Changes in the post-translational modification of lysosomal enzymes during development of Dictyostelium

The form of post-translational modification present on two lysosomal enzymes--acid phosphatase and alpha-mannosidase--changes as part of the developmental program of Dictyostelium discoideum. Prior to 8 h of development, all enzyme molecules are of a single modification type (early form enzyme). Starting at 8 h of development, enzyme molecules with a second type of modification (late-form enzymes) begin to appear in the cell. We separated the early and late forms of these enzymes from each other by chromatography on DEAE-cellulose. We found that the change in protein modification affects the enzymes' in vitro properties. The early and late forms of both of these enzymes differ in thermostability and susceptibility to proteolytic inactivation. We also found that the late form of alpha-mannosidase is preferentially secreted. We suggest that by synthesizing molecules with a second form of modification, the cell confers new characteristics to its lysosomal enzymes.     

Changes in transferrin during the red cell replacement in amphibia

Transferrin, a plasma glycoprotein, carries iron from storage sites to immature erythroid cells for hemoglobin synthesis. The replacement of larval red cells by adult red cells, which occurs during metamorphosis in bullfrogs, requires extensive formation of hemoglobin and new red cells. Large changes in red cell iron storage also occur during the red cell replacement. Both the concentration and the level of iron saturation of plasma transferrin were measured during metamorphosis to determine if there were changes in plasma transferrin which coincided with the changes in red cell iron storage and ferritin content. Plasma transferrin concentrations increased from 0.96 to 2.6 mg/ml during the period when red cell storage iron and ferritin decreased. Plasma iron concentrations also increased when the transferrin concentration increased, suggesting that the additional transferrin may be involved in moving iron from the larval red cell stores. At the end of metamorphosis, the plasma iron concentration decreased to premetamorphic levels but the transferrin concentration remained high, resulting in a decrease in saturation to 18% compared to 45% in the larvae. In addition to differences in iron saturation, adult transferrin had different electrophoretic properties from larval transferrin. The results support the hypotheses that during early ontogeny plasma transferrin and red cell iron storage are coordinated to provide iron for the formation of the first generation of adult red cells and that transferrin may participate in the control of red cell ferritin synthesis.     

Ordered substrate binding and evidence for a thermally induced change in mechanism for E. coli aspartate transcarbamylase

Isotopic exchange kinetics at equilibrium for E. coli native aspartate transcarbamylase at pH 7.8, 30 °C, are consistent with an ordered BiBi substrate binding mechanism. Carbamyl phosphate binds before l-Asp, and carbamyl-aspartate is released before inorganic phosphate. The rate of [¹⁴C]Asp C-Asp exchange is much faster than [³²P]carbamyl phosphate P_i exchange. Phosphate, and perhaps carbamyl phosphate, appears to bind at a separate modifier site and prevent dissociation of active-site bound P_i or carbamyl phosphate. Initial velocity studies in the range of 0–40 °C reveal a biphasic Arrhenius plot for native enzyme: E_a (>15 °C) = 6.3 kcal/ mole and E_a (<15 °C) = 22.1 kcal/mole. Catalytic subunits show a monophasic plot with E_a ? 20.2 kcal/mole. This, with other data, suggests that with native enzyme a conformational change accompanying aspartate association contributes significantly to rate limitation at t > 15 °C, but that catalytic steps become definitively slower below 15 °C. Model kinetics are derived to show that this change in mechanism at low temperature can force an ordered substrate binding system to produce exchange-rate patterns consistent with a random binding system with all exchange rates equal. The nonlinear Arrhenius plot also has important consequences for current theories of catalytic and regulatory mechanisms for this enzyme.     

Changes in the activities of the protoheme-synthesizing system during the growth of yeast under different conditions.

The levels of some enzymatic activities involved in protoheme synthesis have been measured in subcellular fractions obtained at different stages of the growth of the yeast Saccharomyces cerevisiae grown anaerobically and aerobically with glucose (50 or 6 g/ liter), and ethanol (20 g/liter) as the carbon source. The degree of repression of the respiratory system is estimated by the respiratory capacity of whole cells, by the activities of succinate-cytochrome c reductase and cytochrome c oxidase of the mitochondrial particles, and by the cytochrome spectra. The results show that (i) the more porphyrins (cytochromes) that are synthesized by the cells, the lower is the specific activity of δ-aminolevulinic acid (ALA) synthetase and the higher is the specific activity of ALA dehydratase, the activity ratio ALA synthetase/ALA dehydratase decreasing at least 10-fold compared to the repressed cells; (ii) the amount of intracellular ALA found under all conditions tested (from 0.05 to 1.5 mm in the cell sap) correlates well with the measured ALA synthetase activity; its presence argues against a rate-limiting function for ALA synthetase and rather favors such a role for the ALA dehydratase in the formation of heme in yeast; (iii) the rate of porphyrin synthesis measured in vitro is higher in the case of cells with high cytochrome contents; and (iv) the specific activities of succinyl CoA synthetase and protoheme ferrolyase are always present in nonlimiting amounts. Some experiments are described showing that the values of the activities which are calculated from these in situ and in vivo experiments compare well with the values measured in vitro in the acellular extracts. The results concerning the enzymatic activities, together with (i) the excretion of coproporphyrin(ogen) and the accumulation of protoporphyrin + Zn-protoporphyrin in anaerobiosis, (ii) the presence of protoporpho(di)methene (P503) in anaerobic and repressed cells, and (iii) the presence of intracellular ALA under all growth conditions, are discussed in terms of possible control(s) of heme synthesis in yeast.     

