Mammalian and avian liver phosphoenolpyruvate carboxykinase. Alternate substrates and inhibition by analogues of oxaloacetate

Phosphoenolpyruvate carboxykinase from chicken liver mitochondria and rat liver cytosol catalyzes the phosphorylation of alpha-substituted carboxylic acids such as glycolate, thioglycolate, and DL-beta-chlorolactate in reactions with absolute requirements for divalent cation activators. 31P NMR analysis of the reaction products indicates that phosphorylation occurs at the alpha-position to generate the corresponding O- or S-bridged phosphate monoesters. In addition, the enzymes catalyze the bicarbonate-dependent phosphorylation of hydroxylamine. The chicken liver enzyme also catalyze the bicarbonate-dependent phosphorylation of hydroxylamine. The chicken liver enzyme also catalyzes the bicarbonate-dependent phosphorylation of fluoride ion. The kappa cat values for these substrates are 20-1000-fold slower than the kappa cat for oxaloacetate. Pyruvate and beta-hydroxypyruvate are not phosphorylated, since the enzyme does not catalyze the enolization of these compounds. Oxalate, a structural analogue of the enolate of pyruvate, is a competitive inhibitor of phosphoenolpyruvate carboxykinase (Ki of 5 microM) in the direction of phosphoenolpyruvate formation. Oxalate is also an inhibitor of the chicken liver enzyme in the direction of oxaloacetate formation and in the decarboxylation of oxaloacetate. The chicken liver enzyme is inhibited by beta-sulfopyruvate, an isoelectronic analogue of oxaloacetate. The extensive homologies between the reactions catalyzed by phosphoenolpyruvate carboxykinase and pyruvate kinase suggest that the divalent cation activators in these reactions may have similar functions. The substrate specificity indicates that phosphoenolpyruvate carboxykinase decarboxylates oxaloacetate to form the enolate of pyruvate which is then phosphorylated by MgGTP on the enzyme.     

Observations et discussions sur le développement embryonnaire des Phoronida

Resume La fécondation est généralement interne chez les phoronidiens. La segmentation des ceufs est totale, egale (parfois légèrement inégale) et de type radiaire (avec quelquefois une apparence fortuite de segmentation spirale). La gastrula est formée par embolie. La bouehe derive de la zone blastoporale sans formation d'un vrai stomodeum. L'anus est mis en place par perforation de l'ectodersme et représente une néo-formation indépendante du blastopore. Le mesoderme est issu par proliferation cellulaire des regions antérieure et laterales de l'archentéron. Le protoccele est forme par des cellules mésodermiques se disposant le long de la paroi du lobe préoral. Le métaccele est issu probablement suivant les espèces d'une ou deux masses. La formation du mesoderme correspond á une variation de la méthode entéroccelique typique. Les phoronidiens doivent être considérés comme des deutérostomiens, d'après l'ensemble de nos résultats (voir aussi Emig, 1973).

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Observations and discussions on the embryonic Development in Phoronida

Summary Internal fertilization (in metaccelom) generally occurs in Phoronida. The eggs are extruded to the exterior through the nephridia, shed freely into the sea-water or retained in the lophophoral concavity. The cleavage of phoronid eggs is total, equal (or subequal) and radial (with sometimes fortuitous appearance of spiral cleavage patterns). The gastrula is formed by emboly. The mouth is derived from the anterior remnant of the blastopore without a true stomodeum. The anus arises by perforation, as an independent structure of the blastopore. The mesoderm formed by budding originates as isolated cells proliferated from the anterior and lateral surfaces of the archenteron. In the preoral hood appears a protoccel by mesodermal cells lining the walls of the blastoccel. The trunk clom (or metaccel) of Actinotrocha originates from one or two posterior masses of mesodermal cells. It is possible that the mode of formation of this coelom varies in respect to the different species. The mesoderm elaboration is considered as a modified enteroccelous method.The acceptance of Phoronida as deuterostomes is regarded as the logical consequence of the present considerations (see also Emig, 1973): radial cleavage, origin of mesoderm by a derived enteroccelous method, trimetamerous actinotrocha.

