RESUMEN
The rate of autolysis of mu- and m-calpain from bovine skeletal muscle was measured by using densitometry of SDS polyacrylamide gels and determining the rate of disappearance of the 28 and 80 kDa subunits of the native, unautolyzed calpain molecules. Rate of autolysis of both the 28 and 80 kDa subunits of mu-calpain decreased when mu-calpain concentration decreased and when beta-casein, a good substrate for the calpains, was present. Hence, autolysis of both mu-calpain subunits is an intermolecular process at pH 7.5, 0 or 25.0 degrees C, and low ionic strength. The 78 kDa subunit formed in the first step of autolysis of m-calpain was not resolved from the 80 kDa subunit of the native, unautolyzed m-calpain by our densitometer, so autolysis of m-calpain was measured by determining rate of disappearance of the 28 kDa subunit and the 78/80 kDa complex. At Ca2+ concentrations of 1000 microM or higher, neither the m-calpain concentration nor the presence of beta-casein affected the rate of autolysis of m-calpain. Hence, m-calpain autolysis is intramolecular at Ca2+ concentrations of 1000 microM or higher and pH 7.5. At Ca2+ concentrations of 350 microM or less, the rate of m-calpain autolysis decreased with decreasing m-calpain concentration and in the presence of beta-casein. Thus, m-calpain autolysis is an intermolecular process at Ca2+ concentrations of 350 microM or less. If calpain autolysis is an intermolecular process, autolysis of a membrane-bound calpain would require selective participation of a second, cytosolic calpain, making it an inefficient process. By incubating the calpains at Ca2+ concentrations below those required for half-maximal activity, it is possible to show that unautolyzed calpains degrade a beta-casein substrate, proving that unautolyzed calpains are active proteases.
Asunto(s)
Calpaína/metabolismo , Precursores Enzimáticos/metabolismo , Músculo Esquelético/enzimología , Precursores de Proteínas/metabolismo , Animales , Calcio/farmacología , Caseínas/metabolismo , Caseínas/farmacología , Bovinos , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , CinéticaRESUMEN
Glycosyltransferases A and B utilize the donor substrates UDP-GalNAc and UDP-Gal, respectively, in the biosynthesis of the human blood group A and B trisaccharide antigens from the O(H)-acceptor substrates. These enzymes were cloned as synthetic genes and expressed in Escherichia coli, thereby generating large quantities of enzyme for donor specificity evaluations. The amino acid sequence of glycosyltransferase A only differs from glycosyltransferase B by four amino acids, and alteration of these four amino acid residues (Arg-176-->Gly, Gly-235-->Ser, Leu-266-->Met and Gly-268-->Ala) can change the donor substrate specificity from UDP-GalNAc to UDP-Gal. Crossovers in donor substrate specificity have been observed, i.e., the A transferase can utilize UDP-Gal and B transferase can utilize UDP-GalNAc donor substrates. We now report a unique donor specificity for each enzyme type. Only A transferase can utilize UDP-GlcNAc donor substrates synthesizing the blood group A trisaccharide analog alpha-D-Glcp-NAc-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2 )7CH3 (4). Recombinant blood group B was shown to use UDP-Glc donor substrates synthesizing blood group B trisaccharide analog alpha-D-Glcp-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2) 7CH3 (5). In addition, a true hybrid enzyme was constructed (Gly-235-->Ser, Leu-266-->Met) that could utilize both UDP-GlcNAc and UDP-Glc. Although the rate of transfer with UDP-GlcNAc by the A enzyme was 0.4% that of UDP-GalNAc and the rate of transfer with UDP-Glc by the B enzyme was 0.01% that of UDP-Gal, these cloned enzymes could be used for the enzymatic synthesis of blood group A and B trisaccharide analogs 4 and 5.
Asunto(s)
Sistema del Grupo Sanguíneo ABO , Galactosiltransferasas/metabolismo , N-Acetilgalactosaminiltransferasas/metabolismo , Oligosacáridos/biosíntesis , Trisacáridos/biosíntesis , Secuencia de Carbohidratos , Humanos , Datos de Secuencia Molecular , Oligosacáridos de Cadena Ramificada , Especificidad por Sustrato , Uridina Difosfato Glucosa/metabolismo , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
Several hundred molecules of enzyme reaction products were detected in a single spheroplast from yeast cells incubated with a tetramethylrhodamine (TMR) labeled triglucoside, alpha-d-Glc(1-->2)alpha-d-Glc(1-->3)alpha-d-Glc-O(CH2)8CONHCH2- CH2NH- COTMR. Product detection was accomplished using capillary electrophoresis and laser induced fluorescence following the introduction of a single spheroplast into the separation capillary. The in vivo enzymatic hydrolysis of the TMR-trisaccharide involves at least two enzymes, limited by processing alpha-glucosidase I, producing TMR-disaccharide, TMR-monosaccharide, and the free TMR-linking arm. Hydrolysis was reduced by preincubation of the cells with the processing enzyme inhibitor castanospermine. Confocal laser scanning microscopy studies confirmed the uptake and internalization of fluorescent substrate. This single cell analysis methodology can be applied for the in vivo assay of any enzyme with a fluorescent substrate.
