Changes in malate dehydrogenase isoenzymes during differentiation of rabbit bone marrow erythroid cells

• 1.l. Malate dehydrogenase activity was assayed in rabbit bone marrow erythroid cells which had been separated by velocity sedimentation into six fractions corresponding to different stages of development.
• 2.2. During differentiation enzyme activity decreased 10-fold and one of the two isoenzymes present in immature cells was lost.
• 3.3. The most extensive change occurred in orthochromatic erythroblasts immediately after condensation of the nucleus.

     

Stimulation of adenylate cyclase activity by catecholamines and prostaglandins E during differentiation of rabbit bone marrow erythroid cells

After fractionation of rabbit bone marrow into erythroid cells at different developmental stages adenylate cyclase activity of membrane ghosts was assayed in the presence of sodium fluoride, catecholamines or prostaglandins E. Both basal and fluoride-stimulated adenylate cyclase decreased continuously during differentiation. Only catecholamines having beta 2-adrenergic activity stimulated adenylate cyclase and their effect was restricted to the most immature cells, the proerythroblasts and, to a lesser extent, the basophilic erythroblasts. Thus, uncoupling of beta-adrenergic receptors occurs early in erythroblast development and hormone responsiveness is lost before the final cell division. Prostaglandin E receptors and adenylate cyclase remain coupled throughout erythroid cell development.     

Cyclic AMP phosphodiesterase activity in three Morris hepatomas.

Rat liver cAMP phosphodiesterase has been fractionated into four peaks of activity with isoelectrofocusing column chromatography. The major two liver peaks (high Km enzymes) decreased with increasing growth rate while the minor two liver peaks (low Km enzymes) increased in one fast growing Morris hepatoma. There was also less total phosphodiesterase activity in the fast growing hepatoma.     

Messenger RNA turnover during bone marrow erythroid cell differentiation

Incubation of bone marrow cells from anaemic rabbits in the presence of actinomycin D led to a decrease in total protein synthesis and an increase in the relative synthesis of globin. This increase in the proportion of globin was observed with in vivo labelling of cellular proteins and in vitro translation of isolated RNA, which indicates that the messenger RNA for globin is much more stable than the other bone marrow cell messages. This was further shown by pulse-labelling the RNA and characterization of the different species by separation on a cDNA-oligo(dT)-cellulose column. Within 12 h after pulse-labelling the relative levels of globin mRNA had risen 10-fold, while a rapid decrease in the level of the poly(A)-rich RNA fraction was observed. Investigations into the mechanisms of this differential stability indicate that the more metabolically active cells from the early stages of erythropoietic development are more susceptible to inhibitors of RNA synthesis such as actinomycin D and alpha-amanitin. A preliminary study using a lysosomal inhibitor, chloroquine, indicates that there appear to be at least two degradative mechanisms, involving a lysosomal and a non-lysosomal pathway, with selective specificity for different messages.     

Pre-mRNA from erythroid enriched bone marrow cells of the rabbit

Different fractions of cellular RNA from erythroid enriched bone marrow cells of the rabbit, extracted by the temperature fractionation method, were investigated by hybridization to globin cDNA. 97.4% of all globin sequences were found in the 4 degrees C franction (cytoplasmic RNA) 0.11% are in the 40 degrees / 50 degrees C fraction and 2.47% in the 65 degrees C and 85 degrees C franctions (pre-mRNA). This shows a substantial purification of the pre-mRNA fractions from cytoplasmic mRNA. 33% of the globin sequences in the 65 degrees C and 85 degrees C fractions are polyadenylated. The poly(A)+-RNA from the 65 degrees C and 85 degrees C fractions separated in a formamide sucrose gradient showed a clear hybridization to globin cDNA in the region between 9S and 28S and around 4S. In a control experiment in which RNA from baby hamster kidney cells (BHK) was mixed with globin mRNA and separated in the same manner hybridization was observed at the 9S position of the gradient only.     

Cyclic AMP phosphodiesterase activity of murine T and B lymphocytes.

Recent evidence suggests that adenosine 3′,5′-monophosphate (cyclic AMP) may play a role in lymphocyte responses. The nature of this role is yet unclear since it has been reported that increases in intracellular cyclic AMP can both inhibit and enhance immune functions. In an attempt to determine how lymphocyte cyclic AMP metabolism is regulated we have measured cyclic nucleotide phosphodiesterase in populations of murine T and B lymphocytes. Results indicate that the enzyme activity in B cells was over fourfold higher than that found in thymocytes. Further analyses by preparative polyacrylamide gel electrophoresis revealed that these cell populations contained different multiple forms of the enzyme.     

