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1.
Tijdschr Psychiatr ; 63(7): 522-523, 2021.
Artigo em Holandês | MEDLINE | ID: mdl-34523702
2.
Science ; 244(4900): 82-5, 1989 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-2704992

RESUMO

Protein engineering and x-ray crystallography have been used to study the role of a surface loop that is present in pancreatic phospholipases but is absent in snake venom phospholipases. Removal of residues 62 to 66 from porcine pancreatic phospholipase A2 does not change the binding constant for micelles significantly, but it improves catalytic activity up to 16 times on micellar (zwitterionic) lecithin substrates. In contrast, the decrease in activity on negatively charged substrates is greater than fourfold. A crystallographic study of the mutant enzyme shows that the region of the deletion has a well-defined structure that differs from the structure of the wild-type enzyme. No structural changes in the active site of the enzyme were detected.


Assuntos
Fosfolipases A/metabolismo , Fosfolipases/metabolismo , Conformação Proteica , Sequência de Aminoácidos , Animais , Cristalografia , Ativação Enzimática , Cinética , Dados de Sequência Molecular , Mutação , Pâncreas/enzimologia , Fosfolipases A/genética , Fosfolipases A/fisiologia , Fosfolipases A2 , Venenos de Serpentes/análise , Relação Estrutura-Atividade , Suínos
4.
Tijdschr Psychiatr ; 51(2): 75-86, 2009.
Artigo em Holandês | MEDLINE | ID: mdl-19194849

RESUMO

BACKGROUND: Standardised evaluation studies performed in the Netherlands in a large number of inpatient and day-treatment hospitals providing psychotherapy have shown significant symptomatic improvements in patients between the date of entry to the studies and follow-up after one year. However, the work situation of ex-patients hardly changed and a large number of patients were still receiving psychotherapy. AIM: To examine the effectiveness of a specifically designed course of re-integration training. METHOD: A group of 128 patients were assigned randomly either to a re-integration training course aimed at improved functioning at work and improved relationships, or to booster sessions. Outcome measures were symptom level, work status, absence from work, and further psychotherapy. results After two years the number of patients in paid employment remained the same (76%) in the re-integration training course and increased from 67 to 87% in the booster sessions. Attendance was significantly higher in the booster sessions than in the re-integration training. There were no differences in the other outcome measures. CONCLUSION: We conclude that re-integration training was no more effective than the booster sessions. Our hypothesis is that continuity of care (therapists plus programme) explains the favourable result of the booster sessions.


Assuntos
Educação de Pacientes como Assunto/métodos , Transtornos da Personalidade/psicologia , Transtornos da Personalidade/reabilitação , Psicoterapia/métodos , Reabilitação Vocacional/métodos , Atividades Cotidianas , Adulto , Feminino , Humanos , Pacientes Internados , Masculino , Países Baixos , Psicoterapia Breve/métodos , Índice de Gravidade de Doença , Resultado do Tratamento
6.
Tijdschr Psychiatr ; 49(9): 655-9, 2007.
Artigo em Holandês | MEDLINE | ID: mdl-17853375

RESUMO

A 25-year-old woman, known to have schizoaffective disorder, presented with symptoms that had arisen a few weeks earlier. The symptoms indicated that she had a toxic clozapine blood level. The probable cause of the toxicity was a pharmacokinetic interaction between citalopram and clozapine at the level of the cytochrome P450 system. A literature search reveals the importance of monitoring the interactions between selective serotonin reuptake inhibitors and clozapine, a procedure which should, if possible, be accompanied by blood level measurements. Caution is called for, particularly when non-smokers are involved.


