Dithiothreitol: An inhibitor of glucose-6-phosphate-dehydrogenase activity in leaf extracts and isolated chloroplasts

Summary The activity of glucose-6-phosphate dehydrogenase (G-6-P-DH; d-glucose 6-phosphate: NADP oxidoreductase, EC 1.1.1.49) in leaf extracts of barley and spinach can be decreased 20–35% by incubation of the leaf extracts with dithiothreitol (DTT). This inhibition is complete within 2 min at 0°C and is reversible. The DTT-inhibited portion of G-6-P-DH activity in leaf extracts is probably that portion of leaf enzyme inhibited during illumination, and evidence has been obtained that this activity is located in the chloroplasts.     

Histochemische Untersuchungen zum Energie- und Pentosephosphatstoffwechsel bei der epithelialen Wundheilung

Zusammenfassung Im Verlauf des epithelialen Wundverschlusses können zwei Phasen unterschieden werden. In der ersten Phase (0–48 Std) kommt es zur Glykogeneinlagerung in das Epithel des Wundrandes; bei hohen Aktivitäten der Enzyme SDH, LDH, G-6-P-DH und 6-P-G-DH steigt der RNS-Gehalt in den basalen Zellen an. Im Verlauf der zweiten Phase (60–114 Std) nehmen sowohl der Glykogengehalt als auch die Aktivitäten von LDH, G-6-P-DH und 6-P-G-DH ab. Im gleichen Zeitraum bildet sich eine Epithelschicht in der Wundmitte, deren anfänglicher Glykogenreichtum gegen Ende der Wundheilung wieder verschwindet. Hier ist bei hohen Aktivitäten der LDH, G-6-P-DH und 6-P-G-DH ebenfalls ein Anstieg der RNS-Konzentration nachweisbar. Die Befunde zeigen, daß bei der Wundheilung ein Teil der verfügbaren Glucose über den Pentosephosphat-Zyklus für die Nucleinsäuresynthese verwendet wird.

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Histochemical studies of energy and pentosephosphate metabolism during epithelial wound healing

Summary During epithelial wound healing two periods can be distinguished. In the first period (0–48 h) glycogen is accumulated in the epithelium of the peripheral wound zone, and increasing activities of SDH, LDH, G-6-P-DH and RNA concentrations especially in the basal cells can be detected. In the second period (60–114 h) the glycogen content as well as the activities of LDH, G-6-P-DH and 6-P-G-DH decrease. At the same time an epithelial layer is built up in the central wound zone, the glycogen content of which disappears till the end of wound healing. In this layer high activities of LDH, G-6-P-DH and 6-P-G-DH and an increase of RNA concentration can be demonstrated. The findings illustrate, that during wound healing the available glucose partially is metabolized by the pentose-phosphateshunt and is used for the synthesis of nucleic acids.

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Herrn Professor Dr. Dr. F. Timm zum 75. Geburtstag zugeeignet.

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Wesentliche Teile dieser Untersuchung werden von Herrn R. Schroll dem Fachbereich Theoretische Medizin der Universität Tübingen als Inauguraldissertation vorgelegt.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Histochemistry of the carbohydrate metabolism in cysts of Toxoplasma gondii (author's transl)]

Mice were infected with cysts of the ALT strain Toxoplasma by intraperitoneal injection. After 2-8 weeks disseminated cysts could be demonstrated in the brain tissue. All cysts showed identical histochemical characteristics, independent of their sizes or their cell number. The encysted organisms were intensely stained after the PAS-reaction. This polysaccharide is highly diastase and acid resistant. Glycogen synthetase activity could not be demonstrated, but phosphorylase activity was very high. The energy metabolism was characterized by a high lactate dehydrogenase activity, whereas the reaction for succinate dehydrogenase activity only leads to sparse deposits of reaction products. The carbohydrate content is interpreted to be not only a store of energy substrate but also a store of biosynthetic substrate. It is assumed that a part of the liberated glucose at high activities of G-6-P-DH and 6-P-G-DH is metabolized by the hexose monophosphate shunt, the pentoses of which may contribute to nucleic acid synthesis which is necessary for the proliferation of the encysted organisms.     

