Arginase,an s-phase enzyme in a human cell line

Suspension cultures of ‘Chang liver’ cells were synchronized by preincubation in a glutamine-deficient medium or by thymidine blockade. Specific arginase activity varied in the synchronized cultures, being high when the number of S-phase cells was maximal. A relationship between high arginase activity and a high percentage of (S+G₂) cells was also found when unsynchronized cells were separated by velocity sedimentation. The increase in arginase activity near the G₁/S border was totally inhibited in the presence of cycloheximide. The rate of decrease in activity after addition of the drug indicated that the variations in the rate of synthesis of the enzyme, while the rate of degradation was more or less constant, corresponding to 4–6% per h. The role of arginase in cells lacking a urea cycle and the regulation of arginase activity in ‘Chang liver’ cells is discussed.     

DNA POLYMERASES IN FRACTIONATED RAT BRAIN NUCLEI

Abstract— —Adult rat brain nuclei were separated by discontinuous sucrose gradient centrifugation into astrocyte enriched, neuron enriched, and oligodendrocyte/microglia fractions. Nuclear fractions were subjected to velocity sucrose gradient centrifugation and gradient fractions assayed using relatively specific reaction mixtures for DNA polymerase-α, -β and TdT. NEM resistant DNA polymerase activity (DNA polymerase-β) was detected in equivalent amounts in all nuclear fractions. High molecular weight NEM sensitive activity (DNA polymerase-α) was found primarily in the neuron enriched fraction. The significance of the presence of DNA polymerase-α, an enzyme thought to be involved in DNA replication, in a cell incapable of cell division is unknown. TdT was detected in all fractions with increased activity in the neuron enriched fraction. The finding of TdT in thymocytes and neurons further supports the hypothesis that this enzyme is involved in the storage of noninherited information.     

Drug design based on biosynthetic studies: synthesis, biological activity, and kinetics of new inhibitors of 2,3-oxidosqualene cyclase and squalene epoxidase

Various classes of inhibitor of 2,3-oxido squalene cyclase have been synthesized and tested on rat liver and Saccharomyces cerevisiae microsomes, 3T3 fibroblast cultures, and various bacteria, fungi, and yeasts. The compounds include azasqualenes, azasqualanes, bis-azasqualenes, bis-azasqualanes, and N-oxide and ammonium derivatives of squalene. In order to better mimic the transition state involved in the SN2-like opening of 2,3-oxidosqualene, we synthesized squalene N-methyloxaziridine. Other derivatives tested were N-methylimine, aminalic hydroperoxide, and N-methylamide. We also attempted to produce new "suicide" inhibitors of SO cyclase, such as a squalenoid epoxide vinyl ether. Many of the products described inhibited the various cyclases, the best having an IC50 of 0.3 microM on plants and 1.5 microM on rat liver microsomes, and good antibacterial and antifungal activity. In a search for inhibitors of squalene epoxidase, a series of mono- and bifunctional squalenoid acetylenes and allenes were synthesized. Some of them proved to be inhibitors of squalene epoxidase.     

Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene,and expression of inactive mutant enzyme protein in E. coli

Summary A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency. Demonstration of intra-mitochondrial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing. Consequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed. All clones sequenced from the patient exhibited a single base substitution from adenine (A) to guanine (G) at position 985 in the MCAD cDNA as the only consistent base-variation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mutated sequence, revealing their obligate carrier status. Allelic homozygosity in the patient and heterozygosity for the mutation in the parents were established by a modified PCR reaction, introducing a cleavage site for the restriction endonuclease NcoI into amplified genomic DNA containing G⁹⁸⁵. The same assay consistently revealed A⁹⁸⁵ in genomic DNA from 26 control individuals. The A to G mutation was introduced into an E. coli expression vector producing mutant MCAD, which was demonstrated to be inactive, probably because of the inability to form active tetrameric MCAD. All the experiments are consistent with the contention that the G⁹⁸⁵ mutation, resulting in a lysine to glutamate shift at position 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family. We found the same mutation in homozygous form in 11 out of 12 other patients with verified MCAD deficiency.     

