Karl Brand

Mixed function oxidation of hexobarbital and generation of NADPH by the hexose monophosphate shunt in isolated rat liver cells

Abstract Mixed function oxidation of hexobarbital and the generation of NADPH by the hexose monophosphate shunt were studied in isolated rat liver parenchymal cells from phenobarbital-pretreated and untreated animals. In cells isolated from untreated rats, a maximal rate of hexobarbital oxidation of 17 μmol·g−1 liver wet weight·(60 min)−1 was observed, while in cells isolated from phenobarbital-pretreated rats a maximal rate of 29 μmol·g−1 liver wet weight·(60 min)−1 has been obtained. On the basis of the specific radioactivity at carbon atom 1 of glucose 6-phosphate, fructose 6-phosphate and 6-phosphogluconate, determined by enzymatic decarboxylation, a ratio between NADPH formation via the hexose monophosphate shunt and NADH utilization for hexobarbital oxidation of 6:1 in untreated and 9.5:1 in pretreated cells has been obtained. With phenazine methosulfate the stimulation of NADPH generation via the hexose monophosphate shunt exceeded that observed in the presence of hexobarbital by 329 and 160%, respectively, indicating that the capacity of this pathway is sufficient to provide more reducing equivalents than are required for maximal rates of mixed function oxidation.

ENZYME AND METABOLITE PROFILES

Publisher Summary This chapter discusses enzyme and metabolite profiles under steady state and transient conditions in living cells and in model systems. For a number of years, this study has directed attention to newer systems of metabolic control that have greatly clarified concepts of the operation of metabolic pathways. The concentrations of enzymes are controlled by induction, repression, and derepression devices, which involve the operation of genetically controlled protein synthesis. Whereas the mechanism of control of enzyme concentration is only known to a rather superficial extent, the operation of activity control is far better established and can be reasonably approached by means of computer models. Enzyme profiles may not be used for a functional definition of the metabolic state. Enzyme activities are governed by a variety of control mechanisms that can best be recognized by steady state and transient state analyses of metabolites and by analysis of the systems response in titration experiments with pure enzymes under conditions in which the system displays an oscillatory behavior of its overall flux.

Origin of hydrogen required for fatty acid synthesis in isolated rat adipocytes

Abstract Isolated rat adipocytes were incubated in parallel either with [U-14C]-, [1-14C]-, or [6-14C]glucose and unlabeled citrate or with [6-14C]- or [1,5-14C]citrate and unlabeled glucose. Glucose uptake, lactate and pyruvate release, and 14C-incorporation into CO2, fatty acids, glyceride glycerol, and lactate were determined. The results obtained with variously labeled 14C-glucose revealed that the total amount of cytosolic NADH and NADPH was only in slight excess of the hydrogen equivalents required for the synthesis of fatty acids, glyceride glycerol, and lactate. The pentose phosphate pathway was found to provide only about 50–80% of the NADPH required for fatty acid synthesis, indicating, that part of NADPH is generated by transhydrogenation from cytosolic NADH. The isotopic yields of products from variously labeled 14C-citrate were in accord with an operating citrate malate pyruvate-cycle supplying the remaining NADPH required for fatty acid synthesis via malic enzyme. The ratios of the 14C-yields of lactate indicate a randomization of label at the dicarboxylic acid level which is considered as a consequence of an operating malate shuttle.

Untersuchungen über die Verträglichkeit und biologische Wertigkeit von parenteral verabreichten Aminosäure-Mustern

