RESUMEN
The present review focuses on the background and progress that led to discovery of specific inhibition of post-Amadori formation of advanced glycation end products, or AGEs. The "classic" or Hodge pathway begins with glucose condensation with amino groups to form a Schiff base aldimine adduct that undergoes rearrangement to a ketoamine Amadori product. This pathway is considered an important route to AGE formation that has been implicated in glucose-mediated damage in vivo (3-5). We recently described a facile procedure for isolation of proteins rich in Amadori adducts but free of AGEs, thus permitting study of pathways of conversion of Amadori compounds to AGEs. This in turn led to a unique and rapid post-Amadori screening assay for putative "Amadorins," which we define here as inhibitors of the conversion of Amadori intermediates to AGEs in the absence of excess free or reversibly bound (Schiff base) sugar. Our screening assay then led to the identification of pyridoxamine (Pyridorin) as the first member of this class of Amadorin compounds. Rather unexpectedly, the assay also led to the clear demonstration that the well-known AGE inhibitor aminoguanidine, currently in Phase 3 clinical trials for treatment of diabetic nephropathy, has negligible Amadorin activity. In view of the importance of Amadori compounds as intermediates in AGE formation in vivo, the therapeutic potential of Pyridorin is currently being investigated and is now showing highly promising results in different animal models.
Asunto(s)
Productos Finales de Glicación Avanzada/metabolismo , Piridoxamina/metabolismo , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Diseño de Fármacos , Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Productos Finales de Glicación Avanzada/biosíntesis , Productos Finales de Glicación Avanzada/química , Guanidinas/metabolismo , Guanidinas/uso terapéutico , Piridoxamina/química , Piridoxamina/uso terapéutico , Bases de SchiffRESUMEN
Type IV collagen alpha1-alpha6 chains have important roles in the assembly of basement membranes and are implicated in the pathogenesis of Goodpasture syndrome, an autoimmune disorder, and Alport syndrome, a hereditary renal disease. We report comparative sequence analyses and structural predictions of the noncollagenous C-terminal globular NC1 domain (28 sequences). The inferred tree verified that type IV collagen sequences fall into two groups, alpha1-like and alpha2-like, and suggested that vertebrate alpha3/alpha4 sequences evolved before alpha1/alpha2 and alpha5/alpha6. About one fifth of NC1 residues were identified to confer either the alpha1 or alpha2 group-specificity. These residues accumulate opposite charge in subdomain B of alpha1 (positive) and alpha2 (negative) sequences and may play a role in the stoichiometric chain selection upon type IV collagen assembly. Neural network secondary structure prediction on multiple aligned sequences revealed a subdomain core structure consisting of six hydrophobic beta-strands and one short alpha-helix with a significant hydrophobic moment. The existence of opposite charges in the alpha-helices may carry implications for intersubdomain interactions. The results provide a rationale for defining the epitope that binds Goodpasture autoantibodies and a framework for understanding how certain NC1 mutations may lead to Alport syndrome. A search algorithm, based entirely on amino acid properties, yielded a possible similarity of NC1 to tissue inhibitor of metalloproteinases (TIMP) and prompted an investigation of a possible functional relationship. The results indicate that NC1 preparations decrease the activity of matrix metalloproteinases 2 and 3 (MMP-2, MMP-3) toward a peptide substrate, though not to [14C]-gelatin. We suggest that an ancestral NC1 may have been incorporated into type IV collagen as an evolutionarily mobile domain carrying proteinase inhibitor function.
