The activity of hexokinase was studied in several normal and malignant human tissues. The enzyme activity in tumors was significantly higher. Isoenzyme studies on normal gastric mucosa and stomach cancer extracts showed that malignancy is accompanied by a "simplification" of the hexokinase isoenzyme pattern due to "deletion" of the slowest isoenzyme. Preparative polyacrylamide gel electrophoreis was used to isolate hexokinase isoenzymes from normal and malignant tissues. Tumor hexokinase isoenzymes displayed an increased affinity to glucose when compared to the corresponding normal prototypes (Km/glucose, 10(-6) M and 10(-5) M, respectively; Km = Michaelis constant). The molecular weights, subunit composition, and peptide patterns were identical for corresponding isoenzyme pairs from normal and tumor tissues.
Highly purified human ceruloplasmin was isolated from fresh donor blood in the presence of inhibitors of proteolysis and from stored retroplacental blood serum both with and without inhibitors of proteolysis. According to the data of electrophoresis. ultracentrifuge sedimentation velocity and sedimentation equilibrium, all the ceruloplasmin samples were homogeneous, their molecular weight being 130 000.
The dissociation of the samples treated by dodecylsulphate, guanidine · HCl, and urea was studied by means of quantitative analytica and preparative electrophoresis, and sedimentation equilibrium. The dissociation patterns depended on whether inhibitors were used in the isolation procedure. Polypeptides with molecular weights of 130 000, 112 000 and 16 000 (minor component) were obtained, if phenylmethylsulfonyl fluoride and/or 6‐aminohexanoic acid were used; if these compounds were not used, the dissociation yielded additional polypeptides of molecular weights of 100 000 (minor component), 64 000 and 48 000. Under proteolysis‐favouring conditions the relative amount of these polypeptides increased. Prolonged storage of samples under sterile conditions without inhibitors of proteolysis resulted in a decrease of the relative amount of polypeptides with molecular weights of 130 000, 112 000, 100 000, 64 000 and 48 000, contrary to that of the 16 000‐Mr polypeptide, which increased. At the same time new polypeptides appeared with molecular weights of 42 000 and 21 500–23 000.
Spontaneous specific fragmentation of the ceruloplasmin molecule is due to trace amounts of proteases, which seem to originate from blood plasma.
Limited tryptic hydrolysis of the ceruloplasmin globule resulted in the appearance of polypeptides with the same molecular weights which were observed in spontaneous fragmentation.
A conclusion is drawn that the ceruloplasmin molecule in vivo is a single polypeptide chain with at least five bonds which in vitro are the points of specific proteolytic fragmentation, yielding six principal fragments.
Fragments of the natural rat ceruloplasmin (Cp) gene and cDNA copies of rat Cp and transferring (Tf) mRNAs highly labelled by nick translation with 125I-dCTP were used as specific probes for assignment of these genes to the metaphase chromosomes of rat, mouse and man by in situ hybridization. Both Cp and Tf genes were found to be syntenic in rodents, occupying with high probability the regions 9D and 9F1-3 in mice and 7q11-13 and 7q31-34 in rats respectively. The significant increase in silver grain count over chromosome 15 in rats after hybridization with both the Cp and Tf probes suggests the presence of a related pseudogene cluster on this particular chromosome and thus favours its partial homeology to chromosome 7. The localization of silver grains in metaphase chromosome of man indicates subregional assignment of the Tf gene to 3q21. Use of the rat Cp DNA probe does not indicate synteny of the Cp and Tf genes in man and suggests the existence of a related DNA sequence in 15q11-13. The potential and limitations of the in situ hybridization technique with heterologous DNA probes for gene mapping in mammalian species are discussed.
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