Determination of the kinetic parameters in continuous cultivation byDebaryomyces hansenii grown on D-xylose

Summary The kinetic parameters of the yeastDebaryomyces hansenii grown in continous cultivation on D-xylose were determined by different methods. While the values obtained for μ_m by the steady state and the washout methods only gave a 3% difference, the determined K_s values by the steady state and the maximal biomass output methods led a to a 305% difference. The latter method was suggested to overestimate the K_s value.     

Purification of cysteine proteinases from trichomonads using bacitracin-sepharose

Abstract Bacitracin affinity chromatography has been used to purify proteinases of the parasitic protozoon Tritrichomonas foetus . It proved superior to other affinity chromatography methods we have tested for the purification of trichomonad proteinases and should prove a useful procedure for purifying cysteine proteines from these parasites and other parasitic protozoa. The main cysteine proteinases of T. foetus were purified over 100-fold to be free from the majority of other cell proteins. About 90 μg of protein containing 1.56-fold more proteinase activity than was detectable in the original cell lysate was obtained from 10⁹ cells (7.2 mg protein). SDS-PAGE revealed that the eluate contained two main Coomassie blue-staining bands. N-terminal amino acid sequence analysis of these proteins confirmed that one of them was a cysteine proteinase with unusuall features. Cysteine proteinases were also purified from cell lysates of Trichomonas vaginalis and a N-terminal sequence determined. This is the first amino acid sequence information that has been obtained for trichomonad cysteine proteinases. The method was also used to purify proteinases from the medium of T. foetus cultures. Some selectivity in binding of the proteinases to the affinity column was found.     

Enzymology in vivo using NMR and molecular genetics

Models of metabolic flux regulation are frequently based on an extrapolation of the kinetic properties of enzymes measured in vitro to the intact cell. Such an extrapolation assumes a detailed knowledge of the intracellular environment of these enzymes in terms of their free substates and effectors concentrations and possible interaction with other cellular macromolecules, which may modify their kinetic properties. These is a considerable incentive, therefore, to study the properties of enzymes directly in vivo. We have been using non-invasive NMR techniques, in conjunction with molecular genetic manipulation of enzyme levels, to study the kinetic properties of individual enzymes in vivo. We have also developed a novel strategy which has allowed us to monitor, by NMR, the ligand binding properties and mobilities of enzymes in the intact cell. This technique may also allow us to measured the diffusion coefficients of these proteins in the cell. These studies should give new insight into the properties of enzymes in vivo     

Oxide-Based Solid-State Batteries: A Perspective on Composite Cathode Architecture

The garnet-type phase Li₇La₃Zr₂O₁₂ (LLZO) attracts significant attention as an oxide solid electrolyte to enable safe and robust solid-state batteries (SSBs) with potentially high energy density. However, while significant progress has been made in demonstrating compatibility with Li metal, integrating LLZO into composite cathodes remains a challenge. The current perspective focuses on the critical issues that need to be addressed to achieve the ultimate goal of an all-solid-state LLZO-based battery that delivers safety, durability, and pack-level performance characteristics that are unobtainable with state-of-the-art Li-ion batteries. This perspective complements existing reviews of solid/solid interfaces with more emphasis on understanding numerous homo- and heteroionic interfaces in a pure oxide-based SSB and the various phenomena that accompany the evolution of the chemical, electrochemical, structural, morphological, and mechanical properties of those interfaces during processing and operation. Finally, the insights gained from a comprehensive literature survey of LLZO–cathode interfaces are used to guide efforts for the development of LLZO-based SSBs.     

THE SARCOTUBULAR SYSTEM OF FROG SKELETAL MUSCLE : A Morphological and Biochemical Study

In the frog skeletal muscle cell a well defined and highly organized system of tubular elements is located in the sarcoplasm between the myofibrils. The sarcoplasmic component is called the sarcotubular system. By means of differential centrifugation it has been possible to isolate from the frog muscle homogenate a fraction composed of small vesicles, tubules, and particles. This fraction is without cytochrome oxidase activity, which is localized in the mitochondrial membranes. This indicates that the structural components of this fraction do not derive from the mitochondrial fragmentation, but probably from the sarcotubular system. This fraction, called sarcotubular fraction, has a Mg⁺⁺-stimulated ATPase activity which differs from that of muscle mitochondria in that it is 3 to 4 times higher on the protein basis as compared with the mitochondrial ATPase, and is inhibited by Ca⁺⁺ and by deoxycholate like the Kielley and Meyerhof ATPase. We therefore conclude that the "granules" of the Kielley and Meyerhof ATPase, which were shown to have a relaxing effect, are fragments of the sarcotubular system. The isolated sarcotubular fraction has a high RNA content and demonstrable activity in incorporating labeled amino acids, even in the absence of added supernatant.     

Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae. Roles of Arg234 and Arg242 revealed by sequence analysis and site-directed mutagenesis.

Glycogen phosphorylases (GPs) constitute a family of widely spread catabolic alpha1,4-glucosyltransferases that are active as dimers of two identical, pyridoxal 5'-phosphate-containing subunits. In GP from Corynebacterium callunae, physiological concentrations of phosphate are required to inhibit dissociation of protomers and cause a 100-fold increase in kinetic stability of the functional quarternary structure. To examine interactions involved in this large stabilization, we have cloned and sequenced the coding gene and have expressed fully active C. callunae GP in Escherichia coli. By comparing multiple sequence alignment to structure-function assignments for regulated and nonregulated GPs that are stable in the absence of phosphate, we have scrutinized the primary structure of C. callunae enzyme for sequence changes possibly related to phosphate-dependent dimer stability. Location of Arg234, Arg236, and Arg242 within the predicted subunit-to-subunit contact region made these residues primary candidates for site-directed mutagenesis. Individual Arg-->Ala mutants were purified and characterized using time-dependent denaturation assays in urea and at 45 degrees C. R234A and R242A are enzymatically active dimers and in the absence of added phosphate, they display a sixfold and fourfold greater kinetic stability of quarternary interactions than the wild-type, respectively. The stabilization by 10 mm of phosphate was, however, up to 20-fold greater in the wild-type than in the two mutants. The replacement of Arg236 by Ala was functionally silent under all conditions tested. Arg234 and Arg242 thus partially destabilize the C. callunae GP dimer structure, and phosphate binding causes a change of their tertiary or quartenary contacts, likely by an allosteric mechanism, which contributes to a reduced protomer dissociation rate.     

Alteration of tobacco floral organ identity by expression of combinations of Antirrhinum MADS-box genes

Floral organ identity is largely controlled by the spatially restricted expression of several MADS-box genes. In Antirrhinum majus these organ identity genes include DEF, GLO and PLE . Single and double mutant analyses indicated that the type of organ found in a particular whorl is dependent on which combination of these genes is expressed there. This paper reports the ectopic expression of Antirrhinum organ identity genes, alone and in combinations, in transgenic tobacco. Although the phenotypes are broadly in agreement with the genetic predictions, several unexpected features are observed which provide information concerning the action of the organ identity genes. The presumed tobacco homologue of DEF, NTDEF , has been isolated and used to investigate the influence of ectopic expression of the Antirrhinum organ identity genes on the endogenous tobacco genes. Analysis of the spatial and temporal expression patterns of NTDEF and NTGLO reveals that the boundaries are not coincident and that differences exist in the regulatory mechanisms of the two genes concerning both induction and maintenance of gene expression. Evidence is provided which indicates that organ development is sensitive to the relative levels of organ identity gene expression. Expression of the organ identity genes outside the flower or inflorescence produced no effects, suggesting that additional factors are required to mediate their activity. These results demonstrate that heterologous genes can be used to predictably influence floral organ identity but also reveal the existence of unsuspected control mechanisms.     

Sequence analysis of the complete mitochondrial DNA molecule of the hedgehog,Erinaceus europaeus,and the phylogenetic position of the Lipotyphla

The sequence of the mitochondrial DNA (mtDNA) molecule of the European hedgehog (Erinaceus europaeus) was determined. The length of the sequence presented is 17,442 nucleotides (nt). The molecule is thus the largest eutherian mtDNA molecule so far reported. The organization of the molecule conforms with that of other eutherians, but the control region of the molecule is exceptionally long, 1,988 nt, due to the presence of repeated motifs at two different positions in the 3 part of the control region. The length of the control region is not absolute due to pronounced heteroplasmy caused by variable numbers of the motif TACGCA in one of the repetitive regions. The sequence presented includes 46 repeats of this type. The other repeated region is composed of different AT-rich repeats. This region was identical among four clones studied. Comparison of mitochondrial peptide-coding genes identified a separate position of the hedgehog among several mammalian orders. The concatenated protein sequence of the 13 peptide-coding genes was used in a phylogenetic study using the opossum as outgroup. The position of the hedgehog sequence was basal among the other eutherian sequences included: human, rat, mouse, cow, blue whale, harbor seal, and horse. The analysis did not resolve the relationship among carnivores, perissodactyls, and artiodactyls/cetaceans, suggesting a closer relationship among these orders than acknowledged by classical approaches. Correspondence to: U. Arnason