Glycerol Buffer Research Articles

Isolation and characterization ofEuglena nuclei

A novel method for isolatingEuglena gracilis Z. nuclei, based on pretreatment of cells in concentrated glycerol buffer before homogenization, is described. Such a treatment weakens the tough cell pellicle facilitating cell disruption, and avoids nuclear damage induced by detergents and by freezing and thawing the cells in aqueous media. Nuclei, purified by centrifugation in dense sucrose, are obtained with a 30% yield, and only small amounts of cell wall fragments contaminate the nuclear pellets. The purified nuclei retain their ultrastructural characteristics. High molecular weight DNA, as well as undegraded RNA species and histones, can be extracted from these nuclei. Nuclease digestions and spread preparations show an unaltered nucleosomal structure of chromatin. This method has been applied to cell samples at any stage of the cell cycle, including mitosis, since inEuglena the nuclear envelope persists during cell division.

Calcium efflux from Escherichia coli. Evidence for two systems.

Calcium transport into everted membrane vesicles of Escherichia coli was found to have two components, one phosphate-dependent and the other phosphate-independent. In vesicles prepared in a glycerol buffer, calcium/proton exchange was phosphate independent but net uptake of 45Ca2+ required phosphate. In vesicles prepared in a sucrose buffer both phosphate-independent and phosphate-dependent accumulation of 45Ca2+ occurred. Both calcium/proton exchange and phosphate-independent uptake of 45Ca2+ were inactivated by treatment with trypsin, chymotrypsin, or N,N'-dicyclohexylcarbodiimide but not by N-ethylmaleimide. Phosphate-dependent uptake of 45Ca2+ was inhibited by N-ethylmaleimide but not by trypsin, chymotrypsin, or N,N'-dicyclohexylcarbodiimide. Under conditions permitting only phosphate-dependent uptake of 45Ca2+, concomitant uptake of 32Pi was also observed. Both uptake and efflux of the two ions occurred with a 1:1 ratio. These results imply the existence of two calcium transport systems in everted membrane vesicles, one of which catalyzes exchange of calcium ions and protons, the other of which catalyzes cotransport of calcium and phosphate.

A specific cytosolic estrogen receptor in human term placenta

Administration of the antiestrogen ethamoxytriphetol (MER-25) during baboon gestation results in a marked decline in placental progesterone production. Since this effect in primates may be modulated via an estrogen receptor, the present study investigated the possible existence of an estrogen receptor in human placenta. Villous tissue of human, term placentas was homogenized in 0.01M Tris-HCl, ethylenediaminetetraacetic acid, dithiothreitol, glycerol buffer. Cytosol was incubated with 10−8M [3H] 17β-estradiol (E2) in the presence or absence of 10−6M diethylstilbestrol (DES). A single peak of [3H]E2 binding occurred in the 5.2 region after glycerol density gradient centrifugation, which was competed for by DES, E2, and enclomiphene. Scatchard analysis demonstrated E2 binding, which was saturable, of high affinity (Kd = 1.90 × 10−11M) and of low capacity (N = 0.13 × 10−14 moles/mg cytosolic protein). Competition for [3H]E2 binding was DES > E2 > estrone > MER-25 > enclomiphene, whereas androgens, progestins, and corticosteroids were ineffective. The results fulfill the criteria for a specific estrogen receptor. The influence of antiestrogen and, possibly, estrogen upon placental function in baboons may be modulated by an estrogen receptor.

Functional evaluation of human polymorphonuclear leukocytes (PMN) cryopreserved in a dextran glycerol buffer (DGB)

Functional evaluation of human polymorphonuclear leukocytes (PMN) cryopreserved in a dextran glycerol buffer (DGB)

Ultramicro determination of serum triglycerides by bioluminescent assay.

The ultramicro (1-microL samples) assay of serum or plasma triglycerides that we describe here is potentially applicable to 1-nL samples and to isolated cells. This technically simple method involves only three reactions: (a) enzymic hydrolysis with lipase and alpha-chymotrypsin; (b) conversion by glycerol kinase of the liberated glycerol and of adenosine triphosphate added in excess to glycerol-1-phosphate and to adenosine diphosphate; and (c) assay of the residual adenosine triphosphate by the luciferin-luciferase reaction. The assay was optimized with respect to glycerol kinase, buffer, pH, temperature, and adenosine triphosphate. Performance characteristics compare well with those of traditional triglyceride assays.

ATP and Ca2+ induced actomyosin-like volume decrease of glycerinated macronuclei (author's transl)

The addition of 2 mM-3 mM ATP to macronuclei ofParamecium bursaria suspended in a glycerol buffer medium causes a decrease in their volume up to 23% within 3 minutes. The infiltration medium must not only contain Ca2+, but must also be of low ionic strength for ATP to be effective. A slow, careful exchange of the glycerol medium for the contraction solution is also necessary. Ca2+ present alone in the standard contraction buffer can likewise induce a limited volume decrease; in the presence of low concentrations of Ca2+, Mg2+ shows no detectable effect on glycerinated nuclei. When the nuclear volume has been reduced by ATP in the presence of Ca2+, the addition of EGTA induces a reexpansion of the nuclei. Salyrgan, an organic mercurial, either prevents or abolishes the ATP-induced contraction. Other nucleotide triphosphates such as guanosine triphosphate (GTP), inosine triphosphate (ITP) or uridine triphosphate (UTP) likewise induce a volume decrease of the glycerinated macronuclei, but to a distinctly lesser extent than ATP.