Asunto(s)
Colágeno/química , Colágeno/fisiología , Fragmentos de Péptidos/química , Algoritmos , Secuencia de Aminoácidos , Fenómenos Químicos , Química Física , Secuencia de Consenso , Gelatinasas/química , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 3 de la Matriz/química , Metaloendopeptidasas/química , Datos de Secuencia Molecular , Filogenia , Estructura Secundaria de Proteína , Alineación de Secuencia , Relación Estructura-Actividad , Inhibidores Tisulares de Metaloproteinasas/químicaRESUMEN
The glomerular basement membrane (GBM) is damaged in diabetes through complex mechanisms that are not fully understood. Prominent among them is nonenzymatic protein glycation leading to the formation of so-called advanced glycation end products (AGEs). We examined the effects of in vitro glycation of intact collagen type IV in bovine lens capsule (LBM) and kidney glomerular (GBM) basement membranes on their susceptibility to matrix metalloproteinases, using stromelysin 1 (MMP-3) and gelatinase B (MMP-9). Sites of cleavage of unmodified LBM collagen were located in the triple helical region. In vitro glycation by glucose severely inhibited the release of soluble collagen cleavage peptides by MMP-3 and MMP-9. The distribution of AGEs within the three domains of collagen IV (7S, triple helical, and noncollagenous NC1) were compared for LBM glycation using AGE fluorescence, pentosidine quantitation, and immunoreactivity towards anti-AGE antibodies that recognize the AGE carboxymethyllysine (CML). Marked asymmetry was observed, with the flexible triple helical domain having the most pentosidine and fluorescent AGEs but the least CML. The in vivo relevance of these findings is supported by preliminary studies of AGE distribution in renal basement membrane (RBM) collagen IV domains from human kidneys of two insulin-dependent diabetics and one normal subject. Pentosidine and fluorescent AGE distributions of diabetic RBM were similar to LBM, but the CML AGE in diabetic kidney was less in the triple helical domain than in NC1. Our results support the hypothesis that nonenzymatic glycation of collagen IV contributes to the thickening of basement membranes, a hallmark of diabetic nephropathy.
Asunto(s)
Colágeno/metabolismo , Colagenasas/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Adulto , Secuencia de Aminoácidos , Animales , Membrana Basal/metabolismo , Bovinos , Femenino , Glicosilación , Humanos , Glomérulos Renales/metabolismo , Masculino , Metaloproteinasa 9 de la Matriz , Datos de Secuencia MolecularRESUMEN
Nonenzymatic protein glycation (Maillard reaction) leads to heterogeneous, toxic, and antigenic advanced glycation end products ("AGEs") and reactive precursors that have been implicated in the pathogenesis of diabetes, Alzheimer's disease, and normal aging. In vitro inhibition studies of AGE formation in the presence of high sugar concentrations are difficult to interpret, since AGE-forming intermediates may oxidatively arise from free sugar or from Schiff base condensation products with protein amino groups, rather than from just their classical Amadori rearrangement products. We recently succeeded in isolating an Amadori intermediate in the reaction of ribonuclease A (RNase) with ribose (Khalifah, R. G., Todd, P., Booth, A. A., Yang, S. X., Mott, J. D., and Hudson, B. G. (1996) Biochemistry 35, 4645-4654) for rapid studies of post-Amadori AGE formation in absence of free sugar or reversibly formed Schiff base precursors to Amadori products. This provides a new strategy for a better understanding of the mechanism of AGE inhibition by established inhibitors, such as aminoguanidine, and for searching for novel inhibitors specifically acting on post-Amadori pathways of AGE formation. Aminoguanidine shows little inhibition of post-Amadori AGE formation in RNase and bovine serum albumin, in contrast to its apparently effective inhibition of initial (although not late) stages of glycation in the presence of high concentrations of sugar. Of several derivatives of vitamins B1 and B6 recently studied for possible AGE inhibition in the presence of glucose (Booth, A. A., Khalifah, R. G., and Hudson, B. G. (1996) Biochem. Biophys. Res. Commun. 220, 113-119), pyridoxamine and, to a lesser extent, thiamine pyrophosphate proved to be novel and effective post-Amadori inhibitors that decrease the final levels of AGEs formed. Our mechanism-based approach to the study of AGE inhibition appears promising for the design and discovery of novel post-Amadori AGE inhibitors of therapeutic potential that may complement others, such as aminoguanidine, known to either prevent initial sugar attachment or to scavenge highly reactive dicarbonyl intermediates.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Animales , Bovinos , Ensayo de Inmunoadsorción Enzimática , Guanidinas/farmacología , Hemoglobinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Cinética , Piridoxamina/farmacología , Ribonucleasa Pancreática/metabolismo , Ribosa/metabolismo , Albúmina Sérica/metabolismo , Tiamina Pirofosfato/farmacologíaRESUMEN
