Enzymes of cultured human fibroblasts. IV. Enzyme activity in trisomy C strains]

The activity of five enzymes (AIP, AcP, GOT, LDH, MDH) was investigated in four cell strains derived from spontaneous abortuses with C-trisomy (three cell strains with trisomy 7, one--with trisomy 9). Significant differences in the activity of three enzymes were revealed. In all the strains AIP activity was lower and GOT activity--higher than in diploid strains. Lowering of AcP level was found in three strains (two cell strains with trisomy 7, one--with trisomy 9). The data obtained are evaluated as a result of disturbed regulatory interrelations in an abnormal genome.     

Epidermal growth factor stimulates putrescine transport and ornithine decarboxylase activity in cultivated human fibroblasts

Many observations link increased intracellular polyamine levels with increased rates of cell proliferation [6, 7, 10, 23, 25]. EGF stimulates the proliferation of cultivated human skin fibroblasts and KB cells. EGF increases intracellular levels of polyamine through two mechanisms, the stimulation of both ornithine decarboxylase and polyamine transport. It is possible that the stimulation of cell proliferation by EGF may be an effect of increased polyamine concentration. The polyamines putrescine, spermidine, and spermine are actively transported into the cell and appear to share the same transport mechanism.     

Phosphatidylglycerol-modulated protein kinase activity from human spleen. I. Enzyme purification and properties

Protein kinase P (PK-P) is a phospholipid-modulated protein kinase activity previously described in human and murine cells. This paper details the 3300-fold, high yield purification to electrophoretic homogeneity of protein kinase P from human spleen by a three-step chromatographic process. Physical characterization disclosed a protein of Mr 27,000 (by electrophoresis) or 31,700 (by gel filtration and sedimentation) and pI 5.09. Protein kinase P activity was stimulated by phosphatidylglycerol or phosphatidylinositol, with maximal stimulation observed between 200 and 400 micrograms/ml phospholipid. No stimulation was noted using phosphatidic acid or phosphatidylserine. Histone H2B was the best substrate for demonstrating the protein kinase P phospholipid stimulation. Histone H1 was phosphorylated in a phospholipid independent manner. Vinculin and actin were not substrates. Optimum enzyme activity was observed at approximately 35 degrees C and pH 6.95. PK-P was relatively insensitive to the calmodulin and protein kinase C inhibitors W7 and H7, and to the cAMP-dependent protein kinase inhibitor. Kinetic analysis disclosed complex patterns including optimal rather than Michaelis-Menton kinetics for histone and phospholipid concentration, and a steep activation threshold with respect to histone concentration in the presence of phospholipid. Biphasic kinetics for Mg2+-ATP were observed, with the major stimulatory effect of phospholipid being on Vmax rather than Km. These data suggest a model for the mechanism of activation of protein kinase P by phospholipid entailing a direct three-way interaction between substrate, enzyme, and phospholipid micelles rather than allosteric activation by phospholipid.     

Naturally Occurring Enzyme Activity Variation in DROSOPHILA MELANOGASTER. I. Sources of Variation for 23 Enzymes

The genetic component of variation of enzyme activity levels in Drosophila melanogaster was investigated by using 48 second- and 48 third-chromosome isogenic substitution lines derived from natural populations. The results confirm those of our earlier experiments with the same lines and extend them to a number of additional enzymes. All 23 enzymes show a significant genetic component to the variation in one or both sets of lines and only a small part of this variation is accounted for by variation among the lines in the amount of tissue per fly. The magnitude of line effects is, in most cases, considerably larger than the magnitude of environmental and measurement error effects, and the line effects are approximately continuous in distribution. Variation in the geographic origin and karyotype of the chromosomes generally does not contribute to the line component of variation, but allozymes provide an important source of variation for a few of the enzymes. Many of the enzymes show evidence for variation of activity modifiers that are not linked to the structural locus of the enzyme.     

