RESUMO
BACKGROUND: Factor XIII (FXIII) is a transglutaminase that cross-links fibrin and other proteins to improve clot strength and resistance to fibrinolysis. Both congenital and acquired FXIII deficiency may result in a bleeding diathesis, and plasma-derived FXIII has been used to treat many of these clinical conditions. OBJECTIVES: A clinical study was designed and performed to evaluate the safety, pharmacokinetics, and immunogenicity of recombinant FXIII (rFXIII) administration to healthy adult volunteers. PATIENTS AND METHOD: Fifty healthy adult volunteers were enrolled in this randomized, double-blinded, placebo-controlled study. A single dose of rFXIII, ranging from 2 U kg(-1) to 50 U kg(-1), or placebo was administered. Safety was evaluated by capturing adverse events, clinical safety laboratory studies, and clinical score for deep venous thrombosis. Blood samples were taken for pharmacokinetic and immunogenicity analysis throughout the 28-day follow-up period. RESULTS: Recombinant FXIII was well tolerated, with no serious adverse events or dose-related toxicities. Following a single i.v. injection of 50 U kg(-1) rFXIII, the estimated terminal half-life was 270-320 h, the volume of distribution ranged from 40 to 75 mL kg(-1), and FXIII activity increased 1.77% per 1 U kg(-1) rFXIII administered. Increase in circulating A2B2 and decrease in free FXIII-B subunit indicate in vivo formation of FXIII heterotetramer. An immunogenic response to rFXIII or yeast, the production host, was not observed. CONCLUSIONS: Recombinant FXIII was well tolerated at doses of up to 50 U kg(-1) in healthy adult volunteers. The safety, pharmacological and immunological profile of rFXIII suggests it should be studied in patients with congenital FXIII deficiency as well as evaluated as a systemic hemostat in patients with acquired FXIII deficiency or hemorrhage.
Assuntos
Deficiência do Fator XIII/tratamento farmacológico , Fator XIII/química , Fator XIII/farmacocinética , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Adolescente , Adulto , Calibragem , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática , Feminino , Fibrinólise , Humanos , Masculino , Pessoa de Meia-Idade , Placebos , Fatores de Tempo , Trombose Venosa/tratamento farmacológicoRESUMO
The process of heat denaturation of recombinant factor XIII (rFXIII), as well as its C-terminal 24 kDA and 12 kDa elastase-produced fragments starting at Ser514 and Thr628, respectively, was investigated in a wide range of conditions by fluorescence, CD and differential scanning calorimetry (DSC). It was found that the intact protein melts in two distinct temperature regions reflecting unfolding of different parts of the molecule with different stability. The less stable structures unfold in a low temperature transition with a tm of 69 degrees C or lower depending on conditions. Unfolding of the more stable structures was observed at extremely high temperatures, tm > 110 degrees C at acidic pH < 3.5 and tm = 90 degrees C at pH 8.6 with 2 M GdmCL. Thermodynamic analysis of the low and high temperature DSC-obtained heat absorption peaks indicated unambiguously that the first represents melting of three thermolabile independently folded domains while two thermostable domains melt in the second one giving a total of five domains in each a subunit of rFXIII. Both 24 kDa and 12 kDa fragments exhibited a sigmoidal spectral transition at comparatively high temperature where the thermolabile structures are already denatured, indicating that two thermostable domains are formed by the C-terminal portion of rFXIII and correspond to the two beta-barrels revealed by crystallography. The remaining 56 kDa portion forms three thermolabile domains, one of which corresponds to the N-terminal beta-sandwich and the other two to the catalytic core. Fast accessible surface calculations of the X-ray model of rFXIII confirmed the presence of two structural subdomains in the core region with the boundary at residue 332. The thermolabile domains appear to interact with each other intra- and/or intermolecularly resulting in dimerization the a subunits. At acidic pH, where all domains became destabilized but still remained folded, interdomainial interactions seemed to be abolished, resulting in the reversible dissociation of the dimer as revealed by ultracentrifugation analysis.
