Increased demand for NAD+ relative to ATP drives aerobic glycolysis

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Mobility of secondary structure units of horse-muscle acylphosphatase. Relation to antigenicity

The antigenic properties of acylphosphatase are compared with its various sequential characteristics (hydrophobicity, chemical shift of the main-chain 1H-NMR resonances, numbers and intensities of the nuclear Overhauser enhancements, hydrogen-deuterium exchange and sequential arrangement of the secondary structure units). The discussion is based on the complete sequential assignment of the 1H-NMR spectrum and the knowledge of the three-dimensional fold of the protein obtained by NMR spectroscopy from distance geometry calculations. Regions with very different degrees of mobility can be distinguished. It is found that all major antigenic sites are located in the most mobile surface loops.     

Immunoaffinity purification and immunoassay determination of human erythrocyte acylphosphatase

Specific anti-human erythrocyte acylphosphatase antibodies were raised in rabbits, purified by affinity chromatography, and used to develop an enzyme purification procedure based on an immunoaffinity chromatography step. This procedure permitted the rapid purification of the enzyme, with a high final yield and with a specific activity very similar to that found for the enzyme purified by the standard procedure. The noncompetitive enzyme-linked immunoadsorbent assay developed with the affinity-purified antibodies was very specific and sensitive in that a positive reaction could be detected in the presence of antigen amounts of as little as 0.01 ng/ml. By this assay the enzyme content was determined in normal cells, tissues, and organs as well as in blood samples from hemopathy-affected patients. This test could possibly have clinical applications.     

Acylphosphatase increases the rate of ethanol production from glucose in cell-free extracts of Saccharomyces cerevisiae

Addition of acylphosphatase exerted a stimulating effect on the alcoholic fermentation of glucose by Saccharomyces cerevisiae. The rates of glucose degradation and ethanol production by cell-free extracts of the S-288C strain were measured in the absence and in the presence of various levels of this enzyme. Two acylphosphatase isoenzymes were used; one was purified from horse skeletal muscle and the other from human erythrocytes. Both increased the rate of alcoholic fermentation, but that from erythrocytes proved to be the more efficient. This stimulating action is probably due to an "uncoupling effect" of acylphosphatase on the fermentative process, through hydrolysis of 3-phosphoglyceroyl phosphate. This was demonstrated by the fact that alcoholic fermentation was stimulated considerably by a mixture of ADP and inorganic phosphate and by arsenate as well. The possibility of improving the fermentative capacity of microorganisms may have important biotechnological applications.     

Internal blockade of a Ca(2+)-activated K+ channel by Shaker B inactivating "ball" peptide.

Shaker B inactivating peptide ("ball peptide", BP) interacts with Ca(2+)-activated K+ (KCa) channels from the cytoplasmic side only, producing inhibition of channel activity. This effect was reversible and dose and voltage dependent (stronger at depolarized potentials). The inhibition of KCa channels by BP cannot be mimicked by an inactive point mutation of the BP, L7E. BP binds to KCa channels in a bimolecular reaction (dissociation constant of 95 microM at +40 mV). The binding site is probably located in the internal "mouth" or conduction pathway, since both external K+ and internal tetraethylammonium relieve BP-induced inhibition. These results suggest that KCa channels possess a binding site for the BP with some properties similar to the ball receptor found in Shaker B K+ channels.     

Turkey muscle acylphosphatase: purification and comparative studies

Turkey muscle acylphosphatase is strongly bound to anti-(horse muscle acylphosphatase ) antibodies covalently linked to an agarose resin. This permits use of an affinity chromatography step in the purification, which increased the final yield and allowed us to isolate three different molecular forms of the enzyme. Form 1 is a mixed disulfide between the polypeptide chain and glutathione; form 3 is an S-S dimer of the polypeptide chain present in form 1, while form 2, present in a very low amount, consists of a polypeptide chain quite similar in aminoacid composition to that found in form 1. The three molecular forms show very similar kinetic parameters. The comparison of these molecular forms with those isolated from horse muscle showed similar kinetic properties but different structural features.     

INCREASED INCORPORATION OF [G-3H]LEUCINE INTO A POSSIBLE ''RECEPTOR'' PROTEOLIPID IN DENERVATED MUSCLE IN VIVO

Abstract— The incorporation of radioactive leucine into the total proteins and the proteolipids of normal and denervated rat diaphragm has been studied in vivo. Denervation increased the incorporation of isotopically labelled leucine into each of the isolated proteolipids and the effect was particularly marked in a single proteolipid which has been designated a 'receptor' proteolipid. In normal muscle this particular proteolipid was found to have a higher incorporation of isotopically labelled leucine in the area of the muscle rich in endplates compared with an area devoid of endplates. However the stimulatory effect of denervation on the incorporation of radioactive leucine into this proteolipid was considerably more marked in the latter region. An attempt has been made to correlate these findings with the development of the hypersensitivity to ACh characteristic of denervated muscle.     

Identification and description of beta-structure in horse muscle acylphosphatase by nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance spectra of acylphosphatase were searched for signs of beta-structure, i.e. characteristic nuclear Overhauser enhancement patterns displayed in the two-dimensional spectra, typical chemical shifts, coupling constants and slow 2H-H exchange. The results provided identification of the main-chain resonances of amino acid residues involved in the beta-structure. The full sequential assignment of this region was gained by identification of some amino acid spin systems and their alignment with the primary sequence. The assignment of the side-chains was virtually completed subsequently and a list produced of nuclear magnetic resonance (n.m.r.) constraints derived from the spectra. The beta-structure consists of a beta-sheet with four antiparallel chains, one attached parallel chain, three tight turns and a beta-bulge. The conformation of the beta-sheet was determined by distance geometry calculation using the n.m.r. constraints (174 intraresidual, 107 sequential and 226 long-range distances, 32 torsion angles, phi, and 28 hydrogen bonds) as input. Observation of some interactions between the sheet and previously identified alpha-helical regions made it possible to give an outline of the three-dimensional structure of the enzyme.     

The modulation of calcium currents by the activation of mGluRs

Glutamatergic transmission in the central nervous system (CNS) is mediated by ionotropic, ligand-gated receptors (iGluRs), and metabotropic receptors (mGluRs). mGluRs are coupled to GTP-binding regulatory proteins (G-proteins) and modulate different second messenger pathways. Multiple effects have been described following their activation; among others, regulation of fast synaptic transmission, changes in synaptic plasticity, and modification of the threshold for seizure generation. Some of the major roles played by the activation of mGluRs might depend on the modulation of high-voltage-activated (HVA) calcium (Ca²⁺) currents. Some HVA Ca²⁺ channels (N-, P-, and Q-type channels) are signaling components at most presynaptic active zones. Their mGluR-mediated inhibition reduces synaptic transmission. The interference, by agonists at mGluRs, on L-type channels might affect the repetitive neuronal firing behavior and the integration of complex events at the somatic level. In addition, the mGluR-mediated effects on voltagegated Ca²⁺ signals have been suggested to strongly influence neurotoxicity. Rather different coupling mechanisms underlie the relation between mGluRs and Ca²⁺ currents: Together with a fast, membrane-delimited mechanism of action, much slower responses, involving intracellular second messengers, have also been postulated. In the recent past, the relative paucity of selective agonists and antagonists for the different subclasses of mGluRs had hampered the clear definition of the roles of mGluRs in brain function. However, the recent availability of new pharmacological tools is promising to provide a better understanding of the neuronal functions related to different mGluR subtypes. The analysis of the mGluR-mediated modulation of Ca²⁺ conductances will probably offer new insights into the characterization of synaptic transmission and the development of neuroprotective agents.