ZusammenfassungFolgende 3 Aminosäure-Muster wurden als Infusionslösungen auf ihre Verträglichkeit und biologische Wertigkeit an 3 gesunden Versuchs-personen untersucht: 1. das sog. „Rose-Muster“ (R), 2. das Muster nach Knauff (KB) und 3. ein Muster, das einer bestimmten Kartoffel-Ei-Kombination (EK) entspricht. Bei allen Versuchen bleiben die klinisch-chemischen Werte während der gesamten Versuchsdauer im Normalbereich. Die biologische Wertigkeit des „Rose-Musters“ ist die niedrigste, die der beiden anderen Muster nahezu gleich. Hinsichtlich ihrer Verträglichkeit und biologischen Wertigkeit sind die Aminosäure-Muster KB und EK für die parenterale Ernährung am geeignetsten.SummaryThe following 3 amino-acid-patterns are used for parenteral infusion and their compatibility and biological values were studied on 3 healthy test persons: 1. the so called Rose-pattern (R), 2. the Knauff-Braun-pattern (KB), and 3. a pattern corresponding to a definite mixture of potatoe and egg (EK). In all experiments the results of the clinical-chemical tests during the whole period of application were within normal range. The biological value of the “Rose-pattern” was found to be lower than that of the two other amino acid-patterns, which were almost equal. With respect to their compatibility and biological value the aminoacid-pattern KB and EK are proper to parenteral nutrition.

Active center and subunit structure of transaldolase from Candida utilis

Abstract Crystalline transaldolase (type III) isolated from Candida utilis is composed of two identical subunits, as shown by the following lines of evidence. 1. Tryptic digestion of the performic acid oxidized enzyme yields the number of ninhydrin- and arginine-positive peptides expected for identical subunits. 2. All attempts to separate both subunits by molecular weight or charge differences have failed. 3. Cyanogen bromide cleavage and sodium dodecyl sulfate gel electrophoresis of S -carboxymethylated transaldolase revealed four distinct peptides designated C 2 to C 5 according to their decreasing molecular weight and one additional peak, C 1 , in low yield, presumably an aggregate or partially degraded peptide. By chromatography on Sephadex G-100 the maleylated cyanogen bromide digest from 14 C-labeled β-giyceryl-transaldolase could be separated into four peptide peaks which have been analyzed for their amino acid composition. The largest peptide C 2 with a molecular weight of 16,800 was identified as the active site containing fragment. The four fragments together account for all amino acid residues in the entire protein. From transaldolase (type I) containing four methionine residues three cyanogen bromide peptides could be identified. By addition of the individual peptides a molecular weight of 37,100 ± 3500 could be calculated, which is half the molecular weight of the native enzyme. From experimental data presented so far both isoenzymes of transaldolase can be regarded as “half-of-the-sites” enzymes.

Substrate-dependent inactivation of transaldolase activity with tetranitromethane

Abstract Transaldolase (Type III) from Candida utilis was found to be inactivated by tetranitromethane only in the presence of the substrates fructose 6-phosphate and sedoheptulose 7-phosphate. This reaction was prevented by the addition of erythrose 4-phosphate or glyceraldehyde 3-phosphate, which are known to accept dihydroxyacetone from the transaldolase-dihydroxyacetone complex, releasing free transaldolase. These results strongly suggest that tetranitromethane does not react with free transaldolase but only with the Schiff-base intermediate. After 1 min of incubation with the reagent at pH 6.0, 4 moles of nitroformate were produced per mole of inactivated enzyme. The modification, probably a nitration or an oxidation of certain amino acid residues of the complex by tetranitromethane, caused a dissociation of the dihydroxyacetone moiety from the complex without any recovery of the enzymatic activity. The fact that the reaction with tetranitromethane takes place only in the presence of substrates indicates that a substrate-mediated change of conformation occurs in transaldolase. Chemical and spectrophotometric evidence is presented showing that tetranitromethane did not modify tyrosine, cysteine, and tryptophan residues in the inactivated enzyme. From amino acid analyses it appears that histidine, serine, proline, methionine, tyrosine, and phenylalanine residues were not altered by this reagent. The possible mechanisms of modification of the transaldolasedihydroxyacetone complex and the chemical nature of the modification by tetranitromethane are discussed.