Assuntos
Fator XIII/química , Varredura Diferencial de Calorimetria , Fator XIII/efeitos dos fármacos , Fator XIII/genética , Guanidina , Guanidinas/farmacologia , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Fragmentos de Peptídeos/química , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Espectrometria de Fluorescência , Termodinâmica , Ultracentrifugação , Ureia/farmacologiaRESUMO
The X-ray crystal structure of human transglutaminase factor XIII has revealed a cysteine proteinase-like active site involved in a crosslinking reaction and not proteolysis. This is among the first observations of similar active sites in 2 different enzyme families catalyzing a similar reaction in opposite directions. Although the size and overall protein fold of factor XIII and the cysteine proteinases are quite different, the active site and the surrounding protein structure share structural features suggesting a common evolutionary lineage. Here we present a description of the residues in the active site and the structural evidence that the catalytic mechanism of the transglutaminases is similar to the reverse mechanism of the cysteine proteinases.
Assuntos
Cisteína Endopeptidases/química , Fator XIII/química , Transglutaminases/química , Sítios de Ligação , Reagentes de Ligações Cruzadas , Cristalização , Cristalografia por Raios X , Cisteína Endopeptidases/metabolismo , Fator XIII/metabolismo , Modelos Moleculares , Estrutura Molecular , Transglutaminases/metabolismoRESUMO
Platelet accumulation on small- and medium-calibre vascular grafts plays a significant role in graft occlusion. We examined platelet accumulation on the surface of fibrin-coated polyethylene tubing (internal diameter 0.17 cm) during 10 min flow (10 ml/min) at high wall shear rate (764 s-1). Washed platelets labelled with 51Cr were resuspended in Tyrode solution containing albumin, apyrase and red blood cells (hematocrit 40%). When the thrombin that was used to form the fibrin-coated surface was inactivated with FPRCH2Cl before perfusion of the tubes with the platelet: red blood cell suspension, the accumulation of platelets was 59,840 +/- 27,960 platelets per mm2, whereas accumulation on fibrin with residual active thrombin was 316,750 +/- 32,560 platelets per mm2 (n = 4). When the fibrin on the surface was cross-linked by including recombinant factor XIII (rFXIII) in the fibrinogen solution used to prepare the fibrin-coated surface, platelet accumulation, after thrombin neutralization, was reduced by the cross-linking from 46,974 +/- 9702 to 36,818 +/- 7964 platelets per mm2 (n = 12, p < 0.01). Platelet accumulation on tubes coated with D-dimer was ten times less than on tubes coated with D-domain; this finding also supports the observation that cross-linking of fibrin with the formation gamma-gamma dimers reduces platelet accumulation on the fibrin-coated surface. Thrombin-activated platelets themselves were shown to cross-link fibrin when they had adhered to it during perfusion, or in a static system in which thrombin was used to form clots from FXIII-free fibrinogen in the presence of platelets.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Fator XIII/fisiologia , Fibrina , Oclusão de Enxerto Vascular/sangue , Ativação Plaquetária , Adesividade Plaquetária , Produtos de Degradação da Fibrina e do Fibrinogênio , Humanos , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , PolietilenosRESUMO
Vitamin A is clearly an important factor in spermatogenesis. Some of the new data on metabolism of retinoids in the testis has contributed to our understanding of the mechanism(s) involved in the action of vitamin A. It is probable that the requirement of the testis of vitamin A deficient rats for retinol but not retinoic acid involves access of the retinoids to various testicular compartments. Retinol may be required by germinal cells because of a requirement for esterification in order to be successfully transported by the Sertoli cells. Existing evidence suggests that both the Sertoli cells and the germinal cells have specific requirements for retinoids. In the vitamin A deficient rat there appears to be a developmental block at preleptotene spermatocyte and type Al spermatogonia stages. This block is removed by retinol and germinal cell development reinitiates in a synchronous manner. The synchronous testis model offers a number of advantages for the study of molecular events associated with the cycle of the seminiferous epithelium and the development of germinal cells as well as for investigations into the mechanism of action of the retinoids.
