Paul S. Ebert

A simple micro method for the direct determination of δ-amino[14Clevulinic acid production in murine spleen and liver homogenates

Abstract 1. 1. A simple and sensitive radiochemical procedure for the estimation of the activity of δ-aminolevulinate synthetase (succinyl-CoA: glycine succinyltransferase) in homogenates of normal murine spleen and liver is described. 2. 2. Homogenates of spleen or liver are incubated with α- keto [ 14 C] glutarate or [14C]succinate respectively, and the [14-amino14Clevulinic acid generated in the reaction is selectively adsorbed onto a Dowex 50 column run at pH 3.9. Since labeled α-amino acids and unreacted radioactive substrate are not reatained by the column, contamination of α-aminolevulinic acid by these substances does. δ- Amino- [ 14 C] levulinic can be recovered in yields of 97–100%. 3. 3. Maximal δ- amino[ 14 C]levulinic acid production was obtainedin spleen homogenates in the presence of α- keto[ 14 C]glutarate , glycine, EDTA, and pyridoxal phosphate. δ- Amino[ 14 C]leculinic acid utilization was minimal at homogenate concentrations of 2.5% or less. Varying degrees of inhibition of δ-amino14Clevulinic acid production were seen with malate, malonate, arsenite, ATP, CoA, and NAD+. 4. 4. [14]Succinate, glycine, EDTA, and pyridoxal phosphate produced the highest levels of δ- amino[ 14 C]levulinic acid in liver homogenates. δ- Amino[ 14 C]levulinic acid utilization was minimal at homogenate concentrations of 1% or less. Varying degrees of inhibition of δ- amino[ 14 ]levulinic acid production were seen with malate, malonate, arsenite, ATP, and CoA. 5. 5. The sensitivity of the procedure will allow the measurement of δ-amino-levulinate synthetase activity in normal spleen and liver and will permit more detailed investigation of mechanisms controlling heme synthesis. Smaller amounts of tissues can be used than have been previously employed by colorimetric methods. The method can be easily adapted to preparations of mitochondria and blood cells.

Induction of Δ-Aminolevulinic Acid Synthetase During Erythroid Differentiation of Cultured Leukemia Cells

Summary A cloned line of Friend erythroleukemia cells (T-3-Cl-2) can be induced to produce hemoglobin when grown in the presence of 1%-2% DMSO. Such DMSO-treated cells become 30%-60% benzidine-positive in 5-7 days. The control enzyme for hemoglobin synthesis, Δ-aminolevulinic acid (ALA) synthetase becomes elevated after 28 hr in DMSO-treated cells. Furthermore, this synthetase activity increased 2-6 times the level of control cells during the following 3 days. Maximal ALA synthetase activity was found when the culture media contained 2% DMSO. Allylisopropylacetamide alone was found to be as effective an inducer of ALA synthetase as 1% DMSO, and could augment enzyme activity when present with DMSO.

Potentiation of murine sarcoma virus (Harvey) (Moloney) oncogenicity in lactic dehydrogenase-elevating virus-infected mice.

Summary Adult Balb/c mice previously infected with LDV and subsequently challenged with H-MSV or M-MSV showed (i) increased tumor incidence, (ii) reduction in the incidence of tumor regression, (iii) increased incidence of death with tumor, and (iv) reduction of the median survival compared to H-MSV- or M-MSV-infected controls. Extracts of tumor from mice inoculated with H-MSV alone were devoid of focus-forming virus when assayed on 3T3 FL cells while extracts of tumors from mice dually infected with LDV and H-MSV contained 102, 3 FFU/ml of virus in the absence of “helper”virus and 103.0 FFU/ml in the presence of “helper”virus. The mechanism by which LDV potentiate H-MSV and M-MSV oncogenicity in adult mice is discussed.

Plasma lactate dehydrogenase and spleen heme biosynthetic activity following Friend and Rauscher leukemia virus infections.

Abstract The progression of plasma lactate dehydrogenase and heme biosynthetic activity in the spleen was followed in mice infected with Friend and Rauscher viruses. Plasma lactate dehydrogenase increased progressively in both viral diseases and paralleled the increase in spleen weight. Despite the similarity of the spleen weight responses, Friend virus promoted an earlier and more potent erythroid response as measured by porphobilinogen and porphyrin production than did Rauscher virus. The possible origin of the substantial increases in plasma lactate dehydrogenase is discussed.

Correlation of C-Type Virus (WF-1) Production and Heme Synthesis in a Rat Fibroblastic Cell Line

Summary Some characteristics of heme biosynthesis were investigated in normal Wistar/Furth rat embryonic fibroblasts and spontaneously transformed cells producing rat C-type virus (WF-1). The activity of the control enzyme of the heme biosynthetic pathway, δ-aminolevulinic acid synthetase, was increased in mitochondria of the WF-1 line to 4 times the level of the normal embryo line, suggesting a relationship between transformation and heme biosynthetic activity. However, a direct correlation to ALA synthetase activity was observed when five rat fibroblastic cell lines were graded according to increasing levels of virus production suggesting that heme biosynthetic activity is directly related to the rate of virus production. We thank Ismay Wars for excellent technical assistance and Dr. Donald P. Tschudy for helpful suggestions and reviewing the manuscript.

Elucidation of the Nature of the Murine Oncogenic Virus Inhibitor Isolated from JLS-V5 Cell Line

Summary An inhibitor (STAS) prepared from the supernatant of JLS-V5 cells blocked the replication of MLV in vitro when added to MEF cultures as late as 48 hr after but not prior to infection with MLV. Potent antiviral activity against MLV was also observed with inhibitor prepared from JLS—V9 tissue culture supernatant fluid, fetal calf serum (FCS), normal Balb/c mouse plasma, MEM + 10% FCS, saline, or silicotungstic acid (STA). Inhibitor prepared from JLS-V5 supernatant fluid, but substituting ZnSO4 PCA, TCA, ethanol, or ammonium sulfate precipitants instead of STA, showed little or no activity against MLV in vitro. No viracidal activity was obtained when MLV was exposed directly to JLS-V5 STAS. These studies suggest that the active ingredient in JLS-V5 STAS may be silicotungstic acid.