RESUMO
In vitro effects of uric acid (2-6-8 trioxypurine) on purified human neutrophil alkaline phosphatase were studied. A marked activation of the enzyme catalyzed reaction is observed. This activation is dose and pH dependent and not influenced by dialysis. Uric acid can form a stable complex with alkaline phosphatase, whose biochemical characteristics (heat stability, reactivity towards some inhibitors) are significantly modified.
Assuntos
Fosfatase Alcalina/metabolismo , Neutrófilos/enzimologia , Ácido Úrico/farmacologia , Adulto , Fosfatase Alcalina/antagonistas & inibidores , Ativação Enzimática , Humanos , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
An isoelectric focusing technique for human neutrophil alkaline phosphatase isoenzymes is described. After sonication with Zwittergent 3-12, butanol extraction and ultracentrifugation, dialysis of cytosols precedes focusing. Focusing patterns show a heterogeneity with two enzymatic activity areas: a main component at pI 6.7-6.8 with a minor component at pI 4.8-5.0 which is difficult to visualize due to its sensitivity to experimental conditions. The addition of 3 mmol/l ZnCl2 to the agarose gel improved the staining of focused bands and in particular the anodic component.
Assuntos
Fosfatase Alcalina/sangue , Neutrófilos/enzimologia , Compostos de Zinco , Cloretos , Citosol/enzimologia , Detergentes , Diálise , Eletroforese em Gel de Ágar/métodos , Feminino , Humanos , Concentração de Íons de Hidrogênio , Focalização Isoelétrica/métodos , Isoenzimas/sangue , Fígado/enzimologia , Gravidez , Valores de Referência , ZincoRESUMO
We report the case of a healthy young man presenting with atypical neutrophil alkaline phosphatase (NAP) and reduced neutrophil chemotactic activity, but with no susceptibility to infection. NAP activity was low, kinetic parameters were modified and immunoreactive properties and subcellular distribution were abnormal. Neutrophil morphology was normal. A similar pattern was observed in the patient's healthy brother. The profile of the observed anomalies offers some similarity to that previously described in patients with chronic myelogenous leukaemia. However, in the present case, the NAP deficiency with impaired neutrophil function was present in two brothers with no haematological symptoms and is probably related to a non-acquired neutrophil abnormality. This observation of a primary NAP variant reinforces the hypothesis of a direct link between NAP activity and functional properties of neutrophils.
Assuntos
Fosfatase Alcalina/deficiência , Neutrófilos/enzimologia , Neutrófilos/fisiologia , Adulto , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/química , Núcleo Celular/enzimologia , Quelantes , Quimiotaxia de Leucócito , Citoplasma/enzimologia , Dimerização , Ácido Edético/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Microscopia Eletrônica , Neuraminidase/farmacologia , Neutrófilos/ultraestrutura , Ureia/farmacologiaRESUMO
Alkaline phosphatase concentrations are known to increase in blood neutrophils of normal pregnant women. The main kinetic parameters of this enzyme were analysed and compared in a group of 30 women with normal pregnancies and a group of 11 women whose fetuses had trisomy 21 (Down's syndrome = DS). The subjects were studied at an identical stage of gestation. Significant changes occurred in thermal stability and urea resistance in cases of DS pregnancies. We also investigated the inactivation constants for two chemicals: L-p-bromotetramisole, an uncompetitive inhibitor, and sodium thiophosphate, a competitive inhibitor. Ki measured for the two inhibitors were found to be significantly lower in cases of pathological pregnancies. The patterns observed in inhibition constants extend the biochemical characteristics of the atypical isoenzyme expressed in neutrophils of women with DS pregnancies.
Assuntos
Fosfatase Alcalina/antagonistas & inibidores , Síndrome de Down/sangue , Inibidores Enzimáticos/farmacologia , Neutrófilos/efeitos dos fármacos , Complicações na Gravidez/sangue , Gravidez/sangue , Adulto , Ligação Competitiva , Osso e Ossos/enzimologia , Estudos de Casos e Controles , Feminino , Humanos , Rim/enzimologia , Fígado/enzimologia , Idade Materna , Neutrófilos/enzimologia , Fosfatos/farmacologia , Gravidez de Alto Risco , Tetramizol/análogos & derivados , Tetramizol/farmacologiaRESUMO
Intensive studies have been conducted so far on biochemical markers available for screening of chromosome defects in obstetrical monitoring. In this paper we report further data on two protein phosphatases: alkaline phosphatase (a marker of cell maturation) and phosphotyrosine phosphatase (a marker of cell proliferation) assayed in cultured amniotic cells from fetuses with trisomy 18 at 15 weeks of gestation. Comparison with normal fetal cells showed a different behaviour for each enzyme: alkaline phosphatase was very significantly lowered while phosphotyrosine phosphatase remained a normal levels. These results provide a further enlargement of the field of biochemical markers used in the screening tests of trisomy 18.