Changes in DNA-polymerase and thymidine kinase activity during interferon treatment

Treatment of the human glioma cell line, U-251 MG, with human IFN-β resulted in a dose-dependent growth depression and a decreased activity of DNA-polymerase in exponentially growing cells, although paradoxally the number of cells in the S phase increased. In synchronized cells, a S block was confirmed. Both thymidine kinase and DNA-polymerase increased but with a lower rate during IFN treatment. No inhibitory effects on any of the enzymes could be seen when IFN-treated lysate was mixed with control lysate. The possible significance of depressed DNA synthesis during virus infection is discussed.     

Changes in adenylate cyclase and phosphodiesterase activities during the growth cycle of adult rat hepatocytes in primary culture

Long-term primary adult rat hepatocyte cultures show growth-state-dependent changes in adenylate cyclase and cAMP phosphodiesterase activities. Cellular adenylate cyclase activity decreases to undetectable levels within 1 day postplating, reappears on Days 4-5, and becomes maximal on Day 9. Membrane adenylate cyclase and cellular cAMP formation are insensitive to glucagon during log phase (Days 4-8) but not during lag (Day 1) or stationary phase (Day 12). Cyclic AMP phosphodiesterase activities (soluble and particulate) fall approximately equal to 70% by Day 2 but recover as proliferation begins. By contrast, the particulate phosphodiesterase assayed at 100 microM cAMP, decreased during Days 0-2. These observations simulate changes seen during liver proliferative transitions in vivo and, therefore, further support the use of these cultures as a developmental model.     

A microassay for the peptidase activity of enzymes utilizing ribonuclease S peptide as a substrate

A microassay for the peptidase activity of proteins obtained in minute amounts was devised. The method uses ribonuclease S peptide as a substrate. The substrate when cleaved is unable to reconstitute an active ribonuclease S complex. Therefore the loss in activity of the reconstituted complex is a measure of the peptidase activity. The method was previously tested with known peptidases such as clastase (9), chymotrypsin (8), and trypsin. In this work the peptidase activity of a protein related to a sperm-decapitating factor (1) is evidenced.     

Raman study of crystalline peptides containing beta turns

The Raman spectra of crystalline H-ProLeuGlyNH₂ which has a type II β turn, crystalline S-benzylCysProLeuGlyNH₂ which has a type I β-turn, and crystalline gramicidin S which has two β turns and β-sheet structure in its conformation, were investigated. The amide I and amide III bands of the peptides with β turns were generally different from those which are diagnostic for α-helix and β-sheet conformations. The patterns of the amide I and amide III bands, when examined together, indicate that Raman spectra can provide diagnostic evidence for β-turn structure in peptides.     

Changes in nucleosomal core histone variants during chicken development and maturation

The nucleosomal core histones H2A, H2B, and H3 of the chicken can be resolved by polyacrylamide gel electrophoresis in the presence of nonionic detergents into two primary structure variants each, which occur in different relative amounts in various adult tissues. Quantitative analysis of the histone components throughout embryonic development and posthatching maturation of the chicken revealed that the proportions of the three pairs of variants change independently. Thus, the two H2A variants occur in similar proportions throughout embryonic development and in all adult tissues. In contrast, only one variant each of H2B and H3 is detectable at the earliest stages (primitive streak). The second variant of these histones becomes detectable and increases gradually during somite formation (2-12 days of incubation) to reach a plateau at a level of about 3 and 10% of total H2B and H3 histones, respectively. After hatching, the relative amounts of the minor H2B and H3 variants remain at embryonic levels in those tissues which maintain a high mitotic activity such as blood-forming tissues, but increase with different kinetics in tissues which essentially stop cell division in adults (e.g., liver, kidney, etc.). However, while H2B.2 remains a very minor component in all tissues, H3.3 increases at a relatively high rate for more than a year to become the predominant H3 variant in the liver and kidney of older chickens. The changes in chicken core histone variant proportions appear to be related to changes in growth rate rather than cell differentiation. The extensive change of H3 variant proportions in nondividing adult tissues is most likely due to replication-independent incorporation of H3.3 into nucleosomes.     