Immunocytochemical demonstration of tissue-type plasminogen activator in endocrine cells of the rat pituitary gland

We immunocytochemically stained rat pituitary glands using antibodies against plasminogen activators of the tissue type (t-PA) and the urokinase type (u-PA). A large population of endocrine cells in the anterior lobe of the gland displayed intense cytoplasmic immunoreactivity with anti-t-PA. In some areas of the intermediate lobe we found a weak staining, and we observed weakly staining granular structures in the posterior lobe. Controls included absorption of the antibodies with highly purified t-PA. In addition, SDS PAGE followed by immunoblotting of pituitary gland extracts revealed only one band with an electrophoretic mobility similar to that of t-PA when stained with anti-t-PA IgG. No u-PA immunoreactivity was detected in the rat pituitary gland. Sequential staining experiments using antibodies against growth hormone and t-PA demonstrated that the t-PA-immunoreactive cells constitute a large subpopulation of the growth hormone-containing cells. These findings represent the first direct evidence for the presence of t-PA in cell types other than endothelial cells in the intact normal organism. In this article we discuss the implications of the results for a possible role of t-PA in the posttranslational processing of prohormones.     

immunocytochemical localization of urokinase-type plasminogen activator in lewis lung carcinoma

The invasively growing and metasizing Lewis lung carcinoma consistently contained urokinase-type plasminogen activator (u-PA) enzyme activity. When investigated immunocytochemically with antibodies against u-PA, different parts of individual tumors showed a pronounced heterogeneity in staining intensity. Strong staining was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were completely devoid of staining. Immunoreactivity occurred both with a perinuclear cytoplasmic localization in tumor cells and associated with apparently extracellular material. SDS PAGE of tumor extracts, under both reducing and nonreducing conditions, followed by immunoblotting, showed only one immunocytochemically stainable band with an electrophoretic mobility corresponding to that of purified proenzyme to u-PA, while no two-chain u-PA was detected. This indicates that the major part of the activator in Lewis lung carcinoma is present as one-chain pro-u-PA.     

Establishment and Growth Kinetics of the Mutant Ehrlich-Lettré Ascites Cell Strain Hd33 In Permanent Suspension Culture1,2

Cells of a mutant in vivo subline of the Ehrlich-Lettré mouse ascites tumour (ELAT) were converted to growth in suspension culture. Kinetic analysis revealed the selective character of the conversion process; without a detectable adaptation period, a fraction of about 2 × 10^-5 of the explanted cells continued to grow in vitro. the resulting, mutant Ehrlich-Lettré ascites cell strain was designated HD33 and propagated uninterruptedly from 1974 on. the corresponding in vivo ELAT subline HD33 was derived from the HD33 ascites cell strain by intraperitoneal retransplantation. In HD33 cell suspension cultures, the population doubling time, the average intermitotic interval, as determined by videomonitoring, and the average duration of the cell cycle, as determined from percentage of labelled mitoses (PLM) data, were all measured at 15 hr. Cell loss and quiescent compartments were insignificant. the duration of the G₁ phase was effectively zero. Both PLM data and [³H]/[¹⁴C] thymidine double-labetling measurements revealed an S-phase duration of between 11 and 12 hr. the G₂ phase lasted 3–5 hr. The HD33 strain differs from comparable suspension strains of wild-type Ehrlich ascites cells in the insignificant role of density-dependent inhibition in growth, and the striking prolongation of the S phase which is associated with an excessive, cytoplasmic storage of glycogen by the mutant cells.     

Reversal of enzyme regiospecificity with alternative substrates for aspartokinase I from Escherichia coli.

The substrate specificity of aspartokinase I has been examined by using both steady-state kinetic analyses and phosphorus-31 NMR spectroscopic studies. Analogues in which the alpha-amino group is either derivatized or replaced are not substrates or inhibitors for the enzyme, indicating the importance of the alpha-amino group as a binding determinant. The alpha-carboxyl group is not required for substrate recognition, and the alpha-amide or alpha-esters are competent alternative substrates. In addition, beta-derivatized structural analogues, such as the beta-hydroxamate, the beta-amide, or beta-esters, were found to be viable substrates. This was unexpected since the beta-carboxyl group is the usual site of phosphorylation. The nature of the acyl phosphate products obtained from these beta-derivatized alternative substrates has been characterized by coupled enzyme assays, oxygen-18-labeling studies, and phosphorus-31 NMR spectroscopy. These beta-derivatized analogues are capable of productive binding to aspartokinase through a reversal of regiospecificity to make the alpha-carboxyl group available as a phosphoryl acceptor. Many, but not all, of these alpha-acyl phosphates have also been shown to be viable substrates for the next two enzyme-catalyzed steps in this metabolic pathway. This raises the possibility of producing enzyme-generated alternative substrates that can serve as antimetabolites for the downstream reactions in this biosynthetic pathway.