Assuntos
Clozapina/farmacocinética , Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/farmacocinética , Transtornos Psicóticos/tratamento farmacológico , Inibidores Seletivos de Recaptação de Serotonina/farmacocinética , Adulto , Antipsicóticos/efeitos adversos , Antipsicóticos/sangue , Antipsicóticos/farmacocinética , Clozapina/efeitos adversos , Clozapina/sangue , Interações Medicamentosas , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/sangue , Feminino , Humanos , Inibidores Seletivos de Recaptação de Serotonina/efeitos adversos , Inibidores Seletivos de Recaptação de Serotonina/sangue
8.
Structure ; 5(1): 69-79, 1997 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-9016712

RESUMO

BACKGROUND: Acylphosphatase (ACP) is a low molecular weight phosphomonohydrolase catalyzing with high specificity the hydrolysis of the carboxyl-phosphate bond present in acylphosphates. The enzyme is thought to regulate metabolic processes in which acylphosphates are involved, such as glycolysis and the production of ribonucleotides. Furthermore the enzyme is capable of hydrolyzing the phospho-aspartyl intermediate formed during the action of membrane pumps such as (Ca2++Mg2+) ATPase. Although the tertiary structure of a muscle ACP has been determined by NMR spectroscopy, little is known about the catalytic mechanism of ACP and further structures might provide an increased understanding. RESULTS: The structure of 'common type' ACP from bovine testis has been determined by X-ray crystallography to a resolution of 1.8 A. The structure has been refined to an R factor of 17.0 % using all data between 15 and 1.8 A. The binding of a sulphate and a chloride ion in the active centre allows a detailed description of this site. The overall protein folds of common type and muscle ACP are similar but their loops have very different conformations. These differences, in part, are probably caused by the binding of the ions in the active site of the common type form. The phosphate-binding loop of ACP shows some remarkable similarities to that of low molecular weight protein tyrosine phosphatase. CONCLUSIONS: The active site of ACP has been located, enabling a reaction mechanism to be suggested in which the phosphate moiety bound to Arg23 acts as a base, abstracting a proton from a nucleophilic water molecule liganded to Asn41. The transition-state intermediate is stabilized by the phosphate-binding loop. We suggest the catalysis to be substrate assisted, which probably explains why this enzyme can only hydrolyze acylphosphates.


Assuntos
Hidrolases Anidrido Ácido/química , Testículo/enzimologia , Animais , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Masculino , Modelos Químicos , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Acilfosfatase
9.
Structure ; 5(2): 217-25, 1997 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-9032081

RESUMO

BACKGROUND: . The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediates the energy-driven uptake of carbohydrates and their concomitant phosphorylation. In addition, the PTS is intimately involved in the regulation of a variety of metabolic and transcriptional processes in the bacterium. The multiprotein PTS consists of a membrane channel and at least four cytoplasmic proteins or protein domains that sequentially transfer a phosphoryl group from phosphoenolpyruvate to the transported carbohydrate. Determination of the three-dimensional structure of the IIB enzymes within the multiprotein complex would provide insights into the mechanisms by which they promote efficient transport by the membrane channel IIC protein and phosphorylate the transported carbohydrate on the inside of the cell. RESULTS: . The crystal structure of the IIB enzyme specific for cellobiose, IIBcellobiose (molecular weight 11.4 kDa), has been determined to a resolution of 1.8 and refined to an R factor of 18.7% (Rfree of 24. 1%). The enzyme consists of a single four-stranded parallel beta sheet flanked by helices on both sides. The phosphorylation site (Cys 10) is located at the C-terminal end of the first beta strand. No positively charged residues, which could assist in phosphoryl-transfer, can be found in or near the active site. The fold of IIBcellobiose is remarkably similar to that of the mammalian low molecular weight protein tyrosine phosphatases. CONCLUSIONS: . A comparison between IIBcellobiose and the structurally similar low molecular weight protein tyrosine phosphatases provides insight into the mechanism of the phosphoryltransfer reactions in which IIBcellobiose is involved. The differences in tertiary structure and active-site composition between IIBcellobiose and the glucose-specific IIBglucose give a structural explanation why the carbo-hydrate-specific components of different families cannot complement each other.


