Properties of hexokinase bound to glass

Résumé De l'hexokinase soluble a été liée et rendue insoluble par réaction avec une amine aryle derivée du verre poreux. L'enzyme garde sa fonction, mais avec une activité moindre et une constante de Michaelis différente. L'usage prolongé d'une colonne où l'enzyme est enrobé dans du verre montre que l'enzyme se sépare lentement du verre poreux.     

Semiquantitative estimation of phosphatase activity in the Sephadex layer

Zusammenfassung Rasche und einfache semiquantitative Methode zur Bestimmung der Aktivität von spezifischen Phosphatasen bei Reihenuntersuchungen: Detektion des Orthophosphats nach Diffusion des Enzyms durch eine Sephadex G-75-Schicht. Auf die Möglichkeiten ihrer Anwendung zur Bestimmung anderer Enzyme wird hingewiesen.

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The author is indebted to Mrs. J.Korytárová for technical assistance during this work.     

Myosin I: From yeast to human

Myosin I is a non-filamentous, single-headed, actin-binding motor protein and is present in a wide range of species from yeast to man. The role of these class I myosins have been studied extensively in simple eukaryotes, showing their role in diverse processes such as actin cytoskeleton organization, cell motility, and endocytosis. Recently, studies in metazoans have begun to reveal more specialized functions of myosin I. It will be a major challenge in the future to examine the physiological functions of each class I myosin in different cell types of metazoans.     

Chemical approaches to intermediates of enzyme catalysis

Zusammenfassung Die Untersuchung von Enzymsubstratkomplexen auf katalyse-induzierte Änderungen der chemischen Reaktivität hat sich als ein aufschlussreicher experimenteller Zugang zum Mechanismus enzymatischer Reaktionen erwiesen. Ternäre Systeme bestehend aus Enzym, Substrat und einem geeigneten Reagens (Tetranitromethan=TNM) sind auf katalyse-abhängige Reaktionen geprüft worden, die nur im vollständigen System, jedoch nicht mit Reagens und Enzym allein oder Reagens und Substrat allein beobachtet werden. Untersuchungen mit Aldolasen und Aspartat-Aminotransferase haben ergeben, dass katalyseinduzierte Reaktivität sowohl auf dem Substrat-als auch auf dem Enzymteil eines Enzym-Substratkomplexes nachgewiesen werden kann.Im Reaktionsmechanismus von Muskelaldolase (eine Lysinaldolase) und von Hefealdolase (eine Metallaldolase) lässt sich mit TNM ein intermediäres Carbanion des Substrats nachweisen. Die TNM-carbanion-Reaktion lässt sich spektralphotometrisch verfolgen und ist benutzt worden, um carboxypeptidase-behandelte Muskelaldolase und den Effekt von Cofaktoren auf die Hefealdolase zu untersuchen.Katalyse-induzierte Reaktivitätsveränderungen einer Enzymseitenkette sind bei der Aspartat-Aminotransferase beobachtet worden. Ein essentieller Tyrosylrest, der in der Abwesenheit von Substrat nicht mit TNM reagiert, wird ungewöhnlich reaktiv im Laufe der Katalyse und ermöglicht dabei seine selektive Nitrierung.Die katalyse-synchrone oder synkatalytische Aktivierung dieses Aminosäurerests scheint ein integraler Bestandteil des katalytischen Mechanismus von Aspartat-Aminotransferase zu sein und wird vermutlich durch katalyse-induzierte Konformationsänderungen des Enzym-Coenzym-Substratkomplexes hervorgebracht. Mögliche funktioneile Zusammenhänge synkatalytischer Reaktivitätsänderungen funktioneller Gruppen mit der Konformationsflexibilität des Enzymproteins und dem Vorkommen metastabiler Zwischenprodukte in der Enzymkatalyse werden diskutiert.     

Physiological functions of D-amino acid oxidases: from yeast to humans

D-Amino acid oxidase (DAAO) is a FAD-containing flavoenzyme that catalyzes the oxidative deamination of D-isomers of neutral and polar amino acids. This enzymatic activity has been identified in most eukaryotic organisms, the only exception being plants. In the various organisms in which it does occur, DAAO fulfills distinct physiological functions: from a catabolic role in yeast cells, which allows them to grow on D-amino acids as carbon and energy sources, to a regulatory role in the human brain, where it controls the levels of the neuromodulator D-serine. Since 1935, DAAO has been the object of an astonishing number of investigations and has become a model for the dehydrogenase-oxidase class of flavoproteins. Structural and functional studies have suggested that specific physiological functions are implemented through the use of different structural elements that control access to the active site and substrate/product exchange. Current research is attempting to delineate the regulation of DAAO functions in the contest of complex biochemical and physiological networks.     

How to halve ploidy: lessons from budding yeast meiosis

Maintenance of ploidy in sexually reproducing organisms requires a specialized form of cell division called meiosis that generates genetically diverse haploid gametes from diploid germ cells. Meiotic cells halve their ploidy by undergoing two rounds of nuclear division (meiosis I and II) after a single round of DNA replication. Research in Saccharomyces cerevisiae (budding yeast) has shown that four major deviations from the mitotic cell cycle during meiosis are essential for halving ploidy. The deviations are (1) formation of a link between homologous chromosomes by crossover, (2) monopolar attachment of sister kinetochores during meiosis I, (3) protection of centromeric cohesion during meiosis I, and (4) suppression of DNA replication following exit from meiosis I. In this review we present the current understanding of the above four processes in budding yeast and examine the possible conservation of molecular mechanisms from yeast to humans.     

New scanning electron microscopy contribution to the study of yeast protoplasts]

Study by scanning electron microscopy enables us to describe with precision the morphology of protoplasts. We can confirm that the wrinkles more or less perceptible on the protoplasts studied, do belong to the morphology of those just mentioned, that the presence or the absence of globulous component is connected with the physiological activity of the cell and that it is possible to distinguish the different protoplasts thanks to their morphological aspect.     

Vacuolar H+-ATPase: From mammals to yeast and back

Vacuolar H⁺-adenosine triphosphatase (V-ATPase) is composed of distinct catalytic (V₁) and membrane (V₀) sectors containing several subunits. The biochemistry of the enzyme was mainly studied in organelles from mammalian cells such as chromaffin granules and clathrin-coated vesicles. Subsequently, mammalian cDNAs and yeast genes encoding subunits of V-ATPase were cloned and sequenced. The sequence information revealed the relation between V- and F-ATPases that evolved from a common ancestor. The isolation of yeast genes encoding subunits of V-ATPase opened an avenue for molecular biology studies of the enzyme. Because V-ATPase is present in every known eukaryotic cell and provides energy for vital transport systems, it was anticipated that disruption of genes encoding V-ATPase subunits would be lethal. Fortunately, yeast cells can survive the absence of V-ATPase by drinking the acidic medium. So far only yeast cells have been shown to be viable without an active V-ATPase. In contrast to yeast, mammalian cells may have more than one gene encoding each of the subunits of the enzyme. Some of these genes encode tissue- and/or organelle-specific subunits. Expression of these specific cDNAs in yeast cells may reveal their unique functions in mammalian cells. Following the route from mammals to yeast and back may prove useful in the study of many other complicated processes.