Selective stabilization of muscle innervation during development: A mathematical model

The biochemical model presented concerns a critical step of the development of skeletal muscle innervation. After invasion of the muscle by exploratory motor axons, several nerve terminals converge from different motoneurons onto each muscle fibre at a single endplate. During the folloing weeks the redundant innervation disappears: a single nerve ending per muscle fibre becomes stabilized. The model is based on the assumption that the numbers of motoneurons and of muscle fibres remain constant during this evolution and that the selective stabilization of the adult connectivity results from the competition of the active nerve terminals for a postsynaptic retrograde factor μ. At the peak of the multiple innervation, the synthesis of μ by the muscle fiber stops, possibly as a consequence of muscle electrical and/or mechanical activity. The stock of μ becomes limited; a retrograde trans-synaptic diffusion of μ from the muscle to the nerve endings takes place. Within each nerve ending, μ enters into a chemical autocatalytic reaction which results in the production of a presynaptic stabilization factor s. The nerve impulses reaching the nerve terminal initiate this reaction. Any given nerve terminal become stabilized when the concentration of s reaches a threshold value. The mathematical analysis of the model shows that there exists a unique solution which is physically acceptable. Its application and computer simulation predict that only one nerve terminal becomes stabilized per muscle fibre. The model accounts for the experimental observations that the reduction in size of the motor units is not necessarily accompanied by a reduction in the variability of their size. The model also accounts for the acceleration or delay in regression which follows modifications of the chronic activity of the nerve endings and for the variability of the pattern of innervation observed in isogenic organisms. Plausible biochemical hypotheses concerning the factors engaged in the “selective stabilization” of the nerve-endings are discussed.     

A tetrazolium technique for the histochemical localization of ATP: Creatine phosphotransferase

Summary A tetrazolium technique is presented that permits the study of ATP: Creatine phosphotransferase, or creatine kinase, in fixed skeletal muscle tissue sections, within the limits imposed by the properties of the chosen ditetrazole, nitro blue tatrazolium. There is a variation in creatine kinase activity between the muscle fibres. Those with high creatine kinase activity also have high succinate dehydrogenase activity.List of Abbreviations ADP Adenosine-5-diphosphate - ATP adenosine-5-triphosphate - CK creatine kinase - G-6-P glucose-6-phosphate - G-6-P-DH glucose-6-phosphate dehydrogenase - HK hexokinase - NADP nicotinamide adenine dinucleotide phosphate - NBT nitro blue tetrazolium - PMS phenazine methosulphate - SDH succinate dehydrogenase     

The ultrastructure of sensory corpuscles in the skin in the hedgehog snout

The ultrastructure of sensory nerve endings was examined in the snout skin in 3 adult hedgehogs (Erinaceus europaeus). The material was taken intravitally under total anaesthesia and processed in a usual way for the electron microscopy. The corpuscles were evaluated in the individual sections and series sections made through the whole corpuscle. In the superficial layers of the dermis simple sensory corpuscles and free endings were found. The simple sensory corpuscles can be divided into three types. a) Corpuscles containing a greater number of lamellae in the inner core, the lamellae are arranged regularly and are separated by two opposite clefts. The capsule is formed by only several lamellae undoubtedly of fibrocytic origin. b) Corpuscles containing a smaller number of wider lamellae in the inner core situated often at random. The clefts are also irregular and are often closed in the superficial layers of the inner core. The capsule is quite simple mostly formed by a single lamella of fibrocyte which often fails to form a continuous coat of the corpuscle. c) The third type is typical of its inner core being formed by few lamellae arranged irregularly. These corpuscles have no connective tissue capsule and are separated from the environments only by the basement membrane of superficial lamellae of the inner core. The corpuscles of the second type resemble considerably the developmental stages of simple sensory corpuscles as described in the literature in the cat. They are the same in size or smaller than the corpuscles of the first type. The free nerve endings occurred in two forms. a) Flattened (lanciform) nerve terminals. The axon is rich in mitochondria. The sides of the flattened terminal is lined with one to three wide lamellae while the axon reaches as far as the surface of the formation which is covered only with the basement membrane. b) Typical free endings rich in mitochondria which are embedded in the cytoplasm of Schwann cells or occasionally are covered only with the basement membrane. The lanciform endings which are not linked up with the hairs here may represent a transition from free endings to simple sensory corpuscles.     