Asunto(s)
Técnicas Citológicas/instrumentación , Glucosidasas/análisis , Glucósidos/metabolismo , Saccharomyces cerevisiae/enzimología , Trisacáridos/metabolismo , Electroforesis Capilar/instrumentación , Colorantes Fluorescentes , Fluorometría/instrumentación , Fluorometría/métodos , Histocitoquímica/instrumentación , Hidrólisis , Rodaminas , Saccharomyces cerevisiae/citologíaRESUMEN
Alpha-glucosidase I is a key enzyme in the biosynthesis of asparagine-linked oligosaccharides catalyzing the first processing event after the en bloc transfer of Glc3Man9GlcNAc2 to proteins. This enzyme is an inhibitor target for anti-viral agents that interfere with the formation of essential glycoproteins required in viral assembly, secretion and infectivity. Of fundamental mechanistic interest for all oligosaccharide hydrolyzing enzymes is the stereochemical course of the reaction which can occur with either retention or inversion of anomeric configuration. The stereochemistry is used to categorize enzymes and is important in designing mechanism-based inhibitors. To determine the stereochemical course of the alpha-glucosidase I reaction, the release of glucose from a synthetic trisaccharide substrate, Glc(alpha1-2)Glc(alpha1-3)Glc alphaO(CH2)8COOCH3 was directly monitored by 1H NMR spectroscopy. Both the yeast and bovine mammary gland enzymes released beta-glucose concomitant with the formation of the Glc(alpha1-3)Glc alphaO(CH2)8COOCH3 disaccharide product demonstrating that both enzymes operate with inversion of anomeric configuration.
Asunto(s)
Glicósido Hidrolasas/química , alfa-Glucosidasas/química , Animales , Secuencia de Carbohidratos , Bovinos , Femenino , Glucosa/química , Hidrólisis , Espectroscopía de Resonancia Magnética , Glándulas Mamarias Animales/química , Datos de Secuencia Molecular , Oligosacáridos/química , Levaduras/químicaRESUMEN
Mammalian semicarbazide-sensitive amine oxidase (SSAO) enzymes have been classified as EC 1.4.3.6 [amine:oxygen oxidoreductase (deaminating)(copper-containing)]. However, both the identity of the quinone cofactor and the presence of copper remain unconfirmed, and SSAO has proved impossible to purify to homogeneity in sufficient yield to permit cofactor identification. To circumvent this problem, we have partially purified SSAO enzymes from bovine and porcine aortae and have established, with a redox-cycling assay, that no other quinoproteins were present in enzyme preparations. Enzymes were then derivatized with (p-nitrophenyl)hydrazine (p-NPH), which forms a covalent yellow complex with the quinone cofactor. Visible absorbance spectra of derivatized bovine and porcine enzymes (respective lambdamax values 456 and 476 nm at neutral pH, shifting to 580 and 584 nm in 2 M KOH) were consistent with the presence of (2,4,5-trihydroxyphenyl)alanine quinone (TPQ) as cofactor. Resonance Raman spectra were essentially identical to that for pea seedling amine oxidase, a known TPQ-containing enzyme. Extensive digestion of SSAO enzymes, and of porcine kidney diamine oxidase, with pronase E yielded species with identical chromophoric properties characteristic of the dipeptide, TPQ(p-NPH)-Asp. Thermolytic digestion of porcine SSAO gave two cofactor-containing peptides that contained a TPQ consensus sequence, Asn-X-Asp-Tyr-Tyr, where X is a blank cycle corresponding to TPQ. N-terminal sequencing of whole enzymes revealed a membrane-spanning region typical of an extracellular type II glycoprotein. These results confirm the presence of TPQ in mammalian membrane-bound SSAO ectoenzymes.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/química , Coenzimas/química , Dihidroxifenilalanina/análogos & derivados , Amina Oxidasa (conteniendo Cobre)/metabolismo , Secuencia de Aminoácidos , Animales , Aorta/enzimología , Bovinos , Cromatografía Líquida de Alta Presión , Coenzimas/metabolismo , Dihidroxifenilalanina/química , Dihidroxifenilalanina/metabolismo , Hidrólisis , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Espectrometría Raman , PorcinosRESUMEN
Five and nine-tenth kg of Elodea densa (Anacharis), a common aquarium plant, was extracted, and the extract was subjected to column chromatographic procedures that successfully purify the two Ca2(+)-dependent proteinases (calpains) and their protein inhibitor (calpastatin) from a variety of animal tissues. Although these procedures purified a protein having 55- and 16-kDa polypeptides, neither this protein nor any of the other chromatographic fractions contained detectable proteinase or calpastatin activity. Moreover, the purified 55- and 16-kDa polypeptides did not react on immunoblots with polyclonal antibodies that were monospecific for the calpains or calpastatin. We conclude that Elodea densa contains no calpain nor calpastatin at the level of 4 micrograms per g plant protein (1 part per 250,000), which was the sensitivity of our assay.
