Binding of aldolase and triosephosphate dehydrogenase to F-actin and modification of catalytic properties of aldolase.
TL;DR: It was found that fructose-1,6-diphosphate, dihydroxyacetone phosphate, ATP, ADP, and inorganic phosphate cause a marked adsorption hindrance within their physiological concentration ranges, and the binding of triosephosphate dehydrogenase to F-actin is also inhibited most strongly by its substrate glyceraldehydephosphate.
Abstract: The binding of aldolase and triosephosphate dehydrogenase to F-actin was studied in vitro under various conditions. Several monovalent and bivalent cations and anions, most of the glycolytic metabolites, and some nucleotides were examined with respect to their influence on the binding. Desorption of the bound enzymes as well as adsorption hindrance may be due to relatively specific factors (metabolites, ions) and relatively non-specific factors (pH, ionic strength). In the case of aldolase it was found that fructose-1,6-diphosphate, dihydroxyacetone phosphate, ATP, ADP, and inorganic phosphate cause a marked adsorption hindrance within their physiological concentration ranges. In analogy with the effect of fructose-1,6-diphosphate on aldolase, the binding of triosephosphate dehydrogenase to F-actin is also inhibited most strongly by its substrate glyceraldehydephosphate. However, adsorption hindrance of triosephosphate dehydrogenase is also caused by fructose-1,6-diphosphate, ATP, and inorganic phosphate, although higher concentrations of these compounds are needed in comparison with aldolase. Kinetic studies of F-actin-bound aldolase revealed that the properties of the enzyme are modified by the binding. Under the chosen experimental conditions, binding of aldolase to F-actin doubles Vmax and increases Km for fructose-1,6-diphosphate by almost one order of magnitude. Km determinations were performed in the absence of ammonium sulfate. Sulfate and also phosphate anions are strong inhibitors of muscle aldolase. In both cases the inhibition type appears to be competitive. Determinations of Km for fructose-1,6-diphosphate of native muscle aldolase gave values as low as 0.4 μM.
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TL;DR: The three-dimensional intracellular network formed by the filamentous polymers comprising the cytoskeletal affects the way cells sense their extracellular environment and respond to stimuli and can influence ion channel activity at the plasma membrane of cells and conduct mechanical stresses from the cell membrane to internal organelles.
Abstract: Janmey, Paul A. The Cytoskeleton and Cell Signaling: Component Localization and Mechanical Coupling. Physiol. Rev. 78: 763–781, 1998. — The three-dimensional intracellular network formed by the fil...
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TL;DR: The multiple binding capacity of the SDH in conjunction with its GAPDH activity may play a role in the colonization, internalization, and the subsequent proliferation of group A streptococci.
Abstract: The surface of streptococci presents an array of different proteins, each designed to perform a specific function. In an attempt to understand the early events in group A streptococci infection, we have identified and purified a major surface protein from group A type 6 streptococci that has both an enzymatic activity and a binding capacity for a variety of proteins. Mass spectrometric analysis of the purified molecule revealed a monomer of 35.8 kD. Molecular sieve chromatography and sodium dodecyl sulfate (SDS)-gel electrophoresis suggest that the native conformation of the protein is likely to be a tetramer of 156 kD. NH2-terminal amino acid sequence analysis revealed 83% homology in the first 18 residues and about 56% in the first 39 residues with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of eukaryotic or bacterial origin. This streptococcal surface GAPDH (SDH) exhibits a dose-dependent dehydrogenase activity on glyceraldehyde-3-phosphate in the presence of beta-nicotinamide adenine dinucleotide both in its pure form and on the streptococcal surface. Its sensitivity to trypsin on whole organism and its inability to be removed with 2 M NaCl or 2% SDS support its surface location and tight attachment to the streptococcal cell. Affinity-purified antibodies to SDH detected the presence of this protein on the surface of all M serotypes of group A streptococcal tested. Purified SDH was found to bind to fibronectin, lysozyme, as well as the cytoskeletal proteins myosin and actin. The binding activity to myosin was found to be localized to the globular heavy meromyosin domain. SDH did not bind to streptococcal M protein, tropomyosin, or the coiled-coil domain of myosin. The multiple binding capacity of the SDH in conjunction with its GAPDH activity may play a role in the colonization, internalization, and the subsequent proliferation of group A streptococci.
628 citations
TL;DR: An ATPase that accounts for about 0.3% of the total cell protein has been isolated in 90% purity from Acanthamoeba castellanii and shows some activity toward nucleoside triphosphates other than ATP but does not hydrolyze ADP or AMP.
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TL;DR: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified from the breast muscles of 3-week-old chickens and used to raise a specific antiserum in rabbits coupled to an in vitro translation assay to monitor the purification of GAPDH mRNA.