Cyclic AMP phosphodiesterase in Thermomonospora curvata.

Cyclic AMP phosphodiesterase (PDE; EC 3.1.4.17) in Thermomonospora curvata was purified and characterized. Fractionation of cell extracts by ion-exchange and size-exclusion chromatography revealed four PDE isozymes, which differed markedly in molecular weight, theophylline sensitivity, pH optima, and substrate affinity. Although the enzyme was labile after purification, total recovery of PDE activity was fivefold that of the crude extract. PDE biosynthesis appeared sensitive to the growth phase, growth rate, and carbon source. PDE levels in batch cultures peaked and declined rapidly during mid-exponential-phase growth. In continuous culture, maximal PDE and cellulase production occurred at dilution rates yielding mean cell generation times of about 5 and 17 h, respectively. The addition of glucose to cellulose-grown cells caused declines in both cyclic AMP and PDE levels, suggesting that the enzyme was subject to, rather than the agent of, catabolite repression.     

Cyclic AMP phosphodiesterase activity in the midgut of Manduca sexta

Midgut preparations from Manduca sexta larvae exhibit potent cyclic AMP phosphodiesterase activity. The enzyme exhibits a pH optimum at pH 8·8 and the assay reaction is linear for at least 30 min. Enzyme activity is greatest in larvae during the second day of the fourth and fifth instars and decreases at the end of the instar. Midgut cyclic AMP phosphodiesterase activity rises again in the pharate pupa and during pharate adult development. Whether these alterations in emzyme activity reflect humorally controlled morphogenetic changes in the midgut or are involved in the physiological functions of the midgut is as yet not known.     

Cyclic AMP phosphodiesterase activity during the development of the silkworm, Bombyx mori

Cyclic AMP phosphodiesterase activity has been demonstrated in homogenates of the silkworm, Bombyx mori, throughout larval, pupal, and pharate adult life. The enzyme exhibits a pH optimum at pH 8·0, and the enzyme activity was inhibited in the presence of caffeine and theophylline. The enzyme activity increases after hatching, and a relatively higher level of the activity is maintained through the larval stage. The activity, however, falls markedly before pupation and increases sharply once again before emergence.     

Cyclic AMP phosphodiesterase activity in fetal and adult muscle of the rhesus monkey.

Total cyclic AMP phosphodiesterase activity of voluntary skeletal muscle of the rhesus monkey was highest in the 100-day fetal series, decreased near term, and was lowest in the adult series. Kinetic data indicated the existence of two cyclic AMP phosphodiesterase enzymes in both the fetal and adult muscle. The apparent K_m values for the high-affinity phosphodiesterase were similar in the 100-day fetal and adult skeletal muscle, whereas those for the low-affinity enzyme were twofold higher in the fetal series. The V_max of the high K_m enzyme was tenfold higher in the fetal than in the adult series and the low K_mV_max was fourfold higher in the fetal series. Both caffeine and theophylline were competitive inhibitors of the low K_m phosphodiesterase activity and noncompetitive inhibitors of the high K_m enzyme. No difference was observed in the sensitivity of the fetal and adult enzyme preparations to the methylxanthines or to Ro20-1724.     

Cyclic AMP phosphodiesterase in human lymphocytes and lymphoblasts.

Cyclic nucleotide phosphodiesterase activities were examined in lymphocytes from 12 transformed human B cell lines, two T cell lines, six patients with lymphocytic leukemia, and 10 normal donors. A consistent difference bwtween cells from the normal and leukemic state was observed. The cyclic AMP phosphodiesterase activity from normal lymphocytes is inhibited greater than 80% by muM cyclic GMP while this concentration of nucleotide has little or no effect on the enzyme from transformed lymphocytic cell lines or from lymphocytic cells of leukemia patients. The reported lack of cyclic GMP phosphodiesterase in human lymphocytes from several sources is confirmed. The apparent absence of a cyclic GMP degradation mechanism and of cyclic GMP control of cyclic AMP hydrolysis may be related to defective lymphocyte growth control.     

Changes in the lactate dehydrogenase isoenzyme pattern during differentiation of rabbit bone-marrow erythroid cells.