Nonenzymatic glycation (Maillard reaction) of long-lived proteins is a major contributor to the pathology of diabetes and possibly aging and Alzheimer's disease. We report here kinetic studies of the glycation of the model protein ribonuclease A by glucose and ribose leading to the formation of antigenic advanced glycation end products ("AGEs"), detectable by AGE-specific polyclonal antibodies, and pentosidine, an acid-stable fluorescent AGE. As anticipated, the kinetics of glycation by ribose were considerably faster than by glucose, and the rate of AGE formation initially increased with increasing sugar concentrations. However, ribose above 0.15 M appeared to paradoxically slow the kinetics of AGE formation, suggesting ribose inhibits the conversion of "early" Amadori rearrangement products to "late" AGEs and thus favors the accumulation of reactive Amadori intermediates. The facile isolation of such protein intermediates was achieved by an "interrupted glycation" protocol which free and reversibly bound (Schiff base) ribose was removed following a short (24h) initial incubation of 0.5 M ribose at 37 degrees C. The kinetics of buildup of the Amadori intermediates and the kinetics of their post-Amadori conversion to antigenic AGEs were independently studied. A rapid and reversible inhibition of the post-Amadori kinetics by free ribose was verified by direct re-addition of ribose to the isolated, sugar-free intermediate. The pH dependence of the kinetics of antigenic AGE formation from such intermediates was measured and exhibited an unusual bell-shaped profile over the pH range of 5.0-9.5 with a maximum near pH 8.0. Aminoguanidine, a pharmacological AGE inhibitor, was found to moderately or weakly inhibit antigenic AGE formation in such post- Amadori steps. The isolation of the glycated ribonuclease intermediate thus simplifies kinetic and mechanistic studies of AGE formation, permits AGE studies in the absence of complications arising from free or Schiff base bound sugar, and provides a novel methodology for evaluating the mechanism and efficacy of therapeutic agents that may inhibit AGE formation.
Asunto(s)
Glucosa/metabolismo , Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Ribonucleasa Pancreática/metabolismo , Ribosa/metabolismo , Anticuerpos , Arginina/análogos & derivados , Arginina/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Lisina/análogos & derivados , Lisina/metabolismo , Reacción de Maillard , Albúmina Sérica BovinaRESUMEN
Nonenzymatic glycation of proteins by glucose leading to the formation of toxic and immunogenic advanced glycation end products (AGEs) may be a major contributor to the pathological manifestations of diabetes mellitus, aging, and, possibly, neurodegenerative diseases such as Alzheimer's. We tested the in vitro inhibition of antigenic AGE formation on bovine serum albumin, ribonuclease A, and human hemoglobin by various vitamin B1 and B6 derivatives. Among the inhibitors, pyridoxamine and thiamine pyrophosphate potently inhibited AGE formation and were more effective than aminoguanidine, suggesting that these two compounds may have novel therapeutic potential in preventing vascular complications of diabetes. An unexpected finding was that aminoguanidine inhibited the late kinetic stages of glycation much more weakly than the early phase.
Asunto(s)
Productos Finales de Glicación Avanzada/biosíntesis , Productos Finales de Glicación Avanzada/inmunología , Guanidinas/farmacología , Piridoxamina/farmacología , Tiamina Pirofosfato/farmacología , Animales , Antígenos/biosíntesis , Bovinos , Complicaciones de la Diabetes , Diabetes Mellitus/metabolismo , Inhibidores Enzimáticos/farmacología , Glicosilación , Humanos , Técnicas In Vitro , Masculino , Metahemoglobina/química , Metahemoglobina/efectos de los fármacos , Metahemoglobina/metabolismo , Conejos , Ribonucleasa Pancreática/química , Ribonucleasa Pancreática/efectos de los fármacos , Ribonucleasa Pancreática/metabolismo , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/efectos de los fármacos , Albúmina Sérica Bovina/metabolismoRESUMEN