Abstract: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified from the breast muscles of 3-week-old chickens and used to raise a specific antiserum in rabbits. This antiserum was coupled to an in vitro translation assay to monitor the purification of GAPDH mRNA. RNA was isolated from identical breast muscles and consecutively fractionated with several techniques to yield a preparation of GAPDH mRNA which was at least 50% pure. Double-stranded cDNA was made against this purified RNA, inserted into pBR322, and used to transform Escherichia coli. Recombinants were screened by colony filter hybridization with a cDNA probe made against the purified RNA. The hybridization-positive clone with the largest insert, pGAD-28, was then characterized by using pGAD-28-cellulose to select complementary RNA from total poly(A) RNA and then translating the hybridization-selected RNA in vitro. The single translation product was shown to be GAPDH by (1) comigration with pure GAPDH on sodium dodecyl sulfate-polyacrylamide gels, (2) precipitation with specific anti-GAPDH antiserum, (3) cyanylation fingerprinting, and (4) AMP-agarose affinity chromatography. pGAD-28 was mapped with several restriction enzymes and then sequenced by the method of Maxam and Gilbert [Maxam, A. M., & Gilbert, W. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 560]. The 1261-nucleotide insert was found to contain 29 nucleotides of noncoding sequence at the 5' end, the entire coding region, and 230 nucleotides of the 3'-noncoding region including a poly(A) addition signal (AATAAA) and the first five residues of the poly(A) tail.
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TL;DR: In this article, a methodische Teil enthält neben technischen Einzelheiten für die Aufarbeitung im größeren Maßstab eine neue Ableitung zur Errechnung der Salzzugabe für bestimmte Ammonsulfatkonzentrationen and Vorschriften für the Ammonulfat-and Eiweißbestimmung im Routinebetrieb.
Abstract: Extrakt von Kaninchenmuskulatur wird durch steigende Sättigung mit Ammonsulfat in amorphe Fraktionen aufgeteilt, aus denen sich ohne weitere Reinigungsschritte die obengenannten Fermente kristallisieren lassen. Die Reinigung der Rohkristallisate erfolgt durch Waschungen mit Ammonsulfatlösungen und Umkristallisationen. Jedes Ferment wird an Hand seines Absorptionsspektrums, der Umsatzzahl, Kristallform und spezifischen Eigenschaft beschrieben und den entsprechenden reinsten Präparaten anderer Autoren gegenübergestellt. Der methodische Teil enthält neben technischen Einzelheiten für die Aufarbeitung im größeren Maßstab eine neue Ableitung zur Errechnung der Salzzugabe für bestimmte Ammonsulfatkonzentrationen und Vorschriften für die Ammonsulfat- und Eiweißbestimmung im Routinebetrieb.
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TL;DR: The analytical evaluation supports the assumption that the binding of aldolase to F-actin occurs at two different binding sites, and the sigmoid shape of the desorption curve indicates a cooperative mechanism of the binding phenomenon.
Abstract: 1
Only 60–70% of the total activity of aldolase can be extracted from rat or rabbit muscle homogenates with aqueous solutions of relatively low ionic strength. The extraction of aldolase from muscle tissue is only complete in aqueous solutions with an ionic strength greater than 0.2. The fraction of aldolase which is set free at high ionic strength is not located within a special cellular compartment but is present in a bound form and is desorbed in dependence of the ionic strength of the extraction medium.
2
F-actin, myosin, acto-myosin and stroma-protein were prepared from rabbit muscle and the binding of aldolase to each of these structure proteins was studied in vitro. A completely reversible binding of aldolase to F-actin, actomyosin, myosin and stroma protein was found. F-actin possesses by far the highest binding capacity. 100% of the enzyme is bound when 1 mg of aldolase is added to 1 mg of highly purified F-actin. Under identical experimental conditions acto-myosin binds 40%, myosin 25%, and stroma protein only 15% of the aldolase activity.
3
A modified Langmuir isotherm is derived, and the analytical evaluation supports the assumption that the binding of aldolase to F-actin occurs at two different binding sites.
4
The binding of aldolase to F-actin and other structure proteins depends on the ionic strength. At 150 mM KCl, a complete desorption occurs, and 50% of the actin-bound aldolase is set free at a concentration of 80 mM KCl. The sigmoid shape of the desorption curve indicates a cooperative mechanism of the binding phenomenon.
5
Within the physiological range, the pH does not influence the binding of aldolase to F-actin.
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Similar to aldolase, glyceraldehydephosphate dehydrogenase is bound to F-actin, and under the experimental conditions, 1 mg of F-actin binds up to 1.2 mg of glyceraldehydephosphate dehydrogenase.
7
Studies with a purified preparation of myogen reveal that also fructose-6-phosphate kinase, and in a lower degree phosphoglycerate kinase, pyruvate kinase and lactate dehydrogenase can be adsorbed to F-actin. No binding occurs in the case of creatine kinase.
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The possible significance of the binding phenomenon is discussed with respect to the location of the Embden-Meyerhof system at the site of the actin filaments within the isotropic zones of the cross-striated muscle fiber.
265 citations