Grundlagen der Bestimmung von Enzymaktivitten

ZusammenfassungDie optische Methode von Otto Warburg sowie andere physikalische Meßverfahren wie die Fluorimetrie, die Polarimetrie, die Titrimetrie bei stationäremph (ph-Stat) und andere Methoden werden heute bevorzugt zur Aktivitätsbestimmung von Enzymen herangezogen. Diese Verfahren, durch Anschluß registrierender Schreiber erleichtert, haben gegenüber den klassischen chemisch-analytischen Verfahren den Vorteil einer fortlaufenden Darstellung des Reaktionsablaufes und gestatten eine einfache Ablesung der Aktivität eines Enzyms als Maß seiner katalytischen Wirksamkeit.Die Anwendung direkter Meßverfahren wurde von Warburg erweitert durch die Darstellung und Verwendung hochgereinigter, kristallisierter Enzyme, die in zusammengesetzten Tests zur chemischen Kopplung eines Enzyms unbekannter Aktivität an ein sogenanntes Indicator-Enzym eingesetzt werden, dessen Aktivität direkt gemessen werden kann. Durch Einschaltung eines weiteren Hilfsenzyms kann schließlich die analytische Enzymkette verdreifacht werden.Grundbedingung der Aktivitätsbestimmung ist die Proportionalität von Reaktionsgeschwindigkeit und Enzymkonzentration. Sie wird nach der Theorie von Michaelis und Menten entweder bei Substratüberschuß oder bei sehr kleinen Substratkonzentrationen, oder empirisch, unabhängig von dem Ordnungstyp der Reaktion, durch Zeitmessung eines festgelegten Substratumsatzes gefunden. Die enzymatische Aktivität wird für definierte Standardbedingungen in internationalen Einheiten ausgedrückt. Die internationale Einheit ist als die Enzym-Menge definiert, die die Umwandlung von 1 μMol Substrat oder 1 μÄquivalent einer reagierenden Gruppe oder gespaltenen Bindung bzw. das Auftreten von 1 μMol Produkt resp. μÄquivalent unter Standardbedingungen je Minute katalysiert.SummaryThe optical method of Otto Warburg as well as other physical methods like fluorimetry, polarimetry,ph-stat-titrimetry among others, are nowadays preferred for activity determination of enzymes. These procedures which can be facilitated by direct recording have the advantage of a continuous registration of a course of a reaction and a direct determination of enzyme activities.The application of such direct physical methods has been extended by Warburg by the preparation and application of pure enzymes, which can be used in coupled tests for a chemical coupling of an enzyme of unknown activity with a so called indicator enzyme, the activity of which can directly be measured. By use of another accessory enzyme, a three-fold analytical enzyme sequence can be developed.The theoretical basis for enzyme determination is the proportionality of reaction rate and enzyme concentration. According to the theory of Michaelis and Menten, proportionality can be found either with optimal substrate concentration, or with very small substrate levels, and furthermore, empirically by time measurement of a fixed substrate utilization. — The activity of enzymes is expressed for standard conditions in international units. An international unit is the amount of enzyme which catalyzes the reaction of 1 μMole substrate or 1 μEquivalent of a reacting group or split bondage, resp. the production of 1μMole product per minute.

Der Anteil der Glykolyse und des Pentosephosphat-Weges am Glucoseumsatz in normalen und in Glucose-6-phosphat-Dehydrogenase-Mangel-Erythrocyten

