Transport and control of Ca by pigeon erythrocytes. II. Evidence against a simple feedback control of cell Ca and evidence for the involvement of more than one pool

Cells were subjected to a range of ⁴⁵Ca²⁺ influx loads with A23187. We measured cell ⁴⁵Ca²⁺ with time and A23187 dose, and the apparent ⁴⁵Ca²⁺ influxes (≡“J(in,app)”) at Ca²⁺ steady state. We also measured endogeneous exchangeable and total cell Ca²⁺, which were 50 and 17–220 μM respectively. The effects of A23187 and Ca²⁺ on cell ATP, swelling, net Cl⁻ permeability, and cell morphology were measured. These were modest and do not affect our conclusions.J(in,app) 3 × 10⁻⁴ [A23187]^2.9·[Ca²⁺(o)]μmoles/l·min with 92–552 μM [Ca²⁺(o)] (≡ external Ca²⁺ concentration) and 0–7 μM A23187. J(in,app) was increased an order of magnitude by vanadate and is probably much less than the true influx. The least unlikely explanation found for the high [A23187] exponent, 2.9, was that most of the Ca²⁺ crossing the membrane is expelled by the pump before it can move deeper into the cell.Calcium pumping increased rapidly in response to increased influx, but the steady state cell ⁴⁵Ca²⁺ was approximately proportional to J(in,app) rather than approximately constant between 10 and 120 μmoles/l·min with 184 μM [Ca²⁺(o)]. This is not the result expected from a simple feedback mechanism.At high A23187 doses the pump appears fully activated resulting in a linear relation between cell/medium ⁴⁵Ca²⁺ and [A23187]⁻². From the plot we calculated α≡free/total exchangeable Ca²⁺ = 0.38 ± 0.08 (n = 3) and a maximum pump rate, “Pmax” = 78 μmole/l·min. Pmax is underestimated insofar as J(in,app) is less than the true influx.     

Selective affinity chromatography of DNA polymerases with associated 3' to 5' exonuclease activities

The use of 5'-AMP as a ligand for the affinity chromatography of DNA polymerases with intrinsic 3' to 5' exonuclease activities was investigated. The basis for this is that 5'-AMP would be expected to act as a ligand for the associated 3' to 5' exonuclease. The requirements for binding of Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus DNA polymerase delta, all of which have associated 3' to 5' exonuclease activities, to several commercially available 5'-AMP supports with different linkages of 5'-AMP to either agarose or cellulose were examined. The DNA polymerases which possessed 3' to 5' exonuclease activities were bound to agarose types in which the 5'-phosphoryl group and the 3'-hydroxyl group of the AMP were unsubstituted. Bound enzyme could be eluted by either an increase in ionic strength or competitive binding of nucleoside 5'-monophosphates. Magnesium was found to reinforce the binding of the enzyme to these affinity supports. DNA polymerase alpha, which does not have an associated 3' to 5' exonuclease activity, did not bind to any of these columns. These differences can be used to advantage for the purification of DNA polymerases that have associated 3' to 5' exonuclease activities, as well as a means for establishing the association of 3' to 5' exonuclease activities with DNA polymerases.     

Gamma-ray irradiation as a pretreatment for the enzymatic hydrolysis of cellulose

Summary The susceptibility of cellulose to enzymatic hydrolysis can be significantly affected through pretreatment by means of gamma-ray radiation. Experiments were carried out to investigate the effects of this radiation on enzymatic hydrolysis and on the two major structural features of cellulose that most influence hydrolysis, namely, specific surface area and crystallinity.D. H. Beardmore is currently with Phillips Petroleum Company, Bartlesville, OK 74004, U.S.A.     

Pituitary corticotropin-inhibiting peptide: Properties and use in study of corticotropin action

The biological properties of the naturally occurring pituitary peptide α_h^7–38-adrenocorticotropin (ACTH) have been investigated. α_h^7–38-ACTH is devoid of steroidogenic activity but inhibits competitively ACTH-induced steroidogenesis in vitro as well as in vivo. The long-term actions of ACTH on normal and tumor adrenal cells in culture are also antagonized by α_h^7–38-ACTH. The apparent K_i for the inhibition of cyclic AMP production by α_h^7–38-ACTH (301 ± 62 nm) was significantly higher than the apparent K_i for the inhibition of corticosterone synthesis (21.6 ± 6.8 nm). Analysis of the inhibition of ACTH-induced steroidogenesis and cyclic AMP production in normal rat adrenocortical cells indicates that two separate receptors may be involved in mediating these responses.     

Immunological and biochemical analysis of a null mutant of α-glycerolphosphate dehydrogenase from Drosophila melanogaster

Summary A Drosophila null mutant(BO-1-4) of -glycerolphosphate dehydrogenase induced by ethylmethane sulfonate(EMS) was analyzed by double immunodiffusion, enzyme immuno-inactivation, immunoelectrophoresis and two-dimensional electrophoresis. Based on all the immunological evidence, this mutant appears to express no protein that can cross-react with the antiserum specific to -glycerolphosphate dehydrogenase. A protein spot corresponding to -glycerolphosphate dehydrogenase was identified on two-dimensional gels of the soluble fly homogenates. The absence of this protein spot on two-dimensional gels of this null mutant further supported the immunological data. The activities of seven other enzymes in the related metabolic pathways were determined for the mutant and the control Drosophila. The null mutant does not show significant alterations in activities of these enzymes. The relationship between the deficiency of this enzyme and the inability for the sustained flight of the null mutant was discussed in terms of cellular metabolic regulations.Abbreviations used -GPD -glycerolphosphate dehydrogenase (EC 1.1.1.8) - EMS ethylmethane sulfonate - TEMED N,N,N,N-tetramethylene diamine - pI isolectric point - CRM immunological cross-reacting material     

Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells.

Treatment of Ltk^?, mouse L cells deficient in thymidine kinase (tk), with Bam I restriction endonuclease cleaved DNA from herpes simplex virus-1 (HSV-1) produced tk⁺ clones with a frequency of 10^?6/2 μg of HSV-1 DNA. Untreated cells or cells treated with Eco RI restriction endonuclease fragments produced no tk+ clones under the same conditions. The thymidine kinase activities of four independently derived clones were characterized by biochemical and serological techniques. By these criteria, the tk activities were found to be identical to HSV-1 tk and different from host wildtype tk. The tk⁺ phenotype was stable over several hundred cell generations, although the rate of reversion to the tk^? phenotype, as judged by cloning efficiency in the presence of bromodeoxyuridine, was high (1–5 × 10^?3). HSV-1 DNA Bam restriction fragments were separated by gel electrophoresis, and virtually all activity, as assayed by transfection, was found to reside in a 3.4 kb fragment. Transformation efficiency with the isolated fragment is 20 fold higher per gene equivalent than with the unfractionated total Bam digest. These results prove the usefulness of transfection assays as a means for the bioassay and isolation of restriction fragments carrying specific genetic information. Cells expressing HSV-1 tk may also provide a useful model system for the detailed analysis of eucaryotic and viral gene regulation.