Assuntos
Chaperonas Moleculares , Túbulos Seminíferos/fisiologia , Testículo/fisiologia , Vitamina A/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Clusterina , Eletroforese em Gel Bidimensional , Glicoproteínas/genética , Masculino , Dados de Sequência Molecular , Proteínas/metabolismo , Ratos , Túbulos Seminíferos/citologia , Células de Sertoli/fisiologia , Espermatogênese , Deficiência de Vitamina A/patologiaRESUMO
The association of factor XIII a-subunits with fibrin was characterized using recombinant human placental factor XIII (rFXIII) and native fully hydrated fibrin clots formed from purified fibrinogen and thrombin. Binding was assessed using small columns containing fibrin and perfusing them with radioiodinated rFXIII. Results show that thrombin activation of rFXIII led to fibrin binding. The association was partially reversible since much of the bound enzyme could be removed by percolating clots with more buffer. Binding was blocked by antibody directed against the COOH-terminal part of fibrinogen A alpha-chain (A alpha 389-402) and also by the COOH-terminal A alpha-chain peptide fragment A alpha 241-476 (Hi2-DSK).
Assuntos
Fator XIII/química , Fibrina/química , Trombose/metabolismo , Reações Antígeno-Anticorpo , HumanosRESUMO
The three-dimensional structure of the recombinant human factor XIII a2 dimer after cleavage by thrombin has been determined by X-ray crystallography. Factor XIII zymogen was treated with bovine alpha-thrombin in the presence of 3 mM CaCl2, and the cleaved protein was crystallized from Tris buffered at pH 6.5 using ethanol as the precipitating agent. Refinement of the molecular model of thrombin-cleaved factor XIII against diffraction data from 10.0 to 2.5 A resolution has been carried out to give a crystallographic R factor of 18.2%. The structure of thrombin-cleaved factor XIII is remarkably similar to that of the zymogen: there are no large conformational changes in the protein and the 37 residue amino terminus activation peptide remains associated with the rest of the molecule. This work shows that the activation peptide, upon thrombin cleavage, has the same conformation and occupies the same position with respect to the rest of the molecule as it does in the zymogen structure.
Assuntos
Precursores Enzimáticos/metabolismo , Fator XIII/metabolismo , Peptídeos/metabolismo , Conformação Proteica , Trombina/metabolismo , Animais , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Precursores Enzimáticos/química , Fator XIII/química , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Modelos Moleculares , Peptídeos/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Reduced factor XIIIA levels and decreased clot strength have been associated with increased bleeding after cardiopulmonary bypass (CPB). The purpose of this study was to evaluate the relationship between hemostatic factors, including factor XIIIA, and clot strength before, during and after CPB. Factor XIIIA antigen, platelet counts, fibrinogen, factor V activity, tissue plasminogen activator and clot strength (by thromboelastograph) were measured at baseline, after 45 min of CPB, at the end of CPB and 4 h post-operatively in 34 patients. Baseline factor XIIIA antigen was 5.2 +/- 1.4 mg/l. On average, factor XIIIA levels dropped to 64% and clot strength to 77% of baseline values after 45 min on CPB and remained below baseline during the immediate post-operative period. Clot strength was significantly correlated (r = 0.81) with platelet count and fibrinogen but not plasma factor XIIIA levels. Addition of 10 mg/l recombinant factor XIII[a2] significantly increased clot strength. Postoperative bleeding at 2 h was inversely correlated with platelet count, factor XIIIA antigen and clot strength measured at the end of CPB. Maintenance of adequate platelet counts and factor XIIIA levels at the end of CPB may play a role in maintaining clot strength and reducing blood loss.