Micellar solubilization of enzymes in hydrocarbon solvents. Enzymatic activity and spectroscopic properties of ribonuclease in N-octane

Ribonuclease from bovine pancreas has been solubilized in n-octane containing the surfactant di(2-ethyl-hexyl) sodium sulfo-succinate (50 mM) and water (0.55–0.94 M). It is shown that enzymatic activity with cytidine-2′:3′-phosphate and RNA is maintained in the hydrocarbon phase, and that under certain conditions it is even higher than in water solution. Absorption properties and circular dichroism of the enzyme and substrates in this new environment are investigated and compared with those in water solution.     

Changes in the electron transport chain of pea leaf mitochondria metabolizing malate

Pea leaf mitochondria showed complex kinetics for malate metabolism. O₂ uptake increased as malate concentration increased from 0 to 10 mm, reached a plateau between 10 and 20 mm malate, and then increased again up to 40 mm malate. Analysis of the products of malate oxidation by high-performance liquid chromatography revealed that the first phase of O₂ uptake coincided with the synthesis of both pyruvate and oxalacetate (OAA) while the second phase of O₂ uptake at higher malate levels usually occurred with a large increase in OAA formation. The biphasic response in O₂ uptake and the changing ratios of pyruvate and OAA synthesis did not appear to be the direct result of the differing K_m values of malate dehydrogenase and malic enzyme. Rather, they resulted from thermodynamic properties of these two malate oxidases and the kinetics of the two NADH dehydrogenases found in plant mitochondria. At low malate concentrations the rotenone-sensitive NADH dehydrogenase was active and could accept electrons from both malate oxidases. This NADH dehydrogenase became saturated at about 10 mm malate. At higher malate concentrations the rotenone-insensitive NADH dehydrogenase was increasingly important and its increased electron transport capacity was best exploited by malate dehydrogenase. At the higher malate concentrations an increasing portion of the electrons from malate reduce O₂ through the alternative oxidase. Although this coincided with the second phase of malate-dependent O₂ uptake it was not required for this phase to be seen.     

Alteration in lymphocyte recognition repertoire during aging: II. Changes in the expressed T-cell receptor repertoire in aged mice and the persistence of that change after transplantation to a new differentiative environment

Changes in the T-lymphocyte alloreceptor repertoire associated with aging by exploring the frequency of cytotoxic T-lymphocyte precursors (CTLp) available for activation by various major histocompatibility complex (MHC) haplotypes in mice of different ages have been investigated. There was no consistent pattern of change in CTLp frequencies. Thus, for instance, while the frequency of responder C57Bl/6 CTLp for ATH alloantigen decreased with age, the frequency for C3H alloantigen increased. There was no significant change in the overall frequency of splenic CTLp (assessed irrespective of antigen specificity). No evidence was found that CTL produced by activated CTLp of aged mice were less specific in their lytic capacity that CTL produced by CTLp of young mice. However, by assaying responder CTLp cultures at limiting dilution we obtained evidence that the “burst size” (mean lytic capacity per responder well assayed at limiting dilution) was diminished with age of the donor of the CTLp pool. Furthermore, we obtained evidence that the apparent affinity of CTL for their target antigen was consistently decreased when those effector cells were derived from a pool of CTLp of aged mice. All of these changes reflected in mature T cells derived from aged mice were already apparent in the bone marrow stem cell pool of aged individuals and were not due to environmental influences alone, as assessed by the phenotype of T cells derived from young or old bone marrow stem cells transplanted to young or aged recipient mice. A final study has examined evidence for more subtle changes in the T-cell alloreceptor repertoire, reflecting heterogeneity in young or aged mice in the recognition repertoire associated with a given antigenic specificity. By preparing F₁ anti (parent anti-F₁)-suppressor cells directed against CTL from young parental mice (a, b, c), or aged parental mice (x, y, z), we have explored the heterogeneity in the anti-C3H alloreceptor repertoire in individual young or aged C57Bl/6 mice. Suppression by immunized F₁ animals was assessed in tissue culture (inhibition of mixed lymphocyte culture (MLC) responses) or in vivo (inhibition of lethal GvHD induced by inoculation of parental lymphocytes into sublethally irradiated F₁ hybrid mice). Irrespective of the assay system used, the data suggests that the receptor repertoire of aged T lymphocytes uses recognition structures different from those of young individuals, and that there is less individual-to-individual variation in the receptor repertoire of aged mice than in young mice.