RESUMEN
The techniques for measurement of biosynthetic rates and intracellular transit times of an integral membrane protein isoenzyme have now been validated. Thus, induction of placental alkaline phosphatase in cultured HeLa cells by prednisolone and by butyrate is shown to result in its increased biosynthesis as measured by uptake of [35S]methionine into immunoprecipitated cell-surface placental alkaline phosphatase. The cell-surface placental alkaline phosphatase is liberated from the cells by proteolytic cleavage by bromelain, which results in a decrease of the placental alkaline phosphatase subunit mass from 64,000 to 62,000 daltons. The time of transit of new placental alkaline phosphatase molecules from their ribosomal site of synthesis to their terminal cell-surface, bromelain-sensitive site is approximately 55 min. This system may be useful in studies of regulation of intracellular protein processing and transport to the cell surface of proteins destined to become integral membrane proteins.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatasa Alcalina/biosíntesis , Transporte Biológico , Bromelaínas/farmacología , Inducción Enzimática/efectos de los fármacos , Células HeLa/metabolismo , Humanos , Proteínas de la Membrana/biosíntesis , Placenta/enzimología , Propiedades de SuperficieRESUMEN
Quantification of term-placental alkaline phosphatase isoenzyme protein in HeLa TCRC-1 cells grown in the presence and absence of prednisolone indicates that there is a net increase in amount of enzyme-specific protein in prednisolone-stimulated cells. In a similar analysis of HeLa D98AH2 cells, prednisolone treatment causes the appearance of term-placental alkaline phosphatase protein and the loss of the intestinal isoenzyme protein. These results support the interpretation that the response of these cells to corticosteroids is the net accumulation of alkaline phosphatase protein rather than the modification of pre-existing enzyme to a more active state.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Isoenzimas/metabolismo , Prednisolona/farmacología , Proteínas Gestacionales/metabolismo , Células HeLa/efectos de los fármacos , Células HeLa/enzimología , Humanos , Inmunoelectroforesis , Estimulación QuímicaRESUMEN
A rapid enzymatic method is presented which results in the selective release of cell-surface alkaline phosphatase isoenzymes. Treatment of suspensions of human tumor cell lines with the proteolytic enzyme bromelain released certain alkaline phosphatase isoenzymes into low-molecular-weight, catalytically active forms. Cells which expressed term placental or intestinal isoenzymes were equally susceptible to this treatment. A cell line which expressed early placental isoenzyme, however, was unaffected by bromelain as indicated by complete recovery of activity in the treated membrane fraction. Successful solubilization of immunologically reactive enzyme allows quantitation of levels of cell-surface enzyme in response to modulators of gene expression. Moreover, the observed selective solubilization of isoenzymes by bromelain may be of general use in analyses of the physical association between other biologically important surface proteins and the lipid components of the cell membrane.
Asunto(s)
Fosfatasa Alcalina/análisis , Bromelaínas/farmacología , Membrana Celular/enzimología , Células HeLa , Humanos , Inmunoelectroforesis , Isoenzimas/análisis , SolubilidadRESUMEN
Data from four sets of recombinant inbred strains confirm that variation at a single genetic locus is responsible for the previously observed differences in the rate of histidase synthesis in inbred mice. Linkage testing stocks were used to demonstrate linkage with steel (Sl) on chromosome 10.
Asunto(s)
Amoníaco-Liasas/genética , Genes Reguladores , Genes , Histidina Amoníaco-Liasa/genética , Animales , Mapeo Cromosómico , Cruzamientos Genéticos , Femenino , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos/genéticaRESUMEN
Inbred strains of mice fall into two groups with respect to their liver histidase activity levels, high strains having approximately twice as much activity as low strains. Analysis of the F1, F2, and backcross progeny of the mating of a high activity strain (C57BL/6J) and a low activity strain (C3H/HeJ) indicates that the difference between the strains is determined by a single genetic locus with two alleles exhibiting additive inheritance. No differences with respect to various physical and kinetic parameters were found in studies of partially purified histidase from both strains. Quantitation of the amount of enzyme present by immunotitration showed that the amount of enzyme antigen is proportional to the level of enzyme activity in the two strains. Measurements of the relative rates of histidase synthesis by combined radiochemical and immunological techniques showed that the relative rate of synthesis was closely correlated with the amount of enzyme present. Rates of enzyme degradation in the two strains, measured by recovery of activity after irreversible inhibition with nitromethane, were the same.