Assuntos
Proteínas de Bactérias/química , Células Eucarióticas/enzimologia , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , Conformação Proteica , Proteínas Tirosina Fosfatases/química , Animais , Bovinos , Cristalografia por Raios X , Modelos Moleculares , Fosforilação Oxidativa , Homologia de Sequência de Aminoácidos
10.
J Mol Biol ; 216(2): 425-39, 1990 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-2254938

RESUMO

The crystal structure of an engineered phospholipase A2 with enhanced activity has been refined to an R-factor of 18.6% at 2.1 A resolution using a combination of molecular dynamics refinement by the GROMOS package and least-squares refinement by TNT. This mutant phospholipase was obtained previously by deleting residues 62 to 66 in porcine pancreatic phospholipase A2, and changing Asp59 to Ser, Ser60 to Gly and Asn67 to Tyr. The refined structure allowed a detailed comparison with wild-type porcine and Crotalus atrox phospholipase A2. The conformation of the deletion region appears to be intermediate between that in those two enzymes. The residues in the active center are virtually the same. An internal hydrophobic area occupied by Phe63 in the wild-type porcine phospholipase A2 is kept as conserved as possible by local rearrangement of neighboring atoms. In the mutant structure, this hydrophobic pocket is now occupied by the disulfide bond between residues 61 and 91. A detailed description of the second binding site for a calcium ion in this enzyme is given.


Assuntos
Fosfolipases A/metabolismo , Sequência de Aminoácidos , Animais , Deleção Cromossômica , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosfolipases A/química , Fosfolipases A/genética , Fosfolipases A2 , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serpentes , Software , Suínos , Difração de Raios X
11.
J Mol Biol ; 247(2): 149-53, 1995 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-7707365

RESUMO

Crystals of the penicillin binding protein 4 (PBP4) from Escherichia coli have been obtained at 37 degrees C from liquid to liquid diffusion experiments in capillaries. PBP4 was dissolved in a 1.0 M ammonium sulphate solution, buffered at pH 7.2, to a concentration of 5 mg/ml, and was layered on top of a 1.6 to 2.2 M ammonium sulphate solution. Crystals appeared within four to six weeks. They belong to space group C222 with cell dimensions a = 68.5 A, b = 100.5 A and c = 137.0 A, and diffract to at least 2.8 A resolution. There is one molecule with a molecule mass of 49,568 Da in the asymmetric unit.


Assuntos
Proteínas de Bactérias , Proteínas de Transporte/química , Proteínas de Escherichia coli , Escherichia coli/química , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/química , Penicilinas/metabolismo , Peptidil Transferases , D-Ala-D-Ala Carboxipeptidase Tipo Serina , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X , Dados de Sequência Molecular , Proteínas de Ligação às Penicilinas , Homologia de Sequência de Aminoácidos , beta-Lactamases/química
12.
J Mol Biol ; 232(3): 839-55, 1993 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-8355274

RESUMO

The highly homologous bovine and porcine pancreatic phospholipase A2 (85% amino acid residue identity) show a large conformational difference in the loop from residue 59 to 71. In bovine phospholipase A2 residues 59 to 66 adopt an alpha-helix conformation, while residues 67 to 71 are in a surface loop. Residues 59 to 66 in the porcine enzyme have a random coil conformation, and residues 67 to 71 form a short 3(10)-helix. It has been suggested that most probably this conformational difference is caused by the substitution Val63 (bovine) to Phe63 (porcine) in the otherwise invariant loop 59 to 70. To test this hypothesis, a mutant porcine phospholipase A2 was constructed in which residue Phe63 was replaced by a Val. The activity of this F63V mutant towards aggregated substrates was about half the activity of wild-type porcine phospholipase A2, but significantly different from that of the bovine enzyme. The affinity for zwitterionic interfaces was found to be intermediate between porcine and bovine phospholipase. The mutation did not have any effect on the stability of the enzyme towards denaturation by guanidine.HCl. The F63V mutant was crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 79.88 A, b = 65.23 A, c = 52.62 A, with two molecules per asymmetric unit. Its three-dimensional structure was solved by molecular replacement methods, and refined to a crystallographic R-factor of 17.6% for all data between 10 and 2.2 A resolution. In one molecule the 58 to 71 loop is in very weak density, suggesting a high degree of disorder or flexibility. The conformation of the same loop in the other molecule could be determined unambiguously. It shows a conformation which resembles more that of bovine phospholipase A2 than that of porcine phospholipase. It is concluded that indeed the single F63V substitution causes a dramatic conformational change.


