Grace K. Arimura

Enzymes of glycogen metabolism in white blood cells III. Heterogeneity of glycogen phosphorylase from rat chloroma

Summary 1. Glycogen phosphorylase ( α -1,4-glucan:orthophosphate glucosyltransferase (EC 2.4.1.1)) of rat chloroma has been purified and partially characterized. 2. Column chromatography on o -(diethylaminoethyl)-cellulose yields 2 phosphorylase peaks both of which are active without adenosine 5′-phosphate. The identity of these 2 peaks has been ascertained by rechromatography on O -(diethylaminoethyl)-cellulose and electrophoresis on acrylamide gel. 3. There appears to be some distinct differences in the properties of the enzyme from the 2 peaks. 4. The finding of 2 isoenzymes of phosphorylase in rat chloroma represents the second example of heterogeneity of this enzyme, the first example being that of phosphorylase from rabbit heart.

Characteristics of fibrinolysin secreted by cultured rat breast carcinoma cells.

Summary Fibrinolysin secreted by cultured R2426 rat breast carcinoma cells has been purified and partially characterized. Three methods of assay were used: the hydrolysis of [ 125 I]fibrinogen and N-p -tosyl- l -arginine methyl ester (TAME), and clearing of fibrin-agar plates (heated and unheated). Two types of fibrinolytic activity were identified: a plasminogen activator and a direct fibrinolysin. The fibrinolysin had direct hydrolytic action on plasminogen-free fibrinogen, fibrin, and TAME. The products of fibrinogen hydrolysis by the tumor cell fibrinolysin differed from those of human plasmin. It was heat stable (60°C, 12 h), and was inhibited by e-aminocaproic acid and soybean trypsin inhibitor. Ethylenediaminetetraacetate (EDTA) inhibited the action of the tumor cell enzyme on TAME, but not on human fibrinogen. The direct fibrinolytic activity could be separated from plasminogen activator activity by gel filtration on Sephadex G-200, and the molecular weight was estimated to be approx. 110 000 D.

Inhibition by rhodamine 123 of protein synthesis in mitochondria of normal and cancer tissues

The effect of the mitochondrial dye rhodamine 123 (Rho 123) on protein synthesis (PS) activity was investigated in mitochondria isolated from liver and from both chloroma and erythroleukemia tumors. Incorporation of labelled leucine into mitochondrial protein was used to measure the rate of PS. While PS specific activity was much higher in hematopoietic tumors mitochondria as compared to that of liver, the addition of increased concentration of Rho 123 in all tested organelles resulted in increased inhibition of PS to reach 75-82% with 10 micrograms/ml of the dye. Similar results were obtained with 10 micrograms/ml of chloramphenicol, the specific inhibitor of mitochondrial PS. Moreover, under the conditions of the study, the addition of Rho 123 to mitochondria did not trigger any ATPase activity, thus eliminating any competition for the energy source ATP between PS and ATPase. These results demonstrate that, in addition to its known inhibitory action on oxidative phosphorylation, the mitochondrial dye Rho 123 has a potent inhibitory effect on PS in both liver and hematopoietic tumors mitochondria.

Enzymes of glycogen metabolism in white blood cells II. Activation and inactivation of glycogen phosphorylase of rat chloroma

Summary 1. The properties of glycogen phosphorylase (α-1,4-glucan:orthophosphate glucosyl transferase, EC 2.4.1.1) of rat chloroma, a tumor composed entirely of immature granulocytes, and the interconversion of the active and inactive forms of the enzyme have been studied in crude chloroma extract. 2. Striking similarities in the properties of chloroma phosphorylase to liver phosphorylase have been noted. Thus, the following properties are shared by both enzymes: Two forms of enzyme can be identified, an active and an inactive form; the active form exhibits 65–80% of its activity in the absence of adenosine 5′-phosphate, the inactive enzyme is not significantly active even in the presence of the nucleotide; the activity of the enzyme is not enhanced by cysteine; the inactive enzyme shows considerable activity when assayed in the presence of high salt concentration; the enzyme is activated by glucagon. 3. A phosphorylase kinase (ATP:phosphorylase phosphotransferase, EC 2.7.1.38) capable of converting rabbit-muscle phosphorylase b to a and a phosphatase (phosphorylase phosphohydrolase, EC 3.1.3.17) catalyzing the reverse reaction both active at neutral pH have been demonstrated in chloroma tissue.