Structure of the sensory innervation of the anal canal in the pig. A light- and electron-microscopical study

The sensory innervation of the anal canal of the pig was investigated by light and electron microscopy. The distribution of the different types of sensory nerve endings correlates with the histology of different zones: (1) After the rectal mucosa there was a zone lined with nonkeratinized stratified squamous epithelium. (2) A middle zone was lined with keratinized stratified squamous epithelium. Here the dermis already showed a papillary and reticular layer. (3) The last zone showed hairy skin with a high hair density. The following nerve endings were found: Free nerve endings reached the stratum superficiale in nonkeratinized squamous epithelium and the stratum granulosum in the keratinized squamous epithelium. Dermal free nerve endings were found in all zones near the epithelium and two different types were identified as those derived from C-fibers and those from A-delta-fibers. Merkel nerve endings showed different features depending on their location. Few Merkel-like cells were found in the epithelium of the anal crypts. Typical Merkel Tastscheiben were located at the base of epithelial ridges or pegs in zones 2 and 3. The number of Merkel cells varied up to 200. The myelinated afferent fiber supplied 10-15 Merkel cells. Merkel cells were also found regularly in the outermost layer of the external rooth sheath of hair follicles at about the same level as perifollicular nerve endings. Lamellated corpuscles were found in the dermis of all zones except the cranial part of zone 1, where the anal crypts are located. Generally they consisted of a central nerve terminal which may be branched. Each terminal was surrounded by an inner core of concentrically arranged lamellae of the terminal Schwann cell and one or several inner cores were included in a capsule of perineural cells. The size of the corpuscle, the regularity of the inner core and the number of capsular layers depended on the location of the corpuscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Das Verhalten von Nitratreductase,Nitritreductase, Hydrogenase und anderen Enzymen von Ankistrodesmus braunii bei Stickstoffmangel

Zusammenfassung Bei der N-Verarmung von Ankistrodesmus braunii wurde eine intracelluläre Bildung von Nitrit und Nitrat durch heterotrophe Nitrifikation festgestellt, die charakteristisch für den beginnenden N-Mangel ist. Damit wird die hohe Nitrat- und Nitritreductaseaktivität bei N-Mangelalgen verständlich.Die Hydrogenase ist auch bei N-verarmten Algen aktiv, doch kann Nitrit nicht mehr als H-Acceptor verwendet werden. Die Aktivierung des Enzyms ist energieabhängig und durch Antibiotica (Actinomycin C, Puromycin, Gentamycin) hemmbar. Offenbar findet während der Inkubation mit Wasserstoff eine Proteinsynthese statt.Zum Vergleich wurde das Verhalten einiger anderer Enzyme [Glucose-6-phosphat-dehydrogenase, NAD(P)-Reductase, Glyoxylat-reductase, Katalase, Malat-dehydrogenase, Glutamat-dehydrogenase, Isocitratase] bei N-Mangel untersucht.

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Nitrate reductase, nitrite reductase, hydrogenase, and other enzymes in nitrogen-deficient Ankistrodesmus braunii

Summary An oxidation of organic nitrogen compounds leading to an intracellular formation of nitrite and nitrate (heterotrophic nitrification) was found in nitrogen-deficient Ankistrodesmus braunii. This explains the rather high levels of nitrate and nitrite reductases observed in algae after the supply of nitrogen has been exhausted.Hydrogenase is active also in nitrogen-deficient algae which, however, can no longer use nitrite as an acceptor for hydrogen. The activation of hydrogenase is energy-dependent and can be inhibited by means of antibiotics (actinomycin C, puromycin, and gentamycin). Protein synthesis seems to take place during incubation under hydrogen.For comparison, several other enzymes [glucose-6-phosphate dehydrogenase, NAD(P) reductase, glyoxylate reductase, catalase, malate dehydrogenase, glutamate dehydrogenase, and isocitratase) were studied in nitrogen-deficient cells.

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Verwendete Abkürzungen G-6-P-DH Glucose-6-phosphat-dehydrogenase - M-DH Malat-dehydrogenase - Glu-DH Glutamat-dehydrogenase - NAD(P)-R Nicotinamid-adenin-dinucleotid(phosphat)-reductase - CCCP Carbonylcyanid-mchlorophenylhydrazon - DNP 2,4-Dinitrophenol - MB Methylenblau     

Glucose-6-phosphate dehydrogenase isoenzymes in root growth zones ofVicia faba

The specific activity of glucose-6-phosphate dehydrogenase (G-6-PD) in growth zones ofVicia faba roots is increasing with cell maturation and differentiation. Changes in the total activity of G-6-PD are not associated with a change in the number of G-6-PD isoenzymes. Five G-6-PD isoenzymes were found in all root growth zones. Some differences were found in the activity of individual isoenzymes.     