Assuntos
Fosfolipases A/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Modelos Moleculares , Dados de Sequência Molecular , Pâncreas/enzimologia , Fenilalanina/genética , Fosfolipases A/genética , Fosfolipases A2 , Mutação Puntual , Biossíntese de Proteínas , Conformação Proteica , Dobramento de Proteína , Relação Estrutura-Atividade , Suínos , Temperatura , Valina/genética
14.
FEBS Lett ; 364(3): 243-4, 1995 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-7758575

RESUMO

Single crystals of a 'common-type' acylphosphatase from bovine testis have been grown. Crystals belong to space group C2 and have cell dimensions a = 64.6 A, b = 36.5 A, c = 45.2 A and beta = 104.8 and contain one monomer per asymmetric unit. The crystals diffract better than 2.0 A resolution and are well suited for an X-ray structure determination.


Assuntos
Hidrolases Anidrido Ácido/química , Cristalografia por Raios X , Animais , Bovinos , Cristalização , Masculino , Testículo/enzimologia , Acilfosfatase
16.
Nat Struct Biol ; 8(2): 131-5, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11175901

RESUMO

Leukotriene (LT) A(4) hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc enzyme that catalyzes the biosynthesis of LTB4, a potent lipid chemoattractant involved in inflammation, immune responses, host defense against infection, and PAF-induced shock. The high resolution crystal structure of LTA4H in complex with the competitive inhibitor bestatin reveals a protein folded into three domains that together create a deep cleft harboring the catalytic Zn(2+) site. A bent and narrow pocket, shaped to accommodate the substrate LTA(4), constitutes a highly confined binding region that can be targeted in the design of specific anti-inflammatory agents. Moreover, the structure of the catalytic domain is very similar to that of thermolysin and provides detailed insight into mechanisms of catalysis, in particular the chemical strategy for the unique epoxide hydrolase reaction that generates LTB(4).


Assuntos
Aminopeptidases/química , Epóxido Hidrolases/química , Complexos Multienzimáticos/química , Sequência de Aminoácidos , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Epóxido Hidrolases/metabolismo , Humanos , Ligação de Hidrogênio , Inflamação/enzimologia , Leucina/análogos & derivados , Leucina/metabolismo , Leucotrieno A4/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Termolisina/química , Itérbio/metabolismo , Zinco/metabolismo
17.
Biochemistry ; 36(23): 7217-24, 1997 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-9188723

RESUMO

The structural and catalytic importance of the 15-21 and 42-45 loop residues of the acylphosphatase muscular isoenzyme has been investigated by oligonucleotide-directed mutagenesis. Seven mutants involving conserved residues of the two loops have been prepared and characterized for structural, kinetic, and stability features by using different spectroscopic techniques and compared to the wild-type enzyme. The results are discussed in light of the crystal structure of the highly homologous common type acylphosphatase [Thunnissen et al. (1997) Structure 5, 69-79]. A differential role of the two loops has emerged: the 15-21 and the 42-45 loops appear mainly involved in active site formation and enzyme structural stabilization, respectively. These conclusions are supported by a strong impairment of the catalytic efficiency, in terms of enzymatic activity and substrate binding capability, for most of the 15-21 loop mutants. In particular, the Gly15Ala mutant is completely inactive and displays a native-like overall fold, indicating that the correct geometry of the 15-21 loop is an essential requisite for optimal enzymatic catalysis. Instead, the Gly45Ala mutant, though revealing unchanged catalytic properties, shows a considerably reduced conformational stability, as judged by circular dichroism and 1H NMR spectroscopy. This finding confirms previous results relative to Thr42 and Thr46 residues [Taddei et al. (1996) Biochemistry 35, 7077-7083] underlining the structural importance of the 42-45 loop as a linker for the two beta alpha beta units constituting the overall enzyme structure.