The visible spectrum of Co(II)-substituted human carbonic anhydrase I (HCA I) complexed with the unique CO2-competitive inhibitor imidazole undergoes a marked alkaline intensification, with a midpoint near pH 8 [Bauer, R., Limkilde, P., & Johansen, J. T. (1977) Carlsberg Res. Commun. 42, 325-339]. This change was first attributed to the ionization of a nondisplaced water ligand of the active-site metal in a five-coordinate complex. Later proposals favored assigning it to the deprotonation of the bound imidazole itself to give a tetrahedrally coordinated imidazolate anion at high pH. We have determined by isothermal titration calorimetry the pH dependence of the enthalpy of binding of imidazole and its analogues to HCA I and Co(II)HCA I. We devised an indirect strategy whereby the enthalpy of binding of the strong sulfonamide inhibitor methazolamide was determined in the absence and presence of a constant high concentration of the competing imidazole or its analogues. The standard enthalpy of binding of deprotonated methazolamide to the "acid" form of HCA I and Co(II)HCA I was found to be pH independent over the pH range of 6.5-9.5, as expected. It was also identical for both the zinc (-13.5 +/- 1.1 kcal M-1) and the cobalt (-13.7 +/- 0.4 kcal M-1) forms. The standard enthalpy of binding of neutral imidazole (average value -6.1 +/- 0.8 kcal M-1) surprisingly did not show any marked pH dependence, varying by about 1.1 and 2.6 kcal M-1 for the zinc and cobalt enzymes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Calorimetría , Dióxido de Carbono/metabolismo , Inhibidores de Anhidrasa Carbónica/metabolismo , Anhidrasas Carbónicas/metabolismo , Imidazoles/metabolismo , Unión Competitiva , Cobalto/metabolismo , Eritrocitos/enzimología , Humanos , Concentración de Iones de Hidrógeno , Metazolamida/metabolismo , Termodinámica , Zinc/metabolismoRESUMEN
We previously showed that the zinc metalloenzyme carbonic anhydrases (CA I and II isozymes) bind "neutral" amides and related compounds as anions through coordination of their deprotonated amide nitrogen to the active site zinc (Rogers, J. I., Mukherjee, J., and Khalifah, R. G. (1987) Biochemistry 26, 5672-5679). Urethan, the ethyl carbamate ester, was among such compounds. The present study was designed to test whether other N-unsubstituted carbamate esters of pharmacological interest (as sedatives, hypnotics, anxiolytics, and skeletal muscle relaxants) were capable of binding to CA in the same manner. We studied the interaction of human CA I and II with urethan, phenyl carbamate, ethinamate, meprobamate, and methocarbamol. Phenyl carbamate studies were greatly complicated by its uncatalyzed hydrolysis via an elimination mechanism to form cyanate, a powerful CA inhibitor. In general, the compounds display: 1) slow on-off inhibition binding kinetics in the seconds range, 2) maximal inhibitor affinity at alkaline pH, and 3) characteristic three-band visible spectra of their complexes with cobalt-substituted CA I. These properties are shared with the previously studied amide inhibitors and are taken as evidence that the deprotonated carbamate nitrogen coordinates to the active site metal ion. CA I appeared to bind carbamate esters more tightly than CA II, an unusual 1000-fold selectivity being seen in the case of methocarbamol. The inhibition by these drugs is not sufficiently strong to implicate CA I and II in their mechanism of action. However, it does suggest the possible existence of previously unsuspected similarities between binding to CA and to their physiological receptors or targets, particularly the involvement of zinc.
Asunto(s)
Carbamatos/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/sangre , Eritrocitos/enzimología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/sangre , Sitios de Unión , Carbamatos/química , Ésteres , Humanos , Cinética , Matemática , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Espectrofotometría , Relación Estructura-ActividadRESUMEN
We have utilized phosphorus nuclear magnetic resonance, which provides an excellent means of characterizing the physical state of lipids, to investigate the polymorphic phase behavior of pure dielaidoylphosphatidylethanolamine (DEPE). We have observed a sharp isotropic component in the typical bilayer and inverted hexagonal P-31 NMR spectra. This component appears in the spectra of both the bilayer and inverted hexagonal lipid phases after several cycles through the bilayer-to-hexagonal phase transition. The magnitude of the isotropic component increased as a function of the number of cycles through the transition. The appearance of this component was not a function of time at constant temperature, but only a function of the number of cycles through the transition. The isotropic component is stable at all temperatures above the gel-to-liquid crystal transition, but it abruptly disappears when the lipid is cooled below the gel-to-liquid crystal phase transition. It is suggested that this isotropic phase is similar to the isotropic phase observed in dioleoylphosphatidylethanolamine (DOPE) by x-ray diffraction and identified as a cubic phase (Shyamsunder, E., S. M. Gruner, M. W. Tate, D. C. Turner, P. T. C. So, and C. P. S. Tilcock. 1988. Biochemistry. 27:2332-2336).
