Calibration of a flow cytometric assay of glucose-6-phosphate dehydrogenase activity.

The reduction of tetrazolium salts to colored formazans is a reaction which has been exploited both in histo- and cytochemistry. Tetrazolium salts forming fluorescent formazans prove suitable for measuring defined cellular dehydrogenase activities in automated processes. This study considers an important aspect of formazan measurement in flow cytometry, namely, calibration. Calibration is performed by correlating the number (and fluorescence intensity) of formazan-bearing cells measured by flow cytometry with simultaneously performed biochemical analyses of the same material. The method is demonstrated by an example of glucose-6-phosphate dehydrogenase. Using the data of a typical experiment, the enzyme activity is expressed in femtomol of hydrogen transferred per cell during incubation time. Furthermore, through spatially resolved double excitation of formazan and nuclear DAPI fluorescence, an independent analysis of cell cycle and cellular enzymatic activity is established.     

Enzymes of a bacteriolytic lysoamidase preparation. Various properties of bacteriolytic protease L2]

Bacteriolytic proteinase L2 is able to cleave fluorogenic synthetic tripeptide anthranoyl-alanyl-alanyl-phenylalanyl-nitroanilide (Abz-Ala-Ala-Phe-pNA) at the bond between phenylalanine and p-nitroaniline. Optimal conditions of the tripeptide cleavage have been determined: pH 6.7 + 0.1; mu = 2 (by NaCl); t = 40 degrees C; KM = 2.6 x 10(-5) M. Metal cations reduced the enzyme activity. The enzyme was inhibited by EDTA, p-CMB, DIF. The synthetic tripeptide can be used to determine the activity of the L2 enzyme.     

Preferential expression of the maternally inherited X-linked phosphoglycerate kinase allele in human erythrocytes

Summary The stability of allelic gene expression of X-linked phosphoglycerate kinase was studied in seven carriers of a rare genetic variant named PGK München. The enzymatic activities in erythrocytes of five heterozygous females and three hemizygous males were determined repeatedly over a period of 10 years (1975–1984) and shown to remain constant. As the phosphoglycerate kinase activity is lower in cells expressing the PGK München allele, the ratio of the two cell types in all heterozygous females of the PGK München kindred could be calculated from the PGK activity and from the known allozyme activities in erythrocytes of homozygous wild type or hemizygous PGK München carriers. Since the maternal or paternal origin of both alleles is known from the pedigree, the quantitative expression of the maternally derived allozyme in heterozygous women could be determined. In heterozygous carriers the cell pool expressing the maternally inherited allele was significantly increased, independently, of the PGK allele linked to the maternal X chromosome (P<0.001). Our data show that inactivation of one of the two X chromosomes in human female erythropoietic stem cell precursors may be non-random, at least in the kindred and cell populations described here. The results are discussed in the context of random X chromosome inactivation (Lyon hypothesis).Dedicated to J.S., the senior of the family studied, on the occasion of her 80th birthday     

Study of 2'-phosphodiesterase activity in cultured NIH 3T3 cells during activation of cAMP-dependent phosphorylation

A rapid and transient decrease in 2'-phosphodiesterase activity in NIH 3T3 mouse cells was observed after adrenaline addition. The decrease of activity was accompanied by an elevation of intracellular cAMP level. The 2'-phosphodiesterase activity changed similarly when cells sink deeper into the resting state. In the latter case, the fall of the enzyme activity was correlated with elevation of the activity of cAMP-dependent proteinkinase and, moreover, a considerable increase of the intracellular level of 2',5'-oligoadenylate was observed. Phosphorylation of proteins by cAMP-dependent proteinkinase in the cell lysate also produced a pronounced drop of 2'-phosphodiesterase activity. Exogenous 2',5'-oligo (A) treatment of the cells resulted in the rise of 2'-phosphodiesterase activity; actinomycin D prevented this effect. The data presented suggest the involvement of two different mechanisms in regulation of 2'-phosphodiesterase activity: cAMP-dependent phosphorylation and induction of 2'-phosphodiesterase by 2',5'-oligoadenylate.     

Mechanism of action of cAMP-dependent protein kinase. V. Free energy spectra

The catalytic subunit of cAMP-dependent protein kinase from pig brain was shown to catalyse an isotope exchange reaction ATP in equilibrium with ADP. The kinetic parameters of the exchange were determined. The enzyme can also use GTP as the donor substrate; phosphotransferase and "GTPase" reactions were investigated. Based on the kinetic data obtained in this and in the previous paper the free energy profiles of protein kinase catalysed reactions are discussed.     

Dipyridamole-induced interferon production in mouse peritoneal leukocytes

The in vitro explanted mouse peritoneal leukocytes were used for the optimization of the dipyridamole-induced interferon production. After 90-120 min incubation of cells with 30-100 microM dipyridamole, the production of interferon reached 6.4 X 10(4) IU/ml. It was demonstrated that the interferon production phase is preceded by the dipyridamole-dependent increase in cAMP concentration. The possibility of cAMP involvement in the mechanism of interferon production is being discussed.     

Uridine phosphorylase activity among the class mollicutes

Uridine phosphorylase activity has been used to detect mycoplasmas in cell cultures by measuring formation of¹⁴C-uracil from¹⁴C-uridine. In this report we show that all species ofMycoplasma, Acholeplasma, andUreaplasma tested exhibited uridine phorphorylase activity. Among the genusSpiroplasma, serogroups I-1, I-3, I-5, I-7, I-8, IV, XIII, and XIV lacked uridine phosphorylase activity.Present address: Ciba-Geigy, Basel, Switzerland.     

Localization of histone H1 in chromatin. Cross-linking of the N- and C-terminal halves of the molecule with bifunctional reagents

Mutual arrangement of histone H1 molecules was studied in calf thymus nuclei, extended chromatin and chromatin, isolated and kept in 8 M urea. Histone H1 dimers crosslinked with methyl 4-mercaptobutyrimidate were digested with chymotrypsin and crosslinked fragments obtained were analysed by diagonal gel electrophoresis. In all chromatins tested the N- and C-terminal parts of the H1 molecules were crosslinked in all possible combinations, i.e. C-C, C-N and N-N. These and related data obtained earlier indicate, that the proximity of histone H1 molecules in chromatin is determined by the structure of nucleosomal chain itself and not by chromatin superstructure. The results also suggest that the H1A and H1B subfractions of histone H1 are interspersed in extended nucleosomal chains.