Phosphate determination by enzymatic colorimetric assay

A direct colorimetric assay for inorganic phosphate in serum is described. The system is based on utilization of the enzymes, purine-nucleoside phosphorylase and xanthine oxidase, to generate superoxide ions. The superoxide is measured in the presence of an electron mediator compound with 3-(4',5'-dimethyl-2-thiazolyl)-2,4-diphenyl-2H-tetrazolium bromide as the chromogen. The high absorbance of this chromogen between 550 and 660 nm affords useful results with a sample/reagent volume ratio as low as 1:100. A single working reagent is used, and the reaction is complete in 15 min at room temperature. The standard curve is linear for inorganic phosphate concentrations as high as 4.9 mmol/liter. Analytical recovery of phosphate in human sera averages 100%. Within-run precision study gives CV less than or equal to 1.0%. The results of this method compare closely (r greater than 0.99) with those obtained by the semidine method (recommended standard). The method lends itself to automation.     

Isolation of heparan sulfates with antithrombin III affinity and anticoagulant potency from BALB/c 3T3, B16.F10 melanoma, and cutaneous fibrosarcoma cell lines

The heparan sulfates synthesized in vitro by three cell lines were isolated by proteolysis and preparative anion exchange chromatography and purified free of other glycosaminoglycans by selective enzymatic degradation. The isolates from the medium of BALB/c 3T3 fibroblasts, B16.F10 melanoma cells, and a cutaneous fibrosarcoma line, along with that from the detergent-extracted cell layer of the fibroblasts, were affinity-fractionated on columns of matrix-immobilized human antithrombin III. Each heparan sulfate contained subfractions with high affinity for the proteinase inhibitor, ranging from 3-34% of the starting material. The high affinity species possessed measurable anticoagulant activities by a clotting assay (6 to 30 units/mg). Since none of the lines were derived from cell types having any known biological role in vascular homeostasis, we suggest that anticoagulant activity of the glycosaminoglycan is a random property of its primary structure.     

Design of a growth model for yeast

The study of the cell cycle of a yeast strain made it possible to define two parameters:T, the time elapsing between the appearance of two consecutive buds on a mother cell, and Θ, the time elapsing between the appearance of a bud and the beginning of the first mitotic cycle. The influence of these two parameters on the growth rate of the strain is studied.     

A new species of bound ubisemiquinone anion in QH2: cytochrome c oxidoreductase

Using a combination of EPR and low temperature diffuse reflectance spectroscopy, a new species of semiquinone anion has been detected in QH2:cytochrome c oxidoreductase in submitochondrial particles under conditions of oxidant-induced extra reduction of cytochrome b. In contrast to the previously detected semiquinone anion, this new species is insensitive to antimycin but sensitive to treatment with 2,3-dimercaptopropanol and O2. The two species can easily be distinguished on the basis of their respective EPR properties since they differ in g-value, line width, and microwave power saturation behavior. It is concluded that the two species of semiquinone anion are bound to different domains on QH2:cytochrome c oxidoreductase. The existence of two different semiquinone anions in the enzyme strongly supports a mechanism of electron flow as proposed in the Q-cycle.     

Rapid separation of the chloroplast and cytoplasmic fractions from intact leaf protoplasts

Intact protoplasts are ruptured by rapid centrifugation through a narrow-aperture nylon mesh and the intact chloroplasts are then separated from the cytoplasm by sedimentation through a layer of silicone oil below the mesh. Within 6 to 8 s of starting the centrifuge, 90% of the chloroplasts are separated into the pellet fraction which contains only 10 to 15% contamination by mitochondria and peroxisomes and less than 5% contamination by soluble cytoplasm as judged by the distribution of marker enzymes. This technique should allow determination of the distribution of metabolites between the chloroplast and cytoplasmic compartments of intact protoplasts.     

Growth of an ice phase in frozen tissue studied by proton NMR-spectroscopy

Development of an ice phase in frozen tissue is a severe problem in cryo-ultramicrotomy. It is assumed that the growth-rate of such an ice phase is controlled by the mobility of the water molecules in the tissue. In order to verify this, wide-line nmr-spectroscopy has been performed on the protons of frozen whole blood, with and without protective agent, in the temperature range −100 °C- −40 °C. The molecular motion may be characterized by a correlation time, τ, which can be thought of as roughly the mean time between proton jumps. The value of τ can be approximately evaluated from the width of the proton resonance line. It is shown that τ varies rapidly with temperature, in a similar manner to maximum storage time for frozen blood in blood banks. Addition of glycerol increases τ at low temperature, which is consistent with its use as a protective agent in cryobiology. At high temperature, addition of glycerol reduces τ, which is in agreement with reported light-microscope observations on the growth-rate of an ice phase in the same temperature range, nmr-spectroscopy seems to be a useful tool in the further development of cryo-ultramicrotomy. However, to estimate the value in full, more data is required than is presented here.