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32P-labelling anomalies in human erythrocytes. Is there more than one pool of cellular Pi?

ABSTRACT: 1. Human erythrocytes were incubated in autologous plasma containing [32P]Pi, and sampled by a method which avoids washing the cells. 2. In experiments of up to 3 h duration, the specific radioactivity of cellular Pi stabilized at a value below that of extracellular Pi. This can be explained on the basis of a single cellular Pi pool exchanging with a large unlabelled pool of cellular organic phosphates. 3. However, a rapid initial phase of labelling, occurring within 30 s, was inconsistent with the situation described in point 2. A possible explanation is that about 1/4 of cellular Pi occurs in a separate, fast-labelling pool. 4. When the extracellular Pi concentration was doubled, most of the corresponding increase in the steady-state cellular Pi concentration was accounted for by the apparent fast-labelling Pi pool, which also doubled. 5. The observed initial rate of labelling of cellular organic phosphates [which probably occurs through the reaction catalysed by glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12)] was considerably lower than that predicted from the flux through the Embden-Meyerhof pathway. This implies that the enzyme is exposed to Pi whose specific radioactivity is lower than the mean specific radioactivity of cellular Pi, and fails to support earlier suggestions that this enzyme uses extracellular Pi. 6. In 3 h incubations, the rate of organic phosphate labelling was roughly constant throughout, even though the specific radioactivity of cellular Pi had risen slowly to a plateau. Viewed in conjunction with point 5, this again suggests some inhomogeneity in cellular Pi. 7. Cellular Pi and extracellular Pi only reached isotopic steady state after 2 days. At this stage some organic phosphates were probably still incompletely labelled. 8. We conclude that, whatever their physical or technical reasons, such labelling inhomogeneities and slow attainment of isotopic steady state may cause serious misinterpretation of results if ignored during 32P-labelling of intact cells.

SUBMITTER: Kemp GJ

PROVIDER: S-EPMC1133646 | biostudies-other | 1989 Dec

REPOSITORIES: biostudies-other

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Project description:The family of transcription factors with the C2H2 zinc finger domain is expanding in the evolution of vertebrates, reaching its highest numbers in the mammals. The question arises: whether an increased amount of these transcription factors is related to embryogenesis, nervous system, pathology or more of them are expressed in individual cells? Among mammals, the primates have a more complex anatomical structure than the rodents (e.g., brain). In this work, I show that a greater number of C2H2-ZF genes are expressed in the human cells than in the mouse cells. The effect is especially pronounced for C2H2-ZF genes accompanied with the KRAB domain. The relative difference between the numbers of C2H2-ZF(-KRAB) genes in the human and mouse cellular transcriptomes even exceeds their difference in the genomes (i.e. a greater subset of existing in the genome genes is expressed in the human cellular transcriptomes compared to the mouse transcriptomes). The evolutionary turnover of C2H2-ZF(-KRAB) genes acts in the direction of the revealed phenomenon, i.e. gene duplication and loss enhances the difference in the relative number of C2H2-ZF(-KRAB) genes between human and mouse cellular transcriptomes. A higher amount of these genes is expressed in the brain and embryonic cells (compared with other tissues), whereas a lower amount--in the cancer cells. It is specifically the C2H2-ZF transcription factors whose repertoire is poorer in the cancer and richer in the brain (other transcription factors taken together do not show this trend). These facts suggest that increase of anatomical complexity is accompanied by a more complex intracellular regulation involving these transcription factors. Malignization is associated with simplification of this regulation. These results agree with the known fact that human cells are more resistant to oncogenic transformation than mouse cells. The list of C2H2-ZF genes whose suppression might be involved in malignization is provided.

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32P-labelling anomalies in human erythrocytes. Is there more than one pool of cellular Pi?

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