Chemomorphologische Befunde (Glykogen,G-6-P-DH,RNS) am Regenerationsblastem vonLineus sanguineus Rathke (Nemertini)

Zusammenfassung Während der Kopfregeneration vonLineus sanguineus lassen sich zwei Phasen unterscheiden. In derProliferationsphase wird die Schnittwunde durch Epithel verschlossen, danach kommt es durch Einwanderung von Blastocyten zum Aufbau des Blastems. Im Verlauf derDifferenzierungsphase treten Anlagen auf, die sich im weiteren zu den Kopforganen ausbilden. Epidermisproliferation, Blastembildung und Organdifferenzierung (Cerebralorgane, Vorderdarm und Rüssel) sind histochemisch in gleicher Weise charakterisiert: alle diese morphogenetischen Areale zeichnen sich zunächst durch einen hohen Glykogengehalt aus, der sich während der Weiterentwicklung des Regenerats bei zunehmender G-6-P-DH-Aktivität und Anstieg der RNS-Konzentration wieder verliert. Aus diesen Befunden ist abzuleiten, daß die in morphogenetischen Arealen durch Glykogenolyse bereitgestellte Glucose über den Pentose-Phosphat-Zyklus für die Nucleinsäuresynthese verwendet wird.

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Chemomorphological data (glycogen, G-6-P-DH, RNA) in the regeneration blastema ofLineus sanguineus rathke (Nemertini)

Summary The head regeneration ofLineus sanguineus consists of two periods. During the phase ofproliferation the wounds are covered with epidermis, thereafter immigrating blastocytes build up the blastema. During the phase ofdifferentiation anlagen are discernible, which subsequently become the head organs. Proliferation of epidermis, the building of blastema and the differentiation of the organs (cerebral organs, foregut and proboscis) are histochemically characterized in the same way: in the beginning there is a high glycogen content in the morphogenetic areas, which decreases during further development with increasing G-6-P-DH activity and with growing RNA concentration. These results indicate that in morphogenetic areas the glucose resulting from glycogenolysis is metabolized by the pentose-phosphate-shunt and is utilized for the synthesis of nucleic acids.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Analysis of living motor nerve ending of a frog by endocytotic fluorescent marker FM 1-43

In our experiments on motor nerve endings of the frog cutaneous pectoris muscle, using fluorescent marker FM 1-43, the intensity and topography of endocytosis were investigated after the initiation of massive exocytosis of synaptic vesicles by increasing the extracellular potassium concentration. Using FM 1-43, fluorescent spots were shown to appear, looking as accumulations of synaptic vesicles in the active zone region. The forms and sizes of luminous spots and the distances between them were analysed. Considerable variations in brightness and total areas of fluorescent spots per a length unit in different regions of the nerve ending were revealed in addition to a proximal-distal gradient of these parameters along the nerve terminal. Peculiarities of topography and intensities of luminescence in the most terminal regions of the nerve ending are described. The obtained data are discussed in terms of the exo- and endocytosis cycle of synaptic vesicles in the active zone region, and from the point of view of the plasticity of the motor nerve ending and active zones. The factors involved in the transmitter release nonuniformity are analysed.     

Effect of food deprivation on histomorphology and cholinesterase activity of taste buds in ratst

The histomorphology and cholinesterase activity of the taste buds and the gustatory nerve fibre sin well-fed, in protein and protein-calorie deficient rats have been investigated. The nerve fibre arborisation in the taste buds is predominantly nonmyelinated and shows degenerative changes ranging from initial swelling to disintegration, fragmentation and finally complete disappearance with the increasing degree and duration of food deprivation. Coincident with these changes in the nerve fibre, the taste bud also shows various stages of degeneration. By contrast, the cholinesterase activity in the gustatory papillae shows an initial increase during the first week followed by a decline in the activity during the succeeding weeks; a second peak of cholinesterase activity appears during 4–6 weeks. The cholinesterase activity is barely detectable after the 8th week. In the more severely protein calorie restricted groups, the cholinesterase changes are more pronounced and abrupt in onset and show a total disappearance by 4–5 weeks.     

A histochemical analysis of mammalian oxidative skeletal muscle fibres using the enzymes of energetic metabolism

Summary Some histochemical parameters of the three main fibre types of rat vastus lateralis muscle were studied. Succinic dehydrogenase (SDH), creatine kinase (CK), sarcotubular ATPase (SR-ATPase) and mitochondrial ATPase activities were demonstrated in serial sections. The three fibre types, recognised by the distribution pattern of SDH activity, all show high CK activity. However, red Type I oxidative fibres when examined for ATPase and ATPase dependent CK activity, show distinct heterogeneity revealing sub-populations within the same homogeneous fibre type. Three distinct patterns were recognised in the red Type I fibres depending on the distribution of the final reaction product. The present histochemical evidence confirms the fact that subdivision of mammalian skeletal muscle into three fibre types is only approximate and probably more than three types exist.     