1. Differentiation and maturation of rabbit bone-marrow erythroid cells was accompanied by a 15-fold decrease in lactate dehydrogenase activity from approx. 0.1pmol of NADH utilized/min per cell in basophilic cells to 0.007 pmol of NADH/min per cell in reticulocytes. 2. In early cells, cell division takes place with a corresponding decrease in cell volume, but the concentration of lactate dehydrogenase remains almost constant. 3. When cell division ceases, qualitative as well as quantitative changes in the lactate dehydrogenase isoenzyme pattern become apparent and reticulocytes were found to contain almost exclusively the H4 isoenzyme, whereas early erythroblasts contained also the M4 and hybrid isoenzymes. 4. Extracts from a lysosome-enriched subcellular fraction of bone-marrow erythroid cells specifically degraded the M4 isoenzyme in vitro, but the H4 form was stable. It is suggested that lysosomal enzymes are involved in bringing about the observed changes in lactate dehydrogenase isoenzyme patterns in vivo.     

Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase activities of rat mammary tissue.

Cyclic nucleotide phosphodiesterase activity in mammary tissue from rats in midlactation was resolved by DEAE-cellulose chromatography into three functionally distinct fractions: a Ca2+/calmodulin-stimulated cyclic GMP phosphodiesterase, a cyclic GMP-stimulated low-affinity cyclic nucleotide phosphodiesterase, and a high-affinity cyclic AMP-specific phosphodiesterase. The absolute activities and relative proportions of high- and low-affinity enzymes resemble those found, for example, in liver, as distinct from those in excitable tissues. Three functional characteristics are described which are peculiar to mammary-tissue phosphodiesterases. Firstly, the concentration of free Ca2+ required to achieve half-maximal activation of the Ca2+/calmodulin-stimulated phosphodiesterase is somewhat higher than for the analogous enzyme in other tissues; secondly, the activity of this enzyme towards cyclic AMP relative to that towards cyclic GMP is unusually low, and thirdly, the low-affinity cyclic nucleotide phosphodiesterase is inhibited by low concentrations of free Ca2+.     

Changes in ornithine decarboxylase activity during matrix-induced cartilage, bone and bone marrow differentiation.

Subcutaneous transplantation of coarse powders of demineralized rat diaphyseal bone matrix into allogeneic recipients results in new bone formation. The changes in ornithine decarboxylase activity during such bone matrix-induced sequential differentiation of cartilage, bone and bone marrow were investigated. There was a peak in ornithine decarboxylase activity on day 3 corresponding to the appearance of fibroblasts in close contiguity to the bone matrix. This was followed by another peak of enzyme activity on day 8 which was correlated with the onset of proliferation of presumptive osteoblasts and vascular endothelial cells. The peak of ornithine decarboxylase activity on day 3 appears to be a demineralized bone matrix-specific event. Induction of ornithine decarboxylase activity represents one of the early responses to implanted bone matrix.     

Cyclic AMP phosphodiesterase inhibitors depress production of plasminogen activator by Chinese hamster ovary cells.

Oncogenic transformation in a limited number of cell systems has been shown by others to be associated with an increased production of extracellular proteolytic activators that convert the plasma proenzyme, plasminogen, to the active protease, plasmin. In the present study, two cyclic AMP phosphodiesterase inhibitors (theophylline, papaverine) markedly depressed the production of intracellular and extracellular plasminogen activator by Chinese hamster ovary cells of the CHO-Kl line in serum-free medium. Prostaglandin E₁ had a moderately similar effect on the production of only extracellular plasminogen activator. The ability to control experimentally the level of production of plasminogen activator should be of value in elucidating the possible biological role of the proteolytic action of plasmin on the surface of CHO cells, and the cell surface alterations which accompany oncogenic transformation.     

Cyclic AMP phosphodiesterase activities in Xenopus laevis oocytes

Cyclic AMP phosphodiesterase activity has been identified in full-grown Xenopus oocytes in vivo and in vitro. About 50% of the in vitro phosphodiesterase activity was present in the solution fraction and 35% in a partially purified membrane fraction. Both activities exhibited high substrate affinity (Km about 10(-6) M). Sucrose gradient fractionation revealed two forms of phosphodiesterase: a 5 S form (peak I) and a 6.5 S form (peak II). Treatment with trypsin led to the activation of the soluble enzyme with the transformation of peak II into peak I. Ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid, calcium dependent regulator, and Fluphenazine did not influence the enzyme activities suggesting that the oocyte phosphodiesterases were not Ca2+-dependent. Intact oocytes were induced to mature by exposure to progesterone; their phosphodiesterase activities and distribution tested in vitro were comparable to those of untreated oocytes.