Assuntos
Coagulação Sanguínea , Ponte Cardiopulmonar , Transglutaminases/análise , Adulto , Idoso , Plaquetas/fisiologia , Fator V/análise , Feminino , Fibrinogênio/análise , Humanos , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Hemorragia Pós-Operatória/etiologia , Análise de Regressão , Tromboelastografia , Ativador de Plasminogênio Tecidual/análiseRESUMO
When cultured Sertoli cells derived from 20-day-old weanling rats were supplied [3H]retinol bound to serum retinol binding protein-transthyretin complex, [3H]retinol was rapidly incorporated and [3H]retinyl esters were synthesized. Within 28 h after administration, 83% of the labeled retinoids were accounted for as retinyl esters (64% as retinyl palmitate). Sertoli cells derived from vitamin A deficient rats and supplied [3H]retinol in culture under identical conditions likewise incorporated [3H]retinol and synthesized retinyl esters. In contrast to normal Sertoli cells, vitamin A deficient Sertoli cells eventually metabolized virtually all of the cellular [3H]retinol to retinyl esters. The primary metabolic fate of retinol administered to Sertoli cell cultures was the synthesis of retinyl esters under all conditions tested. However, administration of [3H]retinol bound to serum retinol binding protein gave metabolic profiles having a higher proportion of retinyl esters and lower proportions of unresolved polar material than administration of [3H]retinol alone. The kinetics of retinol uptake and intracellular retinyl ester synthesis in cultured Sertoli cells was complex. An initial, rapid phase of [3H]retinol incorporation lasting 30 min was followed by a slower rate of incorporation and a concomitant decrease in the intracellular concentration of [3H]retinol. During the time course the specific activity of [3H]retinyl palmitate eventually exceeded that of intracellular [3H]retinol. These observations suggest that two intracellular pools of retinol may exist in Sertoli cells.
Assuntos
Células de Sertoli/metabolismo , Vitamina A/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Cinética , Masculino , Modelos Biológicos , Ratos , Retinoides/biossíntese , Retinoides/isolamento & purificaçãoRESUMO
Retinyl esters were quantitatively the most significant product formed in short term incubations of isolated rat germinal cells administered 3H-retinol. 3H-retinyl palmitate was found to be the single most abundant metabolite accounting for approximately 80% of the total label 2 h after administration of 3H-retinol bound to bovine serum albumin. Differences were found in the relative activity of retinol uptake and metabolism between the various subpopulations of germinal cells separated by the staput sedimentation technique. The strongest correlation was between the magnitude of 3H-retinyl palmitate synthesis and the distribution of round spermatids. Large pachytene spermatocytes also appeared to correlate with 3H-retinyl palmitate synthesis although this correlation was ambiguous due to the presence of a small population of Sertoli cells and spermatogonia in these fractions. The concentration of cellular 3H-retinol was more than 10 fold greater in germinal cells administered 3H-retinol bound to bovine serum albumin than when 3H-retinol bound to serum retinol-binding protein was administered, and likewise the concentration of cellular 3H-retinyl palmitate was approximately 30 fold higher. In comparison to germinal cells, Sertoli cells administered 3H-retinol bound to serum retinol-binding protein incorporated 20 fold more 3H-retinol and synthesized more 3H-retinyl palmitate. However, both germinal cells and Sertoli cells were found to have incorporated similar concentrations of total label when administered 3H-retinol bound to bovine serum albumin. (ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Células Germinativas/metabolismo , Vitamina A/metabolismo , Animais , Células Cultivadas , Epitélio/metabolismo , Masculino , Ratos , Ratos Endogâmicos , Células de Sertoli/metabolismo , EspermatogêneseRESUMO
Platelet-associated factor XIII provides a means by which to promote clot stabilization and platelet interaction with proteins of the coagulation and fibrinolytic pathways. In addition to its intracellular role within the platelet cytoplasm, activated factor XIII will bind to the surface of activated platelets. These platelets then participate in cell-cell or cell-clot interactions, thereby increasing the local concentration of factor XIIIa. The platelet-associated factor XIIIa may increase the amount of crosslinking in a fibrin clot, thereby contributing to the aging of the clot and the reduction in the degree of platelet binding. Clot resistance to fibrinolysis is enhanced by platelet factor XIIIa-mediated crosslinking of alpha 2-antiplasmin to fibrin. The binding of factor XIIIa to the platelet surface requires the activation of the platelet fibrinogen receptor, glycoprotein IIb-IIIa. Thus, platelet-associated factor XIIIa may be used as a marker of in vivo platelet activation. Since half of the factor XIII present in blood is provided by the platelets, it is not surprising that this form of factor XIII plays an important role in hemostasis.