Asunto(s)
Liposomas , Fosfatidiletanolaminas , Membrana Dobles de Lípidos , Espectroscopía de Resonancia Magnética/métodos , Conformación Molecular , FósforoRESUMEN
The polymorphic phase behavior of dielaidoylphosphatidylethanolamine (DEPE) has been investigated using spectrophotometry and 31P nuclear magnetic resonance (NMR). It has been demonstrated that the bilayer to inverted hexagonal phase transition can be observed by spectrophotometry. The effects of the methanol, ethanol, and propanol on both the gel to liquid crystal transition and the bilayer to inverted hexagonal transition were investigated by spectrophotometry. It was shown that these alcohols shift the gel to liquid-crystalline phase transition to lower temperature, whereas the bilayer to inverted hexagonal phase transition is shifted to higher temperatures by these alcohols. The structural transition between the bilayer and inverted hexagonal phase of pure DEPE was also investigated by 31P-NMR.
Asunto(s)
1-Propanol/farmacología , Etanol/farmacología , Membrana Dobles de Lípidos , Metanol/farmacología , Fosfatidiletanolaminas , Espectroscopía de Resonancia Magnética , Espectrofotometría , TemperaturaRESUMEN
The interactions of carbon-13 enriched butanol with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were studied using C-13 nuclear magnetic resonance. It was found that above the gel to liquid crystal phase transition the resonance from the butanol could be resolved into two signals with similar chemical shifts but different T1 values and line widths. In contrast, only one narrow resonance was observed for ethanol, which has considerably less solubility in the lipids than butanol. Thermodynamic analyses of the effects of butanol on the phase transition temperature predict much greater solubility or butanol when the lipid is above the phase transition temperature than when it is below. It was concluded that the two butanol resonances represent two slowly exchanging populations, the free butanol in the aqueous phase and butanol dissolved in the liquid crystalline region of the lipid. No bound butanol was detected below the gel to liquid crystal phase transition. Relaxation studies were performed on the resonance of the bound butanol in DPPC and DMPC, including measurements of T1, line width, and nuclear Overhauser enhancement. Theoretical analysis of the relaxation parameters indicates that the lipid-bound alcohol has very high mobility within the fluid lipid bilayer. The data are consistent with butanol being present at the aqueous boundary or head group region of the lipid.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/metabolismo , Butanoles/metabolismo , Dimiristoilfosfatidilcolina/metabolismo , Isótopos de Carbono , Fenómenos Químicos , Química Física , Cristalización , Geles , Espectroscopía de Resonancia MagnéticaRESUMEN
Previous studies on the interaction of carbonic anhydrase (CA) with the unique CO2 competitive inhibitor imidazole and related compounds were all interpreted as showing that an ionizable water ligand on the metal of this zinc metalloenzyme is not displaced by inhibitor binding. Internal inconsistencies in the pH dependence of binding and the pH dependence of the visible spectra of complexes with cobalt-substituted enzyme prompted us to reinvestigate this binding. Visible spectroscopy was used to measure the binding of imidazole and 1,2,4-triazole to Co(II)-substituted human CA I and active site carboxymethylated human CA I (CmCA I) and the binding of 1,2,4-triazole to bovine CoIICA II. The limiting visible spectra for these enzyme-inhibitor adducts were also computed and examined for pH dependence. It was shown that the pKa of visible spectral changes can be independently predicted from studies on the pH dependence of binding. After consideration of possible contributions from effects of His-200 ionization in CA I and CmCA I, and His-64 in CA II, the pH effects on binding affinity and spectra were found to be of the correct magnitude to establish linkage between binding and an ionization. It was also shown, however, that pH effects on binding and spectra cannot distinguish whether neutral imidazole binds to both ionization forms of the enzyme (Zn-OH2 and Zn-OH) or whether neutral imidazole and its anion both bind to only the acid form of the enzyme, presumably after displacing the water. These findings have implications to the crystallographic interpretations on the imidazole-enzyme complex and to the catalytic mechanism of CO2 hydration.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/sangre , Imidazoles/farmacología , Animales , Sitios de Unión , Bovinos , Cobalto/farmacología , Humanos , Concentración de Iones de Hidrógeno , Isoenzimas/antagonistas & inhibidores , Isoenzimas/sangre , Cinética , Espectrofotometría , Triazoles/farmacologíaRESUMEN