Nuclear membranes from mammalian liver. VI. Glucose-6-phosphatase in rat liver, a cytochemical and biochemical study

Activities of glucose-6-phosphatase (G-6-Pase) and other phosphatases were determined in nuclei, nuclear membrane and microsomal fractions and subfractions, and condensed chromatin isolated from the liver of adult, newly born and prenatal rats. The purity of the fractions was controlled by electron microscopic morphometry and by measurement of various marker enzymes. The specific G-6-Pase activity of the nuclear membranes was found to be about 60% that of the microsomes. However, when calculated on the basis of the phospholipid content, all fractions had similar activities. Determinations of G-6-Pase enrichments and recoveries were also made. The correspondence of the hydrolysing activities of glucose-6-phosphate, mannose-6-phosphate, and inorganic pyrophosphate, together with various phosphotransferases, showed the same association of the G-6-Pase with these enzymes in the nuclear envelope as in the microsomal membranes. G-6-Pase was also demonstrated in the fractions by cytochemistry, and the activity was localized alongside the cisternal surfaces of both, inner and outer, nuclear membrane. ‘Free’ inner nuclear membrane fragments contained also G-6-Pase. No activity was observed at the nuclear pore complexes. Both, nuclear and microsomal membranes revealed a parallel rapid perinatal increase of G-6-Pase activity climaxing at 23 to 28 h after birth. Triton-X-100 treatment of isolated nuclei, which was found not to selectively release outer nuclear membranes, resulted in a great decrease of G-6-Pase activity as well as in losses of membrane phospholipids. The results clarify the divergence of earlier reports concerning the presence of G-6-Pase in the perinuclear cisterna and add biochemical evidence to the morphologically derived view of the nuclear envelope as being a special form of the ER system.     

Histochemical study on the localization of glucose-6-phosphate dehydrogenase induced by androgen or thyroxine in the convoluted tubules of mouse submandibular gland.

The effects of 5 alpha-dihydrotestosterone (DHT) and thyroxine (T4) on glucose-6-phosphate dehydrogenase (G-6-PDH) activity in mouse submandibular gland were investigated histochemically. A strong positive histochemical reaction for G-6-PDH was observed in the excretory ducts of untreated male and female mice, with a slight reaction in the basal portion of the convoluted tubules (striated ducts) of males. Administration of DHT to female mice increased G-6-PDH activity specifically in the convoluted tubules. T4 increased the enzyme activity in the tubules more than DHT. The induction of G-6-PDH activity by T4 in adrenalectomized mice suggests that T4 has a direct effect on the submandibular gland.     

Histochemical study on the nerve endings in the lung of Rattus rattus rufescens and Francolinus pondicerianus

Presence and the relation of the nerve endings with associated structures in the lund of Rattus rattus rufescens (Indian black rat) and Francolinus pondicerianus (grey partridge or safed teeter) has been studied by cholinesterase technique. Dot, plate, and Vater pacini Corpuscles like nerve endings in the lung of Rattus and dot, and bulb like nerve endings with axis cylinder covered with myelinated sheath in the lung of Francolinus were recorded. The nerve endings were AChE-positive.     

ELECTRON MICROSCOPY OF THE PACINIAN CORPUSCLE

The Pacinian corpuscle has a framework of cytoplasmic lamellae arranged concentrically in the outer zone, and bilaterally in the core. Between these is an intermediate growth zone. The inner core shows an unexpected complexity in that its component lamellae are arranged in two symmetrical groups of nested cytoplasmic sheets. Longitudinal tissue spaces form clefts separating the two groups. The perikarya of the core lamellae lie in or near the intermediate growth zone, and send arms into the clefts. The arms then branch and terminate as lamellae which interdigitate with those of neighboring cells. The single nerve fiber loses its myelin sheath just before it reaches the inner core but retains its Schwann cell cytoplasmic covering for a short additional distance. The Schwann sheath is not continuous with the lamellae of the inner core. Inside the core the fiber contains a striking circumferential palisade of radially disposed mitochondria. The fiber does not arborize. Vascular capillaries penetrate the hilar region of the corpuscle only as far as the myelinated sheath of the nerve, and they have not been seen elsewhere in the corpuscle. There is direct continuity between the clefts of the core and tissue spaces in the vicinity of the capillaries. It is likely that this provides a route whereby metabolites reach the active nerve ending, as well as the cells of the growth zone. The outer zone consists of at least 30 flattened concentric cytoplasmic lamellae separated from one another by relatively wide fluid-filled spaces. Collagenous fibrils are present, particularly on the outer surface of lamellae, and tend to be oriented circularly. The girdle of proliferating cells constituting the growth zone, which is prominent in corpuscles from young animals, is the layer from which the outer lamellae are derived. Osmotic forces probably elevate the lamellae, and maintain turgor pressure.     

