Nucleosidediphosphate Kinase from Ehrlich Ascites Tumor Cells1

Abstract

A phosphate-incorporating protein has been highly purified from the cytosol of Ehrlich ascites tumor cells (EAT cells). The nitrocellulose membrane method was used to follow the progress of the purification by quantitation of the [32P]phosphorylated form of the protein. The purified protein was identified as an NDP-kinase since it exhibited NDP-kinase activity and had enzyme characteristics in common with other NDP-kinases from various mammalian cells. The purified NDP-kinase was found to have a molecular weight of approximately 76,000 daltons. Moreover, the enzyme appears to consist of two distinct polypeptides (18,000 and 20,000 daltons). This enzyme contained 19 amino acids, with high levels of glycine (9.8%) and lysine (9.0%). The enzyme rapidly formed a [32P]phosphoenzyme when incubated with [gamma-32P]ATP in the presence of Mg2+ (1 mM) at the optimum pH of 7.5 even at low temperature (below 4 degrees C). This phosphoenzyme is an enzyme-bound, high-energy-phosphate intermediate, because ATP was formed from it on incubation with ADP in the presence of Mg2+ (1 mM). This finding suggests that the phosphoenzyme functions as an intermediate in NDP-kinase action.

Complete recovery of the phosphoenzyme‐forming activity of nucleoside‐diphosphate kinases after reconstitution of their subunits

“…The enzyme reaction was initiated by the addition of 20pM [y-32P]ATP (3000 cpm/pmol) and incubated for 5 min in an ice bath. The [32P]phosphate incorporation into the fractions was determined by the nitrocellulose membrane method as described in [2-51 290 DEAE-cellulose column chromatography showed the existence of two distinct NDP kinases (F-I, F-II), composed of two similar distinct subunits ( fig.3), in mammalian cells such as mouse NK [2,3], EAT [5] and HeLa S3 cells [4,6]. Although it would seem that the existence of two distinct NDP kinases is common in mammalian cells, there are some dissimilarities between these two enzymes: (i) these two enzyme subunits are differentially phosphorylated when incubated with [Y-~~P]ATP in the presence of divalent cations (Ca2+ and Mg2+) (fig.2); (ii) the [32P]phosphate incorporating (phosphoenzyme-forming) activity of the F-I enzyme is relatively stable as compared with that of the F-II enzyme after incubation for 20 min at 60°C [6]; and (iii) the biochemical properties of the enzyme-associated GTP-binding protein are clearly different in each enzyme [6].…”

“…Using this method, we purified NDP kinases to a high degree from various mammalian cells, such as mouse NK [3], Ehrlich ascites tumor [5] and HeLa S3 cells [4,6]. We found that the purified enzymes from different mammalian cells consist of two distinct subunits [a-subunit (Mr 20000-22000) and P-subunit (Mr l&X000-19000)] [2-61.…”

RELATIONSHIP BETWEEN NUCLEOSIDE DIPHOSPHATE KINASE ACTIVITY AND LIGHT‐LIMITED GROWTH RATE IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1

“…Although NDPK has been measured in a wide range of organisms from bacteria to higher plants to mammalian cells (Parks and Agarwal 1973), we are aware of only one report of a measurement in a unicellular autotroph (Klein and Follmann 1988) and no cases of measurements in marine phytoplankton. There has been speculation about the importance of NDPK in cell growth processes during development (Dickinson and Davies 197 l), including a correlation with growth rate in mammalian tumor cells (Koyama et al 1984) and evidence of relationships between NDPK and growth rate in crustaceans (Berges 1989, Berges et al 1990). In multicellular organisms, however, the relationship between NDPK and growth is complicated by changes in body size and composition during development.…”

Καθαρισμός Ιδιότητες Και Εξειδίκευση Ισομορφών Κινασών Των Διφωσφονουκλεοζιτών Από Ενδοσπέρμιο Καλαμποκιού - Μοριακές Προσεγγίσεις

“…Μη φυτικές NDP κινάσες Η NDP κινάση έχει καθαριστεί μέχρι ομογένειας από διάφορες πηγές (Richard et al, 1975;Agarwal and Parks, 1971;Koyama et al, 1984;Kimura and Shimada, 1988). Στη δεκαετία του 60 και αρχές του 70 υπήρξε ένα ενθουσιώδες κύμα μελετών αλλά μόνον πολύ αργότερα άρχισε να εμφανίζεται μια πιο καθαρή εικόνα του ενζύμου.…”