HETEROGENEITY IN THE THERMALLY‐INDUCED QUENCHING OF THE PHOSPHORESCENCE OF MULTI‐TRYPTOPHAN PROTEINS

Abstract— The steady‐state intensity and lifetime of the tryptophan phosphorescence from a number of globular proteins in 2:1 (v/v) glycerol buffer were monitored as a function of temperature. The phosphorescence lifetimes are essentially independent of the tryptophan local environment in rigid solution at temperatures < 170K, but vary markedly between proteins at temperatures at which the solutions become fluid. The ratio of steady‐state intensity to lifetime P/τ was found to be temperature independent despite the quenching for free tryptophan and the lone residue in myelin basic protein. Heterogeneity in the triplet quenching of the tryptophans in liver alcohol dehydrogenase and alkaline phosphatase were revealed as step‐like decreases in the ratio of P/T followed by plateau regions characterizing the homogeneous behavior of the more resistent tryptophans in the proteins. This heterogeneity exists not only between solvent‐exposed and buried residues, but reflects variations in the flexibility of the structure surrounding distinct buried tryptophans in the globular proteins.

Metabolic changes in rat skin during preservation and storage in glycerol buffer at −196 °C

The penetrating cryoprotective glycerol dissolved in different concentrations in buffer medium effectively protects skin tissue against freeze-thaw injury when progressively cooled to −196 °C followed by a fast warming-up rate. Upon incubation of the tissue after storage, the incorporation of [2- 14C]glycine into the proteins, of [6- 3H]thymidine into DNA, and α-[1- 14C]aminoisobutyric acid transport through the cell membrane are reduced compared to freshly incubated skin. An essential loss in metabolic activity occurs during exposure of the skin to the preserving buffer. Although the length of storage does not seem to affect the final viability, the actual freezing and thawing procedures are particularly damaging to tissue already injured by previous exposure to buffer containing cryoprotective agents.

Some properties of androgen-binding activity in rat testis

High-affinity ( K a ≅ 5 × 10 8 M −1 for testosterone) androgen-binding activity in rat testis was shown to have a rapid dissociation rate constant ( t 1 2 = 3 min , 0 °C, 30% glycerol buffer) using dextran-coated charcoal to separate bound from free hormone. Because of this fact, exchange of endogenous and labeled hormone was complete in the assay incubation time (16 h, 0 °C) and Scatchard plots of the high-affinity binding data were shown to measure total as contrasted to available sites. The binding was highly specific for androgens. Polyacrylamide gel electrophoresis separated high-affinity androgen-binding protein ( R f 0.54 ) from albumin ( R f 0.62 ). Binding site estimates under saturating conditions or by Scatchard analysis of electrophoresis data utilizing [ 3H]dihydrotestosterone agreed reasonably well with estimates made by the charcoal technique using [ 3H]testosterone.

Studies on the Pellicular Complex of the Exocrythrocytic Merozoites of the Avian Malarial Parasite (Plasmodium lophurae)

Plasmodium lophurae grown in monolayers of cells derived from turkey brains was freeze-fractured after fixation in 1.25% gluteraldehyde. The fixed material was placed in an ascending series of glycerol buffer until a 40% concentration was reached. This preparation was then frozen and fractured at -190°C under vacuum. Both fracture surfaces of the specimen were simultaneously shadowed and carbon coated to obtain complementary replicas. The replicas were studied with the JEM 100-B transmission electron microscope.Previous studies on stained ultrathin sections of the merozoites of mature schizonts show the merozoite to be surrounded by two membranes and an innermost microtubular system. In addition to this pellicular system of the merozoites, the entire schizont is surrounded by a loose limiting membrane. Our studies revealed splitting of the two membranes enclosing the exoerythrocytic merozoite. This resulted in the presence of four fracture faces, each with its own distinct topography.

Regulation of Pyruvate Dehydrogenase in Rat Liver Mitochondria by Phosphorylation-Dephosphorylation

