorganellar transfer event occurred a long time ago. The mitochondrial sequence contains regions more homologous to the plastid DNA from tobacco than from Oenothera itself in the regions analysed, suggesting faster sequence evolution in plastids than in mitochondria of Oenothera.     

Signal storage in phytochrome action on nitrate-mediated induction of nitrate and nitrite reductases in mustard seedling cotyledons

Application of nitrate leads to an induction of nitrate reductase (NR; EC 1.6.6.1) and nitrite reductase (NIR; EC 1.7.7.1) in the cotyledons of dark-grown mustard (Sinapis alba L.) seedlings, and this induction can strongly be promoted by a far-red-light pretreatment — operating through phytochrome — prior to nitrate application. This light treatment is almost ineffective — as far as enzyme appearance is concerned — if no nitrate is given. When nitrate is applied, the stored light signal potentiates the appearance of NR and NIR in darkness, even in the absence of active phytochrome, to the same extent as continuous far-red light. This action of previously stored light signal lasts for approx. 12 h.Storage of the light signal was measured for NR and NIR. The process shows enzyme-specific differences. Storage occurs in the absence as well as in the presence of nitrate, i.e. irrespective of whether or not enzyme synthesis takes place. The kinetics of signal transduction and signal storage indicate that the formation and action of the stored signal are a bypass to the process of direct signal transduction. Signal storage is possibly a means of enabling the plant to maintain the appropriate levels of NR and NIR during the dark period of the natural light/dark cycle.Abbreviations cD continuous darkness - cFR continuous far-red light - D darkness - FR far-red light - NIR nitrite reductase (EC 1.7.7.1) - NR nitrate reductase (EC 1.6.6.1) - Pfr phytochrome (far-red absorbing) - Pr phytochrome (red absorbing) - R red light - RG9-light long wavelength far-red light obtained with RG9 glass filter - - Ptot total phytochrome (Pr+Pfr) Professor Wilhelm Nultsch mit guten Wünschen zum 60. Geburtstag     

Systematic investigation of degradation effects on spin-lattice relaxation times in mouse-liver by low resolution NMR

In spite of numerous work on in vitro proton relaxation time investigations of biological tissue, many questions still remain open. In this study we focused on spin-lattice (T1) relaxation time measurements of mouse liver tissue in order to estimate the time-after-excision effects. The post mortem behaviour of excised tissue was measured up to four hours in intervals of about nine minutes. The time course of liver T1 was determined for different temperatures (4 degrees-40 degrees C) for female mice and the effect of starvation (up to 48 hours) on the time course of T1 was investigated for male and female mice at 37 degrees C. We obtained significant differences in liver T1 time course after excision due to different physiological states like sex, starvation and circadian rhythm.     

Rat liver 4-hydroxy-2-ketoglutarate aldolase: purification and kinetic characterization

The enzyme 4-hydroxy-2-ketoglutarate aldolase (4HKG aldolase), which catalyzes the reversible cleavage of 4-hydroxy-2-ketoglutarate to form pyruvate and glyoxylate, was isolated from rat liver. The purification scheme as well as a study of several of the physical and kinetic properties of the enzyme are presented. The effects of anions, various buffers, and possible physiologically relevant effectors on the kinetic parameters of the aldolase were also investigated. It was found that pyruvate analogs inhibited the aldolase. Oxaloacetate was a competitive inhibitor of the aldolase, and in addition caused synergistic inhibition with respect to pyruvate analogs at low substrate concentration. These results are discussed in terms of possible regulation of the aldolase.     

Metal-nucleotide structural characteristics during catalysis by beef heart mitochondrial F1

This study examined the nature of the metal-nucleotide complexes which serve as substrates, products, and intermediates in the beef heart mitochondrial ATPase reaction. The two methods employed involved the use of phosphorothioate ATP analogs as substrates in the presence of Mg2+ or Cd2+ and the use of substitution inert Cr X ATP complexes (the isolated diastereomers of the bidentate complexes) along with the newly synthesized Cr X ITP complexes as inhibitors of both the F1-ATPase and F1-ITPase activities. Little stereoselectivity was observed in the inhibition of F1-ATPase and F1-ITPase activities by the isolated diastereomers of beta,gamma-bidentate CrATP, while the inhibition by the delta,alpha,beta-bidentate CrADP diastereomer was greater than that of the lambda epimer. gamma-Monodentate CrITP was a weak inhibitor of both the ATPase and ITPase activities, whereas beta,gamma-bidentate CrITP failed to show any inhibition at all up to a concentration of 3.2 mM. When adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) was used as the substrate, (VmSp]/(Vm(Rp] with Mg2+ present was 2.7 at 31 degrees C and 3.5 at 13 degrees C. The (Vm/Km(Sp]/(Vm/Km(Rp] ratios with Mg2+ present were 15.3 at 31 degrees C and 73.3 at 13 degrees C. With Cd2+ present, the (Vm(Sp]/(Vm(Rp] ratios were 0.81 and 0.65 at 31 and 13 degrees C, respectively. The (Vm/Km(Sp]/(Vm/Km(Rp] ratios with Cd2+ present were 1.17 at 31 degrees C and 1.34 at 13 degrees C. The large activation energy observed for the isomers of CdATP beta S was not observed for MgATP beta S, MgATP, or CdATP. The Vm for Cd adenosine 5'-O-thiotriphosphate (ATP gamma S) hydrolysis was the largest of all the metal-phosphorothioate nucleotide complexes, while that for MgATP gamma S was the smallest. The results are interpreted in terms of a catalytic model for F1-catalyzed nucleotide hydrolysis describing metal-nucleotide chelation during the reaction.