Most carbonic anhydrase (CA) inhibitors bind at the active site metal and either are anions or are capable of deprotonation to yield anions. Much less is known about the interaction of CA with inhibitors that have hitherto been considered to bind as neutral species. We report a study of the reversible amide inhibition of Co(II)-substituted CA by iodoacetamide and ethyl carbamate (urethane), as well as the ambivalent oxamate, the monoamide of oxalate. Visible cobalt spectral changes indicate coordination of all these inhibitors to the metal. The pH dependence of the affinity of carbonic anhydrase isozyme I (CA I) for ethyl carbamate and iodoacetamide is formally consistent with their binding either as anionic species to the acid form of the enzyme or as neutral species to the basic form of the enzyme. The former view is in better accord with the spectral data. Most strikingly, reversible binding of iodoacetamide and ethyl carbamate leads to uniquely slow kinetics of ligand association and dissociation that could be followed by simple mixing. The slow association kinetics suggest the involvement of energetically unfavorable deprotonation of the amide group preceding final coordination. The complex pH profile for inhibition of CA I by the ambivalent oxamate is consistent with coordination through the carboxylate group at low pH and through the deprotonated amide group at high pH. The visible spectrum of the complex of Co(II)CA I with oxamate shows a parallel dependence on pH, reflecting this dual coordination mode. Similarly, oxamate dissociation kinetics were biphasic and could be correlated with the pH-dependent spectral changes.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Anhidrasas Carbónicas/sangre , Eritrocitos/enzimología , Yodoacetamida/farmacología , Yodoacetatos/farmacología , Sitios de Unión , Unión Competitiva , Inhibidores de Anhidrasa Carbónica/farmacología , Cobalto , Humanos , Concentración de Iones de Hidrógeno , Isoenzimas/antagonistas & inhibidores , Isoenzimas/sangre , Cinética , Matemática , Modelos Teóricos , Unión Proteica , Espectrofotometría , UretanoRESUMEN
The interactions of butanol and mixtures of butanol and ethanol with dipalmitoylphosphatidyl choline (DPPC) liposomes have been investigated by both spectrophotometric measurements and Fourier transform 13C nuclear magnetic resonance spectroscopy. The spectrophotometric experiments indicate that butanol exhibits the same effects on the thermotropic properties of DPPC as the other short chain alcohols, methanol, ethanol and propanol, which have been shown to be characteristic of the alcohol induced transition of the lipid to the interdigitated state. An additive effect of butanol and ethanol on the induction of the interdigitated phase in DPPC was also observed. A decrease in line width and increase in T1 of the choline methyl signal were observed in the 13C-NMR experiments conducted at 32 degrees C when butanol was added to DPPC in increasing amounts suggesting an increase of disorder in the head group region of the lipid. Addition of ethanol to the NMR sample containing butanol produced hysteresis in the heating and cooling curves characteristic of the interdigitated state. In the interdigitated state, the choline methyl signal exhibited a T1 value equal to that when the lipid is in the fluid state. The increase of mobility in the head group region in the interdigitated gel state relative to the bilayer gel can be rationalized by the increase in surface area in that site when the lipid interdigitates.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina , Butanoles , Etanol , Liposomas , 1-Butanol , Espectroscopía de Resonancia Magnética/métodos , Espectrofotometría/métodos , TermodinámicaRESUMEN
The content of carbonic anhydrase isozymes I (CA I) and II (CA II) in red blood cells and bone of osteopetrotic microphthalmic (mi/mi) mice was analyzed. Monospecific rabbit polyclonal antibodies against purified rat carbonic anhydrase I or II detected both isozymes in hemolysates of both normal and mi/mi mice. Total carbonic anhydrase activity measurements of hemolysates from normal or mi/mi mice were identical. A procedure based on bromopyruvate inactivation was devised to measure the relative contributions of CA I and II isozymes to the carbon dioxide hydration activity of hemolyzates. CA II dominates the observed activity of hemolysates of normal and mi/mi mice. Immunohistochemical studies showed that CA II was present in osteoclasts of tibial and calvarial bones of both normal and mi/mi mice. Thus, in contrast to the several cases of inherited osteopetrosis in humans, there is no lack of active CA II in erythrocytes of mi/mi mice. The absence of CA II, therefore, does not appear to play a role in the etiology of osteopetrosis in the mi/mi mouse.