Enzyme activity and bacteriophage infection. I. Breakdown of adenosinetriphosphate

Experiments have been performed on the apyrase activity of E. coli, strain B. Although the dependence on pH and substrate is similar to that of rat tissue, the bacterial extracts are inhibited by Ca⁺⁺ and stimulated by Mg⁺⁺. In bacterial extracts the rate of phosphate release decreases in the course of the reaction, possibly owing to product inhibition. With multiple bacteriophage infection, the apyrase activity of the intact cells increased several fold, and the activity of extracts increased about 30 per cent. It is suggested that the changes could be attributed to an increase in the amount of enzyme although other alternatives cannot be precluded at present.     

Characterization of neuraminidase activity of cultured human fibroblasts.

Investigations have been carried out to establish the enzymatic properties and specificities of the neuraminidase of cultured human fibroblasts. Homogenates of these cells cleaved the actylated derivative of neuraminic acid from fetuin, N-acetylneuraminyllactose and 2-(3' methoxyphenyl)-N-acetyl-alpha-neuraminic acid. Maximum activity occurred between pH 4.2 and 4.6 in sodium acetate buffer. The Km values were 3.6 . 10(-4) M, 3.0 . 10(-3) M and 1.1 . 10(-3) M, respectively, against fetuin, N-acetylneuraminyllactose and 2-(3'methoxyphenyl)-N-acetyl-alpha-neuraminic acid. Against the first two substrates, the rate of hydrolysis fell below the expected value as the cell homogenate was diluted with water or 10 mM NaCl. Dilution with 8 mg/ml bovine serum albumin prevented the deviation and yielded the expected linear decrease. After the first 2-h incubation, the rate of hydrolysis decreased from the initial linear rate. The enzyme(s) was partially or completely inactivated by sonication at 20 kHz, freeze-thaw treatment, incubation at 52 degrees C or storage for 48 h at -70 degrees C. Suspension of the fibroblasts in water for 10 min at room temperature, followed by homogenization with a tissue grinder, yielded preparations that were suitable for the assay of the neuraminidase activity.     

DNA ligase activity in carcinogen-treated human fibroblasts

In an enzymological approach to study DNA repair mechanisms induced by carcinogen-treatment of mammalian cells, we have investigated how DNA ligase activity is affected by the treatment with several compounds producing different DNA lesions. Stationary cultures of human fibroblasts were exposed to various doses of carcinogens (UV-light at 254 nm, N-acetoxy-acetyl-aminofluorene, ethyl-methane sulfonate, N-methylnitro-nitrosoguanidine, mitomycin C and 4-nitroquinoline-N-oxide) at different time-intervals before preparing crude cellular extracts and assaying for ligase activity. Results have shown that: 1. UV-irradiation, AAAF, 4NQO or MMC treatment of cells induces a two-fold increase in the ligase activity compared to control cells within 48 hours following the treatment. 2. A partial purification of the enzyme from these cellular crude extracts by sedimentation through sucrose gradients has shown: a. DNA ligase activity from control cells presents a profile composed of two distinct peaks sedimenting respectively at about 4S and 7S; b. the carcinogen treatment of either repair-proficient human fibroblasts or repair-deficient xeroderma pigmentosum cells (complementation group A) seems to induce a specific increase of the 4S-form of DNA ligase.     

Lysosomal glycosidase activity in cultured human fibroblasts

A study was made of the activity of 3 lysosomal glycosidases -beta-D-galactosidase (K. P. 3.2.1.23), alpha-L-fucosidase (K. P. 3.2.1.51), N-acetyl-beta-D-hexosoaminidase (K. P. 3.2.1.52) depending on the time after subcultivation and duration of the passage of human skin embryonal and postembryonal fibroblasts. It was established that changes in the specific activity of the enzymes should be calculated with reference to the cell rather than to protein whose amount might vary considerably. It was also found that for measuring the specific activity of enzymes, of great importance are the procedures of cell removal from the base layer (by mechanical scraping off or by trypsin solution) and the regimen of the homogenization of cell preparations.     

Akaline phosphatase: activity and variation in human diploid fibroblasts.

A method is introduced for the assay of alkaline phosphatase in homogenates of cultured human skin fibroblasts. In a first group of 11 strains, a four- to fifteen-fold increase of enzyme activity is consistently observed following a period of starvation. In the remaining 31 cell-strains similar specific activities of alkaline phosphatase are found irrespective of medium changes. In regularly fed cultures, an inverse exponential correlation between the specific activity of alkaline phosphatase and the age of the donor has been detected.     