Sodium-Potassium Dependent Adenosine Triphosphatase of Mammalian Reticulocytes and Mature Red Blood Cells.

Summary The level of Na-K-dependent ATPase was found to be considerably higher in reticulocytes than in mature erythrocytes. The fall in enzyme activity in the red cell upon maturation occurring concomitantly with loss of capacity to concentrate aminoacids lends support to the hypothesis that Na-K-dependent ATPase plays an important role in the active transport of aminoacids.

Purification of a colony stimulating factor from culture cell lines propagated from human lung

Colony stimulating factor (CSF) is required for the in vitro growth of marrow myeloid colonies (CFU-C) and is thought to play an important role in the regulation of granulopoiesis in vivo [l-3] . Although CSF has been isolated from multiple murine and human tissues [4-61, the sources of human CSF have been extremely limited. Human urinary CSF has been purified to homogeneity but curiously this glycoprotein stimulates CFU-C growth in murine, but not in human bone marrow [7]. Recently human CSF from the conditioned medium of human placenta and human lung have been partially purified [8,9]. However, major contaminants derived from tissue and serum presented considerable difficulty in purification. To overcome this problem we have attempted and succeeded in propagating cell lines from autopsy lung tissue which secrete CSF into the medium. Serum-free conditioned medium prepared from these cultures provides an excellent source of CSF.

Differentiation of cultured promyelocytic leukemia cells (HL-60) induced by endotoxin-treated human lung conditioned medium

Serum-free conditioned medium prepared from endotoxin-treated human lung tissue contains potent differentiation activity (DA) which induces the differentiation of human promyelocytic leukemia cells (HL-60) to mature functioning granulocytes and macrophages. Upon fractionation, the DA can be separated from colony-stimulating factors (CSFs). The estimated molecular weight of DA is 23,000 d which is similar to CSF-II but is much smaller than CSF-I (50,000 d). The isoelectric point (PI) value obtained from preparative isoelectrofocusing is 5.2, which is higher than CSF-I of 4.1-4.8 but lower than CSF-II of 5.6. Using isopropanol as solvent and trifluoroacetic acid (0.2%) as ion-pair, the DA is eluted at 37% isopropanol from the C-3 column in comparison to 30% for CSF-II and 34% for CSF-I.

Sensitivity of cultured pancreatic carcinoma cells toAcinetobacter glutaminase-asparaginase

SummaryCultured human pancreatic carcinoma cells (MIA PaCa-2) have been shown previously to be very sensitive toE. colil-asparaginase (EC II). The present studies have demonstrated that another enzyme,Acinetobacter glutaminase-asparaginase (AGA) is much more effective in inhibiting cell growth. At the concentration of 0.0025 U/ml of AGA activity the enzyme totally inhibited cell growth, whereas the EC II with the same concentration did not show any effect. The inhibition of cell growth correlated well with inhibition of protein and glycoprotein synthesis. The addition ofl-glutamine at the concentration of 1 mM completely reversed the inhibition of protein synthesis. Similarly, the addition ofl-glutamine at the concentration of 3 mM daily on 3 successive days after adding AGA resulted in significant reversal of growth inhibition. The results of this study indicate that the action of AGA on MIA PaCa-2 is, to a great extent, exerted through itsl-glutaminase activity.

Effect of Monase and Related Compounds on Uptake of 5-Hydroxytryptamine by Platelets.

Summary Monase indole-3(2-aminobu-tyl) acetate, a monoamine oxidase inhibitor, and 8 other structurally related compounds with different substitutions on the indole ring and amine side chain inhibited 5-HT uptake by platelets, degree of inhibition being directly related to concentration of inhibitor without particular relation to structure. C14-Mon-ase which differs from 5-HT by the absence of a hydroxyl group in position 5 of the indole ring and by the presence of a different side chain, was concentrated only slightly by platelets. It was concluded that the structural specificity for 5-HT uptake is largely determined by the hydroxyl group at position 5 of the indole ring.