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Abbréviations des figures a anus - lp lobe préoral - ar archentéron - b blastoccele - ma mésoderme de la région anterieure de l'archentéron - ml mésoderme des régions latérales de l'archentéron - bl blastopore - mes cellule mésodermique - bo bouche - n ebauche des - n éphridiesect mesderme - s sophage - end endoderme - p protocle - est estomac - t ebauche des tentacules - g ebauche du ganglion nerveux - te tentacule - gn glandes nidamentaire - v vestibule - i intestin     

L'histogenèse régénératrice chez les phoronidiens

Summary The regeneration (organogenesis was studied by Emig, 1972 a, b) of Phoronida can be divided into three phases: the first one, cicatrisation, is characterized by a provisional mesodermal scar-tissue, later the old epidermis cover this scar-tissue. The regenerating blastema, second phase, takes place by cellular dedifferentiation processes; each germ layer (ectoderm, mesoderm, endoderm) regenerates itself from its own elements. One exception only seems to be oesophagel regeneration by metaplasia of the prestomacal cells during the asexual reproduction. The differentiation of the amputated structures (third phase) appears submitted to the inductive influence of the mesoderm and to the trophic action of the nervous system (especially the epithelial plexus). The polarity in regeneration sets a problem in Phoronida.

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Ce travail a été effectué dans le cadre du contrat L. A. n 41 au C. N. R. S.     

AMP nucleosidase: kinetic mechanism and thermodynamics

The kinetic mechanism of AMP nucleosidase (EC 3.2.2.4; AMP + H2O----adenine + ribose 5-phosphate) from Azotobacter vinelandii is rapid-equilibrium random by initial rate studies of the forward and reverse reactions in the presence of MgATP, the allosteric activator. Inactivation-protection studies have established the binding of adenine to AMP nucleosidase in the absence of ribose 5-phosphate. Product inhibition by adenine suggests a dead-end complex of enzyme, AMP, and adenine. Methanol does not act as a nucleophile to replace H2O in the reaction, and products do not exchange into substrate during AMP hydrolysis. Thus, the reactive complex has the properties of concerted hydrolysis by an enzyme-directed water molecule rather than by formation of a covalent intermediate with ribose 5-phosphate. The Vmax in the forward reaction (AMP hydrolysis) is 300-fold greater than that in the reverse reaction. The Keq for AMP hydrolysis has been experimentally determined to be 170 M and is in reasonable agreement with Keq values of 77 and 36 M calculated from Haldane relationships. The equilibrium for enzyme-bound substrate and products strongly favors the enzyme-product ternary complex ([enzyme-adenine ribose 5-phosphate]/[enzyme-AMP] = 480). The temperature dependence of the kinetic constants gave Arrhenius plots with a distinct break between 20 and 25 degrees C. Above 25 degrees C, AMP binding demonstrates a strong entropic effect consistent with increased order in the Michaelis complex. Below 20 degrees C, binding is tighter and the entropic component is lost, indicating distinct enzyme conformations above and below 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitor coordination interactions in the binuclear manganese cluster of arginase

Arginase is a manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to form L-ornithine and urea. The structure and stability of the binuclear manganese cluster are critical for catalytic activity as it activates the catalytic nucleophile, metal-bridging hydroxide ion, and stabilizes the tetrahedral intermediate and its flanking states. Here, we report X-ray structures of a series of inhibitors bound to the active site of arginase, and each inhibitor exploits a different mode of coordination with the Mn(2+)(2) cluster. Specifically, we have studied the binding of fluoride ion (F(-); an uncompetitive inhibitor) and L-arginine, L-valine, dinor-N(omega)-hydroxy-L-arginine, descarboxy-nor-N(omega)-hydroxy-L-arginine, and dehydro-2(S)-amino-6-boronohexanoic acid. Some inhibitors, such as fluoride ion, dinor-N(omega)-hydroxy-L-arginine, and dehydro-2(S)-amino-6-boronohexanoic acid, cause the net addition of one ligand to the Mn(2+)(2) cluster. Other inhibitors, such as descarboxy-nor-N(omega)-hydroxy-L-arginine, simply displace the metal-bridging hydroxide ion of the native enzyme and do not cause any net change in the metal coordination polyhedra. The highest affinity inhibitors displace the metal-bridging hydroxide ion (and sometimes occupy a Mn(2+)(A) site found vacant in the native enzyme) and maintain a conserved array of hydrogen bonds with their alpha-amino and -carboxylate groups.     