Assuntos
Fosfatase Alcalina/metabolismo , Âmnio/enzimologia , Cromossomos Humanos Par 18/genética , Proteínas Tirosina Fosfatases/metabolismo , Trissomia/genética , Amniocentese , Âmnio/citologia , Divisão Celular , Células Cultivadas , Feminino , Marcadores Genéticos , Idade Gestacional , Humanos , Cariotipagem , Gravidez , Segundo Trimestre da GravidezRESUMO
A kinetic parameter (the Vm/Km ratio), an index of neutrophil alkaline phosphatase catalytic efficiency, was studied in 36 women with normal pregnancies at 16-20 weeks' gestation. On each blood sample, determinations were achieved on enzyme extracted from maternal granulocytes by monitoring the phosphohydrolytic activity at 400 nm on a spectrophotometer equipped with software for computation of kinetic parameters. Infant sex was recorded at the delivery for all women included in this study. The recent introduction of NAP as a marker for some pathological pregnancies requires a better knowledge of the behaviour of that enzyme in physiological conditions. Data reported focus attention on fetal sex. It appears to be one of the factors involved in variations of kinetic parameters observed in maternal NAP. Sex-linked differences in placental maturation could explain these results.
Assuntos
Fosfatase Alcalina/sangue , Idade Gestacional , Neutrófilos/enzimologia , Caracteres Sexuais , Adulto , Feminino , Humanos , Cinética , Masculino , Gravidez , Análise de RegressãoRESUMO
Quantitative and qualitative analysis of alkaline phosphatases (AP) was performed on amniotic fluid in 59 normal pregnancies and 14 Down's syndrome (DS) pregnancies at 16, 18 and 19 weeks of gestation. In DS cases, intestinal and placental isoenzyme levels were significantly reduced (P<0.001) and the AP electrophoretic pattern was seen to be modified on polyacrylamide gel electrophoresis. A unique component was detected. After extraction and purification of the abnormal isoenzyme, peptide fragments obtained after cyanogen bromide cleavage indicated a hybrid heterodimeric AP composed of intestinal and tissue non-specific subunits, as evaluated by SDS polyacrylamide gel electrophoresis.
Assuntos
Fosfatase Alcalina/metabolismo , Líquido Amniótico/enzimologia , Síndrome de Down/enzimologia , Intestinos/enzimologia , Adulto , Amniocentese , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Isoenzimas/metabolismo , Gravidez , Segundo Trimestre da GravidezAssuntos
Fosfatase Alcalina/biossíntese , Osso e Ossos/enzimologia , Síndrome de Down/enzimologia , Intestinos/enzimologia , Fígado/enzimologia , Neutrófilos/enzimologia , Adolescente , Fosfatase Alcalina/sangue , Criança , Síndrome de Down/sangue , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Estabilidade Enzimática , Feminino , Temperatura Alta , Humanos , Isoenzimas/biossíntese , Isoenzimas/sangue , MasculinoRESUMO
Alkaline phosphatase (AP) has been characterized in the neutrophil polymorphonuclear leukocytes (PMN) from 14 healthy adults. The biochemical properties of the isoenzymes were analyzed by the following methods: enzyme assays, slab gel electrophoresis, thermostability, inhibition pattern using four chemical inhibitors, and immunological specificity. In normal PMNs, two types of AP have been found: a thermolabile isoenzyme identical to the liver/bone/kidney type and a thermostable isoenzyme with respect to its electrophoretic properties, its level of thermostability, its immunological specificity, the thermostable isoenzyme resembles the placental form more closely than the intestinal form. These results are consistent with the proposal that granulocyte AP is a heterogeneous enzyme. Two enzyme forms appear to be expressed in this blood cell.
Assuntos
Fosfatase Alcalina/sangue , Isoenzimas/sangue , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/imunologia , Feminino , Temperatura Alta , Humanos , Fígado/enzimologia , Masculino , Neutrófilos/enzimologia , Placenta/enzimologiaRESUMO
A comparative study of the polymorphonuclear neutrophiles of 62 parents of children suffering from acute leukemia and of 110 couples with normal children revealed a marked increased of Neutrophil Alkaline Phosphatase (p less than 0.05) in the mothers of the leukemic children. The other parameters (periodic acid Schiff and myeloperoxidase) were unchanged.
Assuntos
Fosfatase Alcalina/sangue , Leucemia/genética , Neutrófilos/enzimologia , Doença Aguda , Adulto , Feminino , Humanos , Leucemia/enzimologia , Masculino , Pessoa de Meia-Idade , Reação do Ácido Periódico de Schiff , Peroxidase/sangueRESUMO
The kinetics of neutrophil alkaline phosphatase (AP) inactivation by urea were examined. In standardized conditions (molarity and pH of buffer, magnesium chloride and urea concentration), monitoring of the enzymatic reaction allows identification of neutrophil AP isoenzymes. This study confirms the heterogeneity of AP in normal human neutrophils.
Assuntos
Fosfatase Alcalina/antagonistas & inibidores , Isoenzimas/antagonistas & inibidores , Neutrófilos/enzimologia , Ureia/farmacologia , Adulto , Fosfatase Alcalina/sangue , Feminino , Humanos , Técnicas In Vitro , Isoenzimas/sangue , Cinética , MasculinoRESUMO
A biological [karyotype, leucocyte alkaline phosphatase (L.A.P.), haematimetry] and clinical study was performed on three women carriers of a chronic myaloid leukemia during their pregnancy (3 1/2 months, 6 1/2 months), or just after delivery. The results show : (1) a relation between the acuteness of the illness and a higher rate of L.A.P., probably secondary to pregnancy, and (2) a relation between the clinical evolution and the rate of Ph1 (+) cells.