Assuntos
Plaquetas/química , Ativação Plaquetária , Transglutaminases/fisiologia , Coagulação Sanguínea , Plaquetas/fisiologia , Fibrina/química , Fibrinólise , Fibronectinas/química , Humanos , Adesividade Plaquetária , alfa 2-Antiplasmina/metabolismoRESUMO
Rat heart mitochondria oxidizing pyruvate (in the presence of 20% as much malate) took up nearly the amount of oxygen required for complete oxidation to CO2. Thus pyruvate, a physiological substrate of the citrate cycle, is oxidized through the entire cycle in these mitochondria, and they seem suitable for study of regulation of integrated mitochondrial energy transduction. By addition of graded amounts of hexokinase or pyruvate kinase to the suspending medium (in the presence of excess glucose or phosphoenolpyruvate), a wide range of steady-state values of the ATP/ADP concentration ratio was obtained. At a constant concentration of phosphate, the steady-state rate of oxygen uptake by rat heart mitochondria oxidizing pyruvate was a function of the adenylate energy charge or of the ATP/ADP ratio, and relatively independent of the absolute concentrations of these nucleotides. The oxygen uptake rates typically spanned a range of about 20-fold. At very high values of the ATP/ADP ratio, the rate of oxygen uptake is much lower than the "state 4" rate seen after added ADP has been phosphorylated. This result suggests that "state 4" respiration, at least in these freshly prepared mitochondria, measures the rate at which ADP is made available by ATPase activity, rather than indicating uncoupling of electron transport from phosphorylation. The concentration of orthophosphate affected the rate of oxygen uptake and the pattern of response to the ATP/ADP ratio or the energy charge, but the effects did not seem interpretable in terms of the mass-action expression for hydrolysis of ATP, (ATP)/ (ADP) (Pi).
Assuntos
Nucleotídeos de Adenina/fisiologia , Mitocôndrias Cardíacas/metabolismo , Consumo de Oxigênio , Difosfato de Adenosina/fisiologia , Trifosfato de Adenosina/fisiologia , Animais , Técnicas In Vitro , Masculino , Ratos , Ratos EndogâmicosRESUMO
The kinetics of activation of platelet factor XIII, an a-subunit dimer, were characterized by determining rate constants for activation peptide (AP) release, generation of activity, and exposure of the active-site thiol group. The specificity constant (kappacat/Km) for alpha-thrombin-catalyzed AP release, 1.2 x 10(5) M-1s-1, was found to be similar to that for AP release from the tetramer plasma factor XIII (a2b2) [Janus, T.J., Lewis, S. D., Lorand, L., & Shafer, J. A. (1983) Biochemistry 22, 6269-6272], implying that the b subunits of plasma factor XIII do not hinder alpha-thrombin-catalyzed cleavage of AP from the a subunit. Platelet factor XIIIa activity was generated at a rate approximately twice the rate of AP release. This difference in rates was shown to be consistent with a reaction pathway for activation of platelet factor XIII wherein full factor XIIIa activity is generated when one AP is removed from the dimeric zymogen so that removal of the second AP has no detectable effect on catalytic activity. In accord with this conclusion, the rate constant for exposure of the active-site thiol group, as measured by the incorporation of [1-14C]-iodoacetamide, was about twice that observed for the removal of AP.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Fator XIII/metabolismo , Trombina/farmacologia , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Fator XIII/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , Peptídeos , Proteínas Recombinantes/metabolismoRESUMO
We examined individual and group characteristics associated with the duration of community involvement (i.e., length of residence) in 11 Illinois Oxford Houses for 129 male recovering addicts. Survival analyses indicated that the best predictor of duration of community involvement from demographic items was age (i.e., older age and older age of fellow residents were associated with being more likely to continue residence). Among psychological measures, the best survival predictor was lack of pessimism of the future. Although the relationship between longer length of residence and treatment outcomes are complex, because it is often difficult to keep people involved in treatment programs, knowledge that we can gain about those factors that might lead to greater lengths of stay are of importance.