This has been measured by the glucose uptake, the formation of lactate and pyruvate, the change in the concentration of 2,3-diphosphoglyceric acid and the release of 14CO2 from 14C-1-, 14C-6-, and 14C-uniformly labelled d-glucose.In normal cells at pH 7.6 about 7% of the glucose consumed has been metabolized via the oxidative pentose phosphate pathway, 93% via the glycolytic pathway to lactate. At pH 6.8 glucose metabolism to lactate and pyruvate is diminished by about 45% compared to that at pH 7.6. The amount of the pentose phosphate pathway is increased to 13%. Thus, the synthesis of NADPH is almost equal at both pH-ranges. In the presence of methylene-blue glucose is metabolized almost completely via the oxidative pathway. There is no significant recycling of glucose metabolites in the pentose phosphate pathway.In glucose-6-phosphate dehydrogenase deficient erythrocytes the contribution of the pentose phosphate pathway to glucose metabolism at pH 7.6 is only 1.5%, at pH 6.8 5%. In this pH-range, however, the glucose uptake is markedly decreased. The extent of NADPH synthesis therefore at both pH-ranges is again almost the same. In the presence of methylene-blue glucose metabolism via the pentose phosphate pathway can be stimulated only to a very small extent from 1.5% to 2.5%. The capacity of the glucose-6-phosphate dehydrogenase system in the deficient cells is by a factor of 60 smaller than that of the normal cells.ZusammenfassungDer quantitative Anteil der Glykolyse und des Pentosephosphatweges am Glucoseumsatz in normalen Erythrocyten und solchen, die einen Mangel an Glucose-6-phosphat-Dehydrogenase haben, wurde durch Bestimmung des Glucoseverbrauchs, der Lactat- und Pyruvatbildung, der Änderung des 2,3-Diphosphoglycerinsäurespiegels sowie der 14CO2-Freisetzung aus 14C-1-, 14C-6- und 14C-uniform-markierter Glucose ermittelt. In normalen Zellen werden bei pH 7,6 etwa 7% der umgesetzten Glucose über den Pentosephosphatweg, 93% über die Glykolyse zu Lactat verstoffwechselt. Bei pH 6,8 ist der Glucoseumsatz zu Pyruvat und Lactat um etwa 45% geringer als bei pH 7,6. Der Anteil des Pentosephosphatweges an diesem Umsatz ist jedoch kompensatorisch auf 13% erhöht. Die Menge des durch den oxydativen Glucoseabbau mittels der 2 Dehydrogenasereaktionen synthetisierten NADPH ist in beiden pH-Bereichen annähernd gleich. In Anwesenheit von Methylenblau wird Glucose fast vollständig oxydativ abgebaut. Eine nennenswerte Recyclisierung innerhalb des Pentosephosphatweges findet in normalen Erythrocyten nicht statt. In Erythrocyten mit einem Mangel an Glucose-6-phosphat-Dehydrogenase ist der Anteil des Pentosephosphatweges am Glucoseumsatz bei pH 7,6 nur 1,5%. Bei pH 6,8 erhöht sich dieser Anteil auf 5%, doch ist der Glucoseverbrauch entsprechend reduziert. Der glykolytische Glucosestoffwechsel in den Mangelzellen ist um etwa 20% geringer als in den normalen Zellen. Auch hier wird in beiden pH-Bereichen eine annähernd gleiche Menge NADPH synthetisiert. In Anwesenheit von Methylenblau kann in den Mangelerythrocyten der Anteil des Pentosephosphatweges am Glucoseumsatz nur unwesentlich von 1,5 auf 2,5% stimuliert werden. Die Kapazität des Glucose-6-phosphat-Dehydrogenase-Systems ist in den Mangelzellen um einen Faktor von 60 gegenüber der in normalen Zellen vermindert.

Methoden zur quantitativen Bestimmung des Enzym-Substratkomplexes der Transaldolase

AbstractTransaldolase reacts with fructose-6-phosphate to form a stable transaldolase-dihydroxyacetone complex. This complex intermediate has the structure of a Schiff base. Two methods are described for the quantitative determination of this complex:1.Reduction of the enzymatically active Schiff base with borohydride to an inactive secondary amine (irreversible).2.Addition of cyanide to the Schiff base intermediate forming an inactive aminonitrile derivate (reversible). The amount of the complex present can be calculated by the percentage of inactivation of the original enzyme activity.The number of active sites of transaldolase can be evaluated by measuring the specific incorporation of radioactive compounds with known specific activity into the complex intermediate. With two different methods the number of combining sites of transaldolase from Candida utilis for fructose-6-phosphate has been found to be one.ZusammenfassungTransaldolase reagiert mit Fructose-6-phosphat unter Bildung eines stabilen Dihydroxyaceton-Enzym-Komplexes, der die Struktur einer Schiffsehen Base hat. Für die quantitative Bestimmung dieses Komplexes werden zwei Methoden beschrieben:1.Reduktion der enzymatisch aktiven Schiffsehen Base mit Borhydrid zu einem inaktiven sekundären Aminderivat (irreversibel).2.Addition von Cyanid an die Schiffsche Base unter Bildung eines inaktiven Aminonitrilderivates (reversibel).Aus dem prozentualen Anteil der Inaktivierung läßt sich die vorliegende Menge an Komplex errechnen. Die Zahl der „aktiven Zentren“ der Transaldolase kann aufgrund des spezifischen Einbaus radioaktiver Stoffe in den Komplex ermittelt werden. Mit zwei verschiedenen Methoden konnte übereinstimmend jeweils nur eine Substratbindungsstelle für Fructose-6-phosphat bei der Transaldolase aus Candida utilis nachgewiesen werden.