Asunto(s)
Anhidrasas Carbónicas/análisis , Eritrocitos/enzimología , Isoenzimas/análisis , Osteoclastos/enzimología , Osteopetrosis/enzimología , Animales , Represión Enzimática , Ratones , Tibia/enzimologíaRESUMEN
Human carbonic anhydrase I (EC 4.2.1.1), the low-activity isozyme, has a reactive active-site Histidine-200 that is known to be specifically modified at N tau with haloacetates. Using [1-13C]bromoacetate, we previously introduced a highly sensitive 13C NMR probe into the active site of the enzyme and studied the interaction of the carboxymethyl carboxylate with the active-site zinc, as well as the ionization properties of the carboxymethylated histidine-200 side chain. In the present work, these studies have been extended to metalloderivatives of the enzyme in which the intrinsic zinc has been replaced by Cd2+, Hg2+, and Co2+. In the former two metals, spin-1/2 isotopes (113Cd and 199Hg) in the absence of inhibitory halides were utilized to search for two-bond spin-spin couplings in the spectrum of the 13C-enriched carboxymethyl carboxylate under conditions where coordination exists in the Zn and Co derivatives. The absence of splittings and titration studies of the chemical shift of the resonance both established the absence of coordination. The pH dependence of the carboxylate, which reflects the ionization of the CmHis-200 ring, was observed in the presence and absence of bound inhibitors. Marked differences were seen among the four metalloderivatives in all these properties, suggesting great sensitivity of the active site to the nature of the metal inserted. The data suggest extreme caution in extrapolating results from metal ion substitution studies to the native zinc enzyme, and may reflect functional significance of this sensitivity in the catalysis.
Asunto(s)
Anhidrasas Carbónicas/metabolismo , Metales/metabolismo , Sitios de Unión , Cadmio/metabolismo , Isótopos de Carbono , Inhibidores de Anhidrasa Carbónica/metabolismo , Cobalto/metabolismo , Histidina , Humanos , Isoenzimas/metabolismo , Espectroscopía de Resonancia Magnética , Mercurio/metabolismo , Zinc/metabolismoRESUMEN
Using bromo[1-13C]acetate to modify N tau of His-200 of human carbonic anhydrase isozyme I leads to the introduction of a useful 13C NMR probe into the active site. To complement our previous diamagnetic NMR studies with this probe, we have now succeeded in directly observing the paramagnetically perturbed resonance of the carboxylate in the cobalt-substituted modified enzyme above pH 8. In the pH range 8-10, the resonance undergoes a pH-dependent slow-exchange process, with the more alkaline form having a much smaller pseudocontact shift and a narrower line width. Below pH 8, the resonance apparently undergoes a very large paramagnetic downfield shift that was estimated by extrapolation. An ionization of approximate pK of 6 appears to control this process. Paramagnetic spin-relaxation studies on the resonance under conditions where it was directly observed yielded distance measurements between the carboxylate carbon and the active site cobalt ion. In inhibitor complexes, this distance was in the range of 5-7 A. In the absence of inhibitors, the distance was approximately 3.0-3.2 A at pH 7.9, consistent with the coordination of the carboxylate to the metal. However, at pH 10, the distance was increased to 4.8 A. These distance determinations were aided by relaxation measurements of a paramagnetically shifted proton resonance at 60-65 ppm downfield assigned by others to a proton of a ligand histidine of metal and confirmed by us to be 5.2 +/- 0.1 A from the metal. Our findings provide a molecular basis for the observed changes in catalytic properties that accompany the carboxymethylation.
Asunto(s)
Anhidrasas Carbónicas/metabolismo , Isoenzimas/metabolismo , Acetatos/farmacología , Sitios de Unión , Inhibidores de Anhidrasa Carbónica/metabolismo , Cobalto , Eritrocitos/enzimología , Histidina/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , MatemáticaAsunto(s)
ortoaminobenzoatos/metabolismo , Adulto , Biotransformación , Femenino , Humanos , Cinética , Masculino , Unión Proteica , Factores de Tiempo , Distribución TisularRESUMEN
Metabolites of tolfenamic acid appearing in human urine have been isolated and their structures determined by C-13 nuclear magnetic resonance and gas chromatography-mass spectrometry. Comparative studies on tolfenamic, mefenamic, and flufenamic acids in conjunction with the metabolites have permitted complete C-13 NMR assignments for this series of compounds. Five metabolites identified included three that were monohydroxylated, one that was both methoxylated and hydroxylated, and another in which the methyl group was oxidized to a carboxyl group. The information presented on the fenamate standards and the metabolites represents an excellent basis for structural elucidation of other fenamates and their metabolites.