Asunto(s)
Calpaína/metabolismo , Plantas/enzimología , Actinas/metabolismo , Animales , Calcio/farmacología , Proteínas de Unión al Calcio/metabolismo , Bovinos , Cromatografía DEAE-Celulosa , Filamentos Intermedios/efectos de los fármacosRESUMEN
Limited proteolysis of caldesmon has been used in studying the structure-function relationship of this protein. Digestion with alpha-chymotrypsin yields three major fragments of 110, 80 and 40 kDa. Only the 40 kDa fragment preserves functional properties of the parent molecule: it binds to F-actin, causes inhibition of actomyosin ATPase and binds to calmodulin in a Ca2+-dependent manner. Its further degradation produces an 18 kDa polypeptide that also retains all these properties. Neither F-actin nor calmodulin binding induces dramatic changes in susceptibility to chymotryptic cleavage and the sites of cleavage of caldesmon.
Asunto(s)
Proteínas de Unión a Calmodulina/análisis , Adenosina Trifosfatasas/metabolismo , Animales , Sitios de Unión , Calcio/metabolismo , Calmodulina/metabolismo , Pollos , Quimotripsina/metabolismo , Electroforesis en Gel de Poliacrilamida , Molleja de las Aves/enzimología , Peso Molecular , Fragmentos de Péptidos/análisis , Conejos , Relación Estructura-ActividadRESUMEN
Caldesmon induces inhibition of MG2+-ATPase activity of actomyosin and relaxation of skinned fibers of chicken gizzard smooth muscle without influencing the level of myosin light chain-1 phosphorylation. Both these effects are reversed by calmodulin at a high molar excess over caldesmon in the presence of Ca2+.
Asunto(s)
Actomiosina/metabolismo , ATPasa de Ca(2+) y Mg(2+)/antagonistas & inhibidores , Proteínas de Unión a Calmodulina/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/fisiología , Animales , Calcio/farmacología , Calmodulina/farmacología , Proteínas de Unión a Calmodulina/antagonistas & inhibidores , Molleja No Aviar , Cinética , Miosinas/metabolismo , FosforilaciónRESUMEN
Filamin binds to F-actin and influences the myosin-actin interaction. At relatively low concentrations, filamin activates actomyosin Mg2+-ATPase, whereas higher concentrations of filamin exert an inhibitory effect. Activation of ATPase activity occurs under conditions where a loose meshwork of actin filaments is present and inhibition is associated with the appearance of closely apposed bundles of actin filaments. Maximum activation (about fourfold) of actomyosin ATPase activity by filamin occurs between 30 and 65 mM KCl, at pH 6.5, and at temperatures not less than 30 degrees C. ATPase activation requires higher concentrations of filamin in the presence than in the absence of tropomyosin. Filamin does not activate Mg2+-ATPase activity of acto-subfragment-1 and has only a slight effect on the Mg2+-ATPase of acto-heavy meromyosin, but it inhibits the activity of both these systems under conditions similar to those that inhibit actomyosin ATPase activity.
Asunto(s)
Actomiosina/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/farmacología , Proteínas Contráctiles/farmacología , Proteínas de Microfilamentos/farmacología , Músculos/enzimología , Actinas/metabolismo , Animales , ATPasa de Ca(2+) y Mg(2+) , Pollos , Proteínas Contráctiles/aislamiento & purificación , Activación Enzimática , Filaminas , Cinética , Magnesio/farmacología , Cloruro de Magnesio , Proteínas de Microfilamentos/aislamiento & purificación , Microscopía Electrónica , ConejosRESUMEN
It was found that thin filaments from chicken gizzard muscle activate skeletal muscle myosin Mg2+-ATPase to a greater extent than does the complex of chicken gizzard actin and tropomyosin. The protein factor responsible for this additional activation has been now identified as the high Mr actin binding protein, filamin.