RESUMO
The coupled-optical-phonon-mode theory of Barker and Hopfield of two mode interaction in isotropic crystals has been extended to monoclinic crystals. The analytical expressions for the calculation of the dielectric tensor elements in the ac crystal plane have been derived. It has been shown that the interaction dielectric model is a generalized expression of the dielectric tensor for monoclinic case when no interaction between modes is present. Also, the results of Barker and Hopfield are obtained from this more general theory, when an isotropic case is considered. In order to be able to investigate real crystals, meaning extending the interactions to a large number of pairs of modes, but at the same time to make the fitting procedure possible, a model dielectric function taking into account the interaction between modes with closest frequencies has been derived. The validity of the model obtained has been tested on a Tutton salt single crystal of K(2)Co(SO(4))(2).6H(2)O. The recorded spectra from the ac crystal plane were fitted in order to obtain best fit parameters. The comparison between the experimentally recorded spectra and the model reflectance function give good results and verify this model to be applicable.
Assuntos
Complexos de Coordenação/química , Raios Infravermelhos , Modelos Químicos , Fenômenos Ópticos , Sais/química , Sulfatos/química , Cristalização , Eletricidade , Íons , Movimento (Física) , SoluçõesRESUMO
The neurotoxic activity of ammodytoxin A (AtxA), a phospholipase A(2) from Vipera ammodytes ammodytes venom, has been investigated by protein engineering. With the aim of obtaining AtxA as a non-fused protein in the bacterial cytoplasm and avoiding problems with incomplete cleavage in vivo of the initial Met preceding the first residue (Ser1), a double mutant (S1A/E4Q) was prepared and expressed in Escherichia coli. Immunoblotting of the bacterial lysate showed that the mutant was synthesized at a low level not exceeding 0.5% of total cell protein. Analysis of the potential secondary structure of the mutant mRNA in the translation initiation region suggested that the Ala1 (GCC) and Leu2 (CUG) codons used are likely to be involved in a hairpin structure with the Thr13 (ACG) and Gly14 (GGG) codons, hindering effective translation at the ribosome. To weaken this structure (by DeltaG of about 20 kJ/mol) the same double mutant was prepared using another mutagenic oligonucleotide with silent mutations in the Ala1 (GCU) and Leu2 (UUG) codons. The mutant was successfully produced at a level of approximately 15% of total protein, with the initial Met completely removed in the bacterial cell. Such an approach could be important in solving similar problems in bacterial production of other toxic proteins.
Assuntos
Escherichia coli/genética , RNA Mensageiro/química , Venenos de Víboras/genética , Sequência de Aminoácidos , Sequência de Bases , Expressão Gênica , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Fosfolipases A/antagonistas & inibidores , RNA Mensageiro/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Venenos de Víboras/metabolismoRESUMO
Quadruple (Y115K/I116K/R118M/N119L) and double (Y115K/I116K) mutants of ammodytoxin A, a presynaptically toxic phospholipase A(2) from Vipera ammodytes ammodytes venom, were prepared and characterized. The enzymatic activity of the quadruple mutant on phosphatidylcholine micelles was threefold higher than that of AtxA, presumably due to higher phospholipid-binding affinity, whereas the activity of the double mutant was twofold lower. The substantial decrease by more than two orders of magnitude in the lethal potency of both mutants, together with their decreased binding affinity for neuronal receptors, indicates involvement of the amino acid region 115-119 in neurotoxicity. The similar decrease of toxicity for the two mutants points to the importance of the residues Y115 and I116.
Assuntos
Fosfolipases A/química , Fosfolipases A/metabolismo , Venenos de Víboras/química , Venenos de Víboras/toxicidade , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Ligação Competitiva , Córtex Cerebral , Fosfolipases A2 do Grupo II , Dose Letal Mediana , Camundongos , Micelas , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Mutação/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolipases A/genética , Fosfolipases A/toxicidade , Conformação Proteica , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/toxicidade , Alinhamento de Sequência , Suínos , Venenos de Víboras/antagonistas & inibidores , Venenos de Víboras/genética , Venenos de Víboras/metabolismoRESUMO
A cDNA encoding a novel human putative member of the papain family of cysteine peptidases has been cloned. The protease, named cathepsin P, is synthesized as a preproprotein. The presumed propeptide of 38 amino acids is followed by a 242-residue mature protein. The mature protease region is 30% identical to human papain-like cathepsins, with all the residues important for catalysis conserved. No similarity was observed in the propeptide region. On the contrary, the proenzyme shares 51-87% residues with some precursors of cysteine proteases from other species that have not yet been characterized. They all show a nearly completely conserved "CYTRED motif" in the propeptide region, not present in other members of the family, and could therefore constitute a distinct subfamily.
Assuntos
Catepsinas/genética , Clonagem Molecular , Sequência de Aminoácidos/genética , Catepsina K , Sequência Conservada/genética , Cisteína Endopeptidases/genética , Humanos , Dados de Sequência Molecular , Família Multigênica , Papaína/genética , Precursores de Proteínas/genéticaRESUMO
The mRNA of a novel human cathepsin P is expressed at high levels in lung, liver and heart. Using antibodies raised against recombinant cathepsin P produced in Escherichia coli, a single protein band of 33 kDa was detected by immunoblotting an extract of human liver. By immunofluorescence, positive signals were observed in hepatocytes and Kupffer cells of liver, and the distal tubule cells of kidney showing mainly perimembranous distribution, indicating a role, as yet unknown, for this novel putative protease that is distinct from other cathepsins of the papain family.