Sur la structure des parois de l'appareil circulatoire dePhoronis psammophila Cori (Phoronida,Lophophorata)

Resume Trois types de parois ont été décrits dans l'appareil circulatoire du tronc dePhoronis psammophila. La succession des diverses couches de chaque type est la suivante: 1. cellules péritonéales — lame basale — rares cellules endothéliales; 2. cellules myoépithéliales — lame basale — rares cellules endothéliales; 3. une couche de muscles circukires, puis une de muscles longitudinaux — épaisse lame basale — endothélium continu.

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On the wall structure of the circulatory system inPhoronis psammophila Cori (Phoronida, Lophophorata)

Summary Three types of wall structure of blood vessels have been described. It consists of the following distinct layers, from exterior to interior: 1. peritoneal cells — thin basal lamina — some endothelial cells; 2. myoepithelial cells resting on a basal lamina — some endothelial cells; 3. circular and longitudinal muscle layers of myoepithelial cells — thick basal lamina — continuous endothelial lining.

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Abréviations C capillaire - ce cavité coelomique - ce cellule endothéliale - cm cellule myoépithé'liale - cp cellule péritonéale - fc fibre musculaire circulaire - fl fibre musculaire longitudinale - gs globule sanguin - lb lame basale - m myofibrille - mf myofilament - tvp tissu vasopéritonéal - V vaisseau sanguin     

Phylogenèse des Phoronida

Phylogenese of Phoronida. Lophophorata and the Archimerata concept The main phylogenetic characteristics of Phoronida and other Lophophorates are discussed: 1. Archimeric segmentation of the body; 2. Egg cleavage of radial (or biradial) type, coeloblastula and gastrulation by invagination (emboly); 3. Mesoderm formation by a derived enteroccelous method (primitive stage of enteroc? ly); 4. Bringing of the anus anteriorly to lie rocoelous method (primitive stage of enterocoely); 4. Bringing of the anus anteriorly to lie sence of a true lophophore; 6. Larva not of trochophoral type, but actinotroch related to Tornaria-Dipleurula; 7. Nervous system basi-epithelial with primitive neurulation in Phoronopsis, without any orthogons; 8. Adult nervous ganglion neo-formed, not issuing from the apicale plate; in Phoronida this ganglion is located in the prosome and not in the mesosome; 9. Lack of cephalization. All these characteristics are closely related to that of the primitive phyla of the Chordata assemblage. The only exception is the presence of protonephridia with solenocytes in actinotroch, but such organs are also described in Cephalochordata. The Lophophorata (Phoronida, Brachiopoda, Bryozoa) are undoubtedly a primitive group on the Chordata trend and the Phoronida appear to be the most phylogenetically evolved phylum of this group with predominating position. The validity of placing the Lophophorata within the Echinoderm-Hemichordata assemblage is demonstrated. The term Archic?lomata appears not suitable and its substitution by Archimerata, assemblage at the base of the Chordata, is here proposed. The Archimerata concept brings together the Lophophorata, Echinodermata and Hemidiordata and is considered as a phylogenetic stage and a natural systematic unit.     

Passive myocardial mechanical properties: meaning,measurement, models

Passive mechanical tissue properties are major determinants of myocardial contraction and relaxation and, thus, shape cardiac function. Tightly regulated, dynamically adapting throughout life, and affecting a host of cellular functions, passive tissue mechanics also contribute to cardiac dysfunction. Development of treatments and early identification of diseases requires better spatio-temporal characterisation of tissue mechanical properties and their underlying mechanisms. With this understanding, key regulators may be identified, providing pathways with potential to control and limit pathological development. Methodologies and models used to assess and mimic tissue mechanical properties are diverse, and available data are in part mutually contradictory. In this review, we define important concepts useful for characterising passive mechanical tissue properties, and compare a variety of in vitro and in vivo techniques that allow one to assess tissue mechanics. We give definitions of key terms, and summarise insight into determinants of myocardial stiffness in situ. We then provide an overview of common experimental models utilised to assess the role of environmental stiffness and composition, and its effects on cardiac cell and tissue function. Finally, promising future directions are outlined.     

Structure collisions between interacting proteins

Protein-protein interactions take place at defined binding interfaces. One protein may bind two or more proteins at different interfaces at the same time. So far it has been commonly accepted that non-overlapping interfaces allow a given protein to bind other proteins simultaneously while no collisions occur between the binding protein structures. To test this assumption, we performed a comprehensive analysis of structural protein interactions to detect potential collisions. Our results did not indicate cases of biologically relevant collisions in the Protein Data Bank of protein structures. However, we discovered a number of collisions that originate from alternative protein conformations or quaternary structures due to different experimental conditions.