Growth factor-mediated regulation of aromatase activity in human skin fibroblasts.

We investigated the effects of various hormones and growth factors on aromatase activity in cultured human skin fibroblasts. Several potential trophic factors were tested for their ability to modify basal aromatase activity or the response to dibutyryladenosine 3',5'-cyclic monophosphate and dexamethasone because (i) no endogenous ligand has been identified that is responsible for stimulating aromatase activity in the periphery, and (ii) dexamethasone and cAMP analogs can increase this enzyme's activity in fibroblasts. The effect of insulin and insulin-like growth factors were examined in closer detail because of the clinical association between insulin and hyperandrogenism. Pituitary hormones and hypothalamic releasing factors, such as human ACTH (10 nM), beta-endorphin (10 nM), beta-lipotropin (10 nM), alpha-MSH (10 nM), gamma 3-MSH (10 nM), ovine luteinizing hormone (10 ng/ml), ovine follicle-stimulating hormone (10 ng/ml), ovine thyroid-stimulating hormone (10 ng/ml), rat growth hormone (10 ng/ml), rat prolactin (10 ng/ml), rat corticotropin-releasing factor (10 nM), luteinizing hormone-releasing factor (10 nM), thyrotropin-releasing factor (10 nM), human growth hormone-releasing factor (10 nM), and somatostatin (10 nM), have no significant effects on aromatase activity. Porcine inhibin A (10 ng/ml) and porcine activin AB (10 ng/ml), two ovarian hormones with structural transforming homology to transforming growth factor-beta, also have no effect on aromatase activity. Although basic fibroblast growth factor (1-100 ng/ml), acidic fibroblast growth factor (1 ng/ml), epidermal growth factor (1 ng/ml), platelet-derived growth factor (1 ng/ml), tumor necrosis factor (1 ng/ml), and transforming growth factor-beta 1 (1 ng/ml) have no effect on basal aromatase activity in human skin fibroblasts, all of these growth factors inhibited the ability of dibutyryladenosine 3',5'-cyclic monophosphate to stimulate aromatase activity. In contrast, both insulin (100 pg/ml-10 ng/ml) and insulin-like growth factor-1 (1-100 ng/ml) had no effect on cAMP-stimulated aromatase but potentiated the action of dexamethasone (100 nM). Thus, there is a clear distinction between the effects of dexamethasone and cAMP on peripheral aromatase. On the basis of the results presented here, it is interesting to speculate that the hyperandrogenism that is often associated with insulin resistance may be due to a combination of growth factor-mediated inhibition of aromatase activity and the failure of peripheral tissues to respond to insulin and metabolize androgens to estrogens.     

Regulation of cyclic nucleotide phosphodiesterase activity in human lung fibroblasts.

Cyclic nucleotide phosphodiesterase activity (3', 5'-cyclic-nucleotide 5'-nucleotidohydrolase, 3.1.2.17) was studied in homogenates of WI-38 human lung fibroblasts using 0.1--200 microgram cyclic nucleotides. Activities were observed with low Km for cyclic AMP(2--5 micron) and low Km for cyclic GMP (1--2 micron) as well as with high Km values for cyclic AMP (100--125 micron) and cyclic GMP (75--100 micron). An increased low Km cyclic AMP phosphodiesterase activity was found upon exposure of intact fibroblasts to 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase activity in broken cell preparations, as well as to other agents which elevate cyclic AMP levels in these cells. The enhanced activity following exposure to 3-isobutyl-1-methylxanthine was selective for the low Km cyclic AMP phosphodiesterase since there was no change in activity of low Km cyclic GMP phosphodiesterase activity or in high Km phosphodiesterase activity with either nucleotide as substrate. The enhanced activity due to 3-isobutyl-1-methylxanthine appeared to involve de novo synthesis of a protein with short half-life (30 min), based on experiments involving cycloheximide and actinomycin D. This activity was also enhanced with increased cell density and by decreasing serum concentration. Studies of some biochemical properties and subcellular distribution of the enzyme indicated that the induced enzyme was similar to the non-induced (basal) low Km cyclic AMP phosphodiesterase.