Kontrollmechanismen der Glycolyse

Control mechanisms of enzymic reactions are generally based on interactions between activators, inhibitors, substrates and products with enzyme proteins or on induction and repression of enzyme synthesis. All main types of control can be recognized in glycolysis. They are the basis of the network which controls the over-all glycolytic function and operates according to the feed-back principle. — Enzyme profiles may not be used for a functional definition of the metabolic state. Enzyme activities are governed by a variety of control mechanisms, which can best be recognized by steady-state and transient state analysis of metabolites and by an analysis of the systems response in titration experiments with pure enzymes under conditions whereby the system displays oscillatory behaviour of its over-all flux. The important parameter for the definition of the metabolic state is the net-flux through the system, since this parameter, along with the steady-state levels of the meabolites, gives the steady-state flux pattern, reveals the kinetic state of enzymic reactions and points to control points of metabolism. Continuous glycolytic oscillations in a cell-free extract ofSaccharomyces carlsbergensis have been observed over a period of 22 hours with a constant frequency of 0.17 min−1 and a rate of 7.2 nMol ethanol per mg protein per min. Titration of such an extract by pure yeast enzymes reveals the gain (FDP, ADP) and damping components (ATP), which are fed back to the enzymes PFK and PK, respectively, PFK, PGK and PK operating as the control units. On the basis of the titration data as well as metabolite and enzyme activity phase relationship, the mechanism of this oscillation can be understood as a crossed feed-back interaction. Furthermore, it is discussed as the biochemical model of a physiological clock mechanism.Zusammenfassung1.Die Mechanismen der Kontrolle enzymatischer Reaktionssequenzen lassen sich allgemein auf Wechselwirkungen von Aktivatoren, Inhibitoren, Substraten und Produkten mit Enzymproteinen sowie auf Induktion oder Repression der Enzymsynthesen zurückführen. Alle Kontrolltypen werden im Verlaufe der Glycolyse beobachtet. Sie sind die Grundlage des Kontrollnetzes, das den Ablauf der Glycolyse bestimmt und nach dem Rückkopplungsprinzip operiert.2.Das stationäre Verhalten der Fließgleichgewichte der Glycolyse kann durch Bestimmung von Netto-Fluß und stationären Intermediatkonzentrationen adequat in Form der Metabolit- und Flußprofile für den Hin- und Rückfluß jeder enzymatischen Reaktion beschrieben werden. Derartige Profile kennzeichnen den Kontrolltyp jeder enzymatischen Reaktion. Metabolit- und Flußprofile können als Grundlage mathematischer Modelle der Glycolyse benutzt werden. Das Verhalten dieser Modelle unter stationären und transienten Bedingungen steht in Übereinstimmung mit den experimentellen Beobachtungen.3.Die Untersuchung von Übergangszuständen ergänzt die Analyse stationärer Zustände. Sie führt unabhängig zur Aufdeckung von Kontrollpunkten der glycolytischen Sequenz und erfaßt allgemein den Bereich sowie die Güte biochemischer Kontrollmechanismen.4.Der Mechanismus der stationär oder transient oszillierenden Glycolyse konnte im zellfreien Extrakt durch Metabolitanalysen, durch Titrationen mit Intermediaten und Enzymen weitgehend aufgeklärt werden. Er beruht auf der spezifischen Kontrolle der Phosphotransferasen durch gekreuzte Rückkopplung und stellt das biochemische Modell zellulärer Uhrenmechanismen dar.