Assuntos
Serviços Comunitários de Saúde Mental , Grupos de Autoajuda , Transtornos Relacionados ao Uso de Substâncias/reabilitação , Sobrevida/psicologia , Adulto , Estudos de Coortes , Humanos , Relações Interpessoais , Masculino , Instituições Residenciais , Apoio Social , Resultado do TratamentoRESUMO
After treatment for substance abuse, whether it is in hospital-based treatment programs, therapeutic communities, or recovery homes, many patients return to former high-risk environments or stressful family situations. Returning to these settings without a network of people to support abstinence increases chances of a relapse. As a consequence, substance abuse recidivism following treatment is high for both men and women. Alternative approaches need to be explored, and there are some promising types of recovery homes. From a public health perspective, a series of studies conducted at DePaul University suggests that one type of recovery home for alcohol abuse recovery has much potential. For example, within this self-help communal living setting, recovering alcoholics were able to maintain employment, thereby reducing their need for government subsidies. Maintaining employment for recovering alcoholics may promote increased personal responsibility, which may impact self-efficacy beliefs. These pilot studies, then, raised both theoretical and practical issues needing further evaluation.
Assuntos
Lares para Grupos , Grupos de Autoajuda , Centros de Tratamento de Abuso de Substâncias , Transtornos Relacionados ao Uso de Substâncias/reabilitação , Chicago , Humanos , Projetos PilotoRESUMO
Factor XIII is the terminal enzyme of the coagulation cascade which serves to rapidly crosslink the adjacent gamma-chain C-termini of fibrin clots. In vivo, this process is initiated by the proteolytic action of thrombin which simultaneously converts both soluble fibrinogen to fibrin and activates zymogen FXIII; fibrin then spontaneously polymerizes to form a gel which activated FXIII stabilizes through crosslinking. Due to the kinetic complexity and the difficulty of investigating gel phase reactions, methods employing pre-activation of recombinant human Factor XIII (rFXIII[A'2]) were developed to effectively decouple these reactions. By utilizing these methods, the kinetic parameters of gamma-chain crosslinking in fibrin gels could be determined by both initial rate and integrated rate techniques under physiologically relevant conditions. The crosslinking of the gamma-chain of fibrin gels could be described by apparent Michaelis kinetics with K(m)(app) = 6.2 microM, kcat = 1872 min-1, and Ksp = 302 min-1 microM-1 for a fibrin gamma-chain monomer of M(r) = 170000 Da. In contrast, both the crosslinking rates of alpha-chains within fibrin gels (Ksp = 0.38 min-1 microM-1: Bishop et al. (1993)) and the crosslinking of a soluble synthetic peptide containing the unique gamma-chain fibrin crosslinking site (Ksp = 0.030 min-1 microM-1) could not be shown to saturate and gave apparent first-order rates with respect to rFXIII[A'2]. These observations coupled with the large differences in the turnover rates (approximately 10(4)) suggest two likely mechanisms for FXIII[A'2]-substrate interactions: (1) random (or independent) binding of non- or weakly interacting substrate pairs imposes a high entropic barrier (i. e., delta Gbinding) to the formation of a productive catalytic complex, e.g., for soluble gamma-chain peptides and the flexible alpha-chains within fibrin, and (2) binding to an oriented substrate pair effectively lowers the entropic barrier to formation of a Michaelis complex and thus greatly enhances the rate of catalysis, e.g., for gamma-chain pairs within the fibrin fibrils.