Comparative spatial and temporal localisation of perlecan, aggrecan and type I, II and IV collagen in the ovine meniscus: an ageing study

This is the first study to immunolocalise perlecan in meniscal tissues and to demonstrate how its localisation varied with ageing relative to aggrecan and type I, II and IV collagen. Perlecan was present in the middle and inner meniscal zones where it was expressed by cells of an oval or rounded morphology. Unlike the other components visualised in this study, perlecan was strongly cell associated and its levels fell significantly with age onset and cell number decline. The peripheral outer meniscal zones displayed very little perlecan staining other than in small blood vessels. Picrosirius red staining viewed under polarised light strongly delineated complex arrangements of slender discrete randomly oriented collagen fibre bundles as well as transverse, thick, strongly oriented, collagen tie bundles in the middle and outer meniscal zones. The collagen fibres demarcated areas of the meniscus which were rich in anionic toluidine blue positive proteoglycans; immunolocalisations confirmed the presence of aggrecan and perlecan. When meniscal sections were examined macroscopically, type II collagen localisation in the inner meniscal zone was readily evident in the 2- to 7-day-old specimens; this became more disperse in the older meniscal specimens. Type I collagen had a widespread distribution in all meniscal zones at all time points. Type IV collagen was strongly associated with blood vessels in the 2- to 7-day-old meniscal specimens but was virtually undetectable at the later time points (>7 month).     

A MICRO-SCALE PROCEDURE FOR THE PREPARATION OF SUBCELLULAR FRACTIONS FROM INDIVIDUAL AUTONOMIC GANGLIA

Abstract— A microscale modification for the preparation of subcellular fractions employing milligram and submilligram amounts of neuronal tissue (brain nuclei and autonomic ganglia) is described.
Electron microscope characterization and enzymic studies were carried out on the six subcellular fractions of sympathetic ganglia of cat thus prepared.
The synaptosomal preparations obtained from individual ganglia were poorer in their nerve ending content than those obtained from brain by previous investigators. The highest RSA for AChE was found in layer L₂ which was rich in membranes and vesicle components. ChAc activity was also highly concentrated in layers L₂ and L₃ (membranes, nerve ending-like particles, mitochondria and 'ghosts'). MAO activity was particularly high in the layers L₄ and L₅ which contained a large number of mitochondria. Layer L₁ (membrane fragments) and particularly layer L₆ which contained mainly collagen fibres, were low in activity of all three enzymes.
After preganglionic denervation, both ChAc and AChE activities were significantly reduced in the purest nerve ending fraction, L₃ while MAO activity was practically unchanged.     

Regulation of pyruvate kinase from Propionibacterium shermanii

Pyruvate kinase from Propionibacterium shermanii was shown to be activated by glucose-6-phosphate (G-6-P) at non-saturating phosphoenol pyruvate (PEP) concentrations but other glycolytic and hexose monophosphate pathway intermediates and AMP were without effect. Half-maximal activation was obtained at 1 mM G-6-P. The presence of G-6-P decreased both the PEP_0.5V and ADP_0.5V values and the slope of the Hill plots for both substrates. The enzyme was strongly inhibited by ATP and inorganic phosphate (P_i) at all PEP concentrations. At non-saturating (0.5 mM) PEP, half-maximal inhibition was obtained at 1.8 mM ATP or 1.4 mM P_i. The inhibition by both P_i and ATP was largely overcome by 4 mM G-6-P. The specific activity of pyruvate kinase was considerably higher in lactate-, glucose- and glycerol-grown cultures than that of the enzyme catalysing the reverse reaction, pyruvate, phosphate dikinase. It is suggested that the activity of pyruvate kinase in vivo is determined by the balance between activators and inhibitors such that it is inhibited during gluconeogenesis while, during glycolysis, the inhibition is relieved by G-6-P.Abbreviations PEP phosphoenolpyruvate - G-6-P glucose-6-phosphate - P_i inorganic phosphate