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Purine nucleotide synthesis in normal and leukemic blood cells

Purinnukleotidsynthese in normalen und leukämischen Blutzellen

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Zusammenfassung

Die erste irreversible und geschwindigkeitsbestimmende Reaktion in der Purinde-novo-Synthese ist die Bildung von β-Phosphoribosylamine aus Glutamin und Phosphoribosylpyrophosphat (PRPP), katalysiert durch das Enzym PRPP-Glutamin-Amidotransferase (EC 2.4.2.14).

In der vorliegenden Arbeit wird nachgewiesen, daß normale Granulozyten, Erythrozyten und Knochenmarkszellen dieses Enzym nicht besitzen und deshalb auf die externe Zufuhr von Purinbasen, bzw. auf den Reutilisierungsstoffwechsel (“salvage-pathway”) angewiesen sind. Im Vergleich zu normalen Lymphozyten aus Milz und peripheren Blut besitzen die leukämischen Zellen eine deutlich gesteigerte Enzymaktivität und damit eine erhöhte Purin-de-novo-Synthese.

Bei der Untersuchung der kinetischen Eigenschaften zeigt die PRPP-Amidotransferase im Rohhomogenat für PRPP eine sigmoidale Substratsättigungskurve während es für Glutamin eine hyperbole Kinetik aufweist.

Im Vergleich zum normalen Lymphozytenenzym — ist die PRPP-Amidotransferase in allen leukämischen Zelltypen bei wesentlichen niedrigeren PRPP-Konzentrationen gesättigt — und wird bei physiologischen AMP-Konzentrationen in geringerem Ausmaß gehemmt.

Das partiell gereinigte Enzym (ca. 10 – 15fach) — sowohl von normalen Milzlymphozyten — als auch von allen leukämischen Zellen zeigt für beide Substrate nun eine hyperbole Substratsättigungskurve. Bei der doppelt reziproken Auftragung nach Lineweaver-Burk betragen die Km-Werte für PRPP 0,14 mM und für Glutamin 2,0 mM. Es wurden nun sowohl für das partiell gereinigte Enzym aus leukämischen Zellen als auch für das Lymphozytenenzym gleiche Km-Werte gefunden.

In Anwesenheit verschiedener AMP-Konzentrationen weist die Substratsättigungskurve für PRPP wieder eine sigmoidale Kinetik auf. Das partiell gereinigte Lymphozytenenzym wird nun durch AMP im gleichen Ausmaß gehemmt, wie das Enzym aus leukämischen Zellen.

Nach den hier vorgelegten Befunden ist die Steigerung der Aktivität des Schlüsselenzyms der Purinsynthese (PRPP-Amidotransferase) ein metabolischer Parameter der maligne transformierten Leukämiezellen. Zugleich wird verdeutlicht, daß hier ein Punkt besonderer Angreifbarkeit durch die Chemotherapie liegt.

Summary

The present study was undertaken to obtain more precise information about the purine biosynthetic pathway in human blood cells. 5′-phosphoribosyl-1-pyrophosphate (PP-ribose-P) amidotransferase was found in cell-free extracts from all leukemic cells and normal lymphocytes and therefore these cells could synthesize the first intermediate of the purine-de-novo-synthesis. Normal leucocytes, erythrocytes and bone marrow cells lack this enzyme system and have an absolute requirement for externally supplied purines via salvage pathway. Leukemic blast cells show different enzyme activities independent of their cell count.

Kinetic studies with the crude enzymes showed sigmoidal substrate velocity curves for PP-ribose-P, whereas glutamine shows hyperbolic kinetics. The leukemic cell enzymes from all four donor types (ALL, CLL, AML and CML) are rapidly saturated with low concentrations of PP-ribose-P and less inhibited by the physiological feedback inhibitor, adenosine 5′monophosphate.

The crude enzymes of normal spleen lymphocytes and leukemic cells were further purified (10 to 15-fold) and substrate velocity curves for PP-ribose-P and glutamine show now hyperbolic kinetics and double reciprocal plots were linear with and apparent Km for PP-ribose-P of 0.14 mM and for glutamine 2.0 mM. In the presence of different concentrations of AMP, the PP-ribose-P substrate velocity plot changed from a hyperbolic to a sigmoidal curve; no difference in the degree of the inhibition between both partially purified enzymes (normal spleen lymphocytes and leukemic cells from all four donor types) could now be observed.

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Authors and Affiliations

  1. Department of Medicine, University of Freiburg/Breisgau, Germany

    H. Becher, M. Weber & G. W. Löhr

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  1. H. Becher
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  2. M. Weber
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  3. G. W. Löhr
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This study was supported by the Deutsche Forschungsgemeinschaft Be 458/3

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Becher, H., Weber, M. & Löhr, G.W. Purine nucleotide synthesis in normal and leukemic blood cells. Klin Wochenschr 56, 275–283 (1978). https://doi.org/10.1007/BF01489173

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