Assuntos
Fibrina/metabolismo , Transglutaminases/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Reagentes de Ligações Cruzadas , Fibrina/química , Humanos , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Especificidade por Substrato , Transglutaminases/químicaRESUMO
Mild acid hydrolysis of a small (Mr = 6 kDa) pectic polysaccharide isolated from tomato leaves, an inducer of the synthesis and accumulation of two proteinase inhibitors in excised tomato plants, yielded a alpha-D-polygalacturonic acid polymer with degree of polymerization = 20 that retained proteinase inhibitor-inducing activity. Enzymic and acid hydrolysis of this polygalacturonan yielded a series of alpha-1,4-D-galacturonic acid oligomers with degrees of polymerization from 2 to 6 which were purified to homogeneity and assayed for proteinase inhibitor-inducing activity in young excised tomato plants. All of the oligomers exhibited activity. The hexagalacturonide possessed the highest activity and the trimer the lowest. The evidence supports a possible role for plant cell wall fragments as systemic messengers that regulate the expression of proteinase inhibitor genes in plant leaves in response to pest attacks.
Assuntos
Ácidos Hexurônicos , Pectinas/isolamento & purificação , Plantas/análise , Ácidos Urônicos/análise , Carboidratos/análise , Cromatografia DEAE-Celulose , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Oligossacarídeos/análiseRESUMO
The synthesis and accumulation of proteinase inhibitor I in excised tomato leaves can be induced with oligosaccharides obtained by fungal endo-alpha-1,4-polygalacturonase digestion of a pectic polysaccharide (M(r) 5000-10,000) isolated from tomato leaves. Active oligosaccharides were also released from isolated tomato leaf cell walls by endopolygalacturonases partially purified from tomato plants. It is suggested that oligosaccharides, released from plant cell wall pectic polysaccharides by either endogenous or exogenous endopolygalacturonases at a wound or infection site, may have hormone-like roles in regulating plant defense responses in unwounded tissues many centimeters away from the site of release.
RESUMO
Factor XIII is the terminal enzyme of the clotting cascade. A cDNA sequence encoding human placental factor XIII was expressed in Saccharomyces cerevisiae with the yeast ADH2-4c promoter. Expression levels were a strong function of the noncoding flanking DNA content of the construction. When the terminal 3'-flanking noncoding DNA was removed, expression increased approximately 50-fold. The protein was produced in quantity by high-yield fermentation and purified to homogeneity. The recombinant protein was cleaved by thrombin at the same activation site as purified human placental FXIII and exhibited 100% enzymatic activity. At high thrombin concentrations rFXIIIa was cleaved into inactive 54- and 25-kDa polypeptides. The identity of these cleavage sites and the blocked N-terminus to that of the human protein was revealed by amino acid microsequencing. A time course of thrombin activation was performed and the relative distribution of the thrombin-cleaved subunits to the uncleaved zymogen subunits determined; the results were consistent with the half of the sites catalytic model for transglutaminase activity proposed by Chung et al. (Chung, S. I., Lewis, M. S., & Folk, J. E. (1974) J. Biol. Chem. 249, 940-950, 1974) and Hornyak et al. (Hornyak, T. J., Bishop, P. D., & Shafer, J. A. (1989) Biochemistry 28, 7326-7332). Equilibrium and velocity sedimentation analysis indicated that rFXIII exists as a 166-kDa nondissociating dimer that behaves as a compact particle of 8.02 S. Thus, all of the properties of rFXIII thus far examined are consistent with those reported for human platelet and placental FXIII.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Fator XIII/genética , Saccharomyces cerevisiae/genética , Aminoácidos/análise , Cromatografia DEAE-Celulose , Cromatografia em Gel , DNA/genética , DNA/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Fator XIII/isolamento & purificação , Fator XIII/metabolismo , Feminino , Expressão Gênica , Humanos , Peso Molecular , Placenta/enzimologia , Gravidez , Regiões Promotoras Genéticas , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Crystals of human recombinant factor XIII from the yeast Saccharomyces cerevisiae have been grown from solutions of ammonium sulfate at pH 5.8. The crystals are orthorhombic, with space group P2(1)2(1)2 and unit cell dimensions gamma a = 101.2, b = 182.7, and c = 93.4 A. The asymmetric unit consists of one a2 dimer of molecular mass 166 kDa. A 3.5-A resolution data set for the native protein has been collected. Practical resolution limits for these crystals have not been determined, but reflections have been observed to a Bragg spacing of 2.8-A resolution.