Assuntos
Hidrolases Anidrido Ácido/química , Músculos/enzimologia , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Animais , Sítios de Ligação , Catálise , Dicroísmo Circular , Cristalografia por Raios X , Drosophila , Estabilidade Enzimática , Escherichia coli , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Acilfosfatase
18.
Nature ; 347(6294): 689-91, 1990 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-2215698

RESUMO

Phospholipases A2 play a part in a number of physiologically important cellular processes such as inflammation, blood platelet aggregation and acute hypersensitivity. These processes are all initiated by the release of arachidonic acid from cell membranes which is catalysed by intracellular phospholipases A2 and followed by conversion of arachidonic acid to prostaglandins, leukotrienes or thromboxanes. An imbalance in the production of these compounds can lead to chronic inflammatory diseases such as rheumatoid arthritis and asthma. Inhibitors of phospholipase A2 might therefore act to reduce the effects of inflammation, so structural information about the binding of phospholipase A2 to its substrates could be helpful in the design of therapeutic drugs. The three-dimensional structure is not known for any intracellular phospholipase A2, but these enzymes share significant sequence homology with secreted phospholipases, for which some of the structures have been determined. Here we report the structure of a complex between an extracellular phospholipase A2 and a competitively inhibiting substrate analogue, which reveals considerable detail about the interaction and suggests a mechanism for catalysis by this enzyme.


Assuntos
Ácidos Láuricos/metabolismo , Compostos Organofosforados/metabolismo , Fosfolipases A/metabolismo , Sequência de Bases , Sítios de Ligação , Catálise , Dissulfetos , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/química , Fosfolipases A2 , Conformação Proteica , Difração de Raios X
19.
Protein Eng ; 5(7): 597-603, 1992 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-1480613

RESUMO

Tyr52 and Tyr73 are conserved amino acid residues throughout all vertebrate phospholipases A2. They are part of an extended hydrogen bonding system that links the N-terminal alpha-NH3(+)-group to the catalytic residues His48 and Asp99. These tyrosines were replaced by phenylalanines in a porcine pancreatic phospholipase A2 mutant, in which residues 62-66 had been deleted (delta 62-66PLA2). The mutations did not affect the catalytic properties of the enzyme, nor the folding kinetics. The stability against denaturation by guanidine hydrochloride was decreased, however. To analyse how the enzyme compensates for the loss of the tyrosine hydroxyl group, the X-ray structures of the delta Y52F and delta Y73F mutants were determined. After crystallographic refinement the final crystallographic R-factors were 18.1% for the delta Y52F mutant (data between 7 and 2.3 A resolution) and 19.1% for the delta Y73F mutant (data between 7 and 2.4 A resolution). No conformational changes occurred in the mutants compared with the delta 62-66PLA2, but an empty cavity formed at the site of the hydroxyl group of the former tyrosine. In both mutants the Asp99 side chain loses one of its hydrogen bonds and this might explain the observed destabilization.


Assuntos
Fosfolipases A/química , Estabilidade Enzimática , Ligação de Hidrogênio , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fosfolipases A/genética , Fosfolipases A2 , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Relação Estrutura-Atividade , Difração de Raios X
20.
Biochem Soc Trans ; 21(1): 39-40, 1993 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8449320

RESUMO

Large single crystals (up to 1 mm in each dimension) of the B800-850 antenna complex from Rhodopseudomonas acidophila strain 10050 have been grown in the presence of beta-octyl-glucoside. These crystals have the space group R32 and unit cell dimensions of a = b = 119.9 A and c = 297.0 A. Recently we have improved our crystallization procedures so that all crystals now diffract reliably to beyond 3.5 A, with some diffracting to below 3 A. A range of isomorphous heavy atom derivatives have been prepared and we are now engaged in locating the heavy atom sites within the unit cell.


Assuntos
Proteínas de Bactérias/química , Rodopseudomonas/química , Apoproteínas/química , Proteínas de Bactérias/isolamento & purificação , Bacterioclorofilas/química , Cristalização , Difração de Raios X
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