Abstract The percentage of total pyruvate dehydrogenase in the active nonphosphorylated form was measured in intact rat liver mitochondria after rapid separation of the mitochondria through silicone oil and extraction with glycerol buffer at -10°. The contents of ATP, ADP, Mg2+, and Ca2+ in the mitochondrial matrix of separated mitochondria were determined in parallel experiments. Mitochondria were incubated with glutamate and malate as substrates in a variety of metabolic states. These were induced by addition of oligomycin, uncoupler, the divalent cation ionophore A23187, and other inhibitors in order to alter the phosphorylation state of the intramitochondrial adenine nucleotides and the contents of magnesium and calcium. Interconversion between the active nonphosphorylated form of pyruvate dehydrogenase and the inactive phosphorylated form appeared to be dominated by regulation of pyruvate dehydrogenase kinase activity. Under conditions of controlled respiration, the percentage of pyruvate dehydrogenase in the active form varied directly with the ADP content and inversely with the ATP:ADP ratio. Regulation of pyruvate dehydrogenase interconversion by pyruvate dehydrogenase phosphatase activity could only be demonstrated under severe conditions of magnesium and calcium depletion. It is concluded that the normal content of Mg2+ and Ca2+ in isolated mitochondria is sufficient to provide optimal activation of pyruvate dehydrogenase phosphatase and that release of Mg2+ by conversion of MgATP2- to ADP, Pi, and Mg2+ during active respiration has a negligible effect on pyruvate dehydrogenase phosphatase activity. The over-all steady state activity of pyruvate dehydrogenase appears to be determined by pyruvate and ADP which inhibit pyruvate dehydrogenase kinase, and thereby prevent phosphorylation and inactivation of pyruvate dehydrogenase. Regulation of pyruvate dehydrogenase activity is thus achieved without an unnecessary expenditure of ATP for enzyme phosphorylation and dephosphorylation.

Protection ofAcholeplasma LaidlawiiB by Superoxide Dismutase

SummaryIrradiation of Acholeplasma laidlawii B cells in air in the presence of superoxide dismutase produced a protective effect that had two components. The smaller component manifested itself immediately after the irradiation and depended on the buffer used. In Tris NaCl, pH 7·6, it amounted to an increase in D0 of 7·5 per cent or less; in phosphate glycerol buffer, pH 7·0, the increase amounted to 10·5–18·9 per cent, depending on the enzyme concentration. The larger component was elicited over a 4-hour period after irradiation and amounted to an increase in D0 of 17–52 per cent, depending on the enzyme concentration. A major portion of this component was also elicited by the enzyme when added to the cells 10 min after irradiation. Inactivated enzyme was generally ineffective. Mechanistically, these results require reactions involving the superoxide radical both during and after the irradiation and implicate sulphydryl oxidation as well as peroxidation of membrane phospholipids.

Separation of serum proteins by using a simple thin layer polyacrylamide gel electrophoresis

Although the thin layer polyacrylamide gel electrophoresis is a highly suitable and satisfactory method for the analysis of serum proteins, the procedure is not so simple as that of cellulose acetate membrane electrophoresis.The authors have been studying on the micro-electrophoresis of proteins and enzymes in serum and have accomplished a procedure for “Simple polyacrylamide gel film electrophoresis” of high resolution. The procedure and some of the results were reported.It is based on thin layer electrophoresis on a sheet coated by the thin layer of polyacrylamide gel (1mm or 2mm thick), which has been previously prepared and kept in moist condition. Therefore, preparation of thin layer could be omitted from the procedure.As the sheets, cellophane of a quality comparable to dialysis tubing, 124 PD cellophane (Du Pont Co.), was selected. A polyester film used for drawing with a smooth surface and a rough back was also found suitable in regard to the firm adhesion of the gel layer to the sheet, since the gel layer could be formed on the rough side.Various buffer conditions of gel layer could be obtained by using monomer buffer mixture solution which was prepared by dissolving 9.5g of acrylamide and 0.5g of BIS (N, N'-methylenebisacrylamide) in 200ml of 10% glycerol buffer solution. The gelation was performed by adding catalysts to monomer buffer mixture solution.Sera were applied without dilution into the sample grooves previously prepared on the gel layer surface. 3μl or 10-15μl of serum was sufficient for each groove on the gel layer with 1mm or 2mm thickness, respectively.The gel layer on a supporting glass plate bridged between the two electrode compartments and the ends of the gel were connected by wet filter papers to the buffer solution in the compartments.To prevent over-heating, the electrophoresis was carried out at 5°C to 10°C. The voltage was adjusted to deliver a constant current of 0.5 to 1.0mA per cm width of the gel layer and the current was applied for 120min.Various staining methods for enzyme and protein components could be applied after electrophoresis.The stained gel layer was inserted between the sheets of cellophane, and the gel layer was plastized by leaving at room temperature for two days.The procedure was also found to be a useful tool for the estimation of molecular weights of polypeptide chains by using the gel layer containing sodium dodecyl sulfate (SDS).

Purification of Protochlorophyllide Holochrome

Phototransformable protochlorophyllide holochrome was prepared from etiolated bean leaves. The detergent Triton X-100 in the presence of glycerol and tricine-KOH buffer (pH 8) enhanced the extractability, specific activity, and phototransformability of the holochrome. Purification was achieved by polyethylene glycol-6000 precipitation and hydroxyl-apatite, DEAE-cellulose, and agarose chromatography. The presence of Triton X-100 permitted removal of the carotenoid contamination from the holochrome. The 678-nm absorption maximum of newly formed chlorophyllide a holochrome shifts to 672 nm in a temperature-dependent manner. The purified holochrome contains 0.24 g of protein per mumole of protochlorophyllide. Estimation of the molecular weight of the holochrome by gel filtration on agarose revealed the presence of aggregates of approximately 550,000 and 300,000. There are at least 2 chromophores per 550,000 molecular weight.