Immunochemical subfractionation of a microsomal fraction of rat liver with antibody-coated Staphylococcus aureus cells

The procedure for immunochemical adsorption of vesicles with specific antigen on their outer surfaces was improved. When microsomal vesicles were mixed with Staphylococcus aureus cells coated with the antibody against NADPH-cytochrome c reductase, more than 90% of the enzyme activity was adsorbed on the cell, whereas, only about 10% of the activity was adsorbed on cells coated with the same amount of anti-ovalbumin antibody. NADH-cytochrome c reductase and aldehyde dehydrogenase activities were adsorbed on the cell to the same extent as was NADPH-cytochrome c reductase activity. Under this condition, there was no adsorption of the activities of the marker enzymes of lysosomes and Golgi apparatus, whereas large amounts of the activities of the plasma membrane enzymes were adsorbed. The specific activity of NADPH-cytochrome c reductase in the adsorbed vesicles from the microsomal fractions increased considerably. In contrast, marker enzymes of the Golgi or of the plasma membranes could be enriched in unadsorbed vesicles from the Golgi fractions.     

Purification of a fibrolast-inhibitory factor from Actinobacillus actinomycetemcomitans Y4

Abstract A factor showing inhibitory activity against human gingival fibrolasts was extracted from the cytosol fraction of Actinobacillus actinomycetemcomitans Y4. The activity markedly inhibited the proliferation of human gingival fibrolasts, but had no effect on cell viability or gross morphology. No such activity was found in cytosol fractions from either Porphyromonas gingivalis 381 or Escherichia coli HB101. The extract from A. actinomycetemcomitans Y4 was then purified by anion-exchange chromatography, hydroxyapatite chromatography and gel-filtration chromatography to give a single band on SDS-PAGE with an apparent molecular mass of 65 kDa. The purification ratio was 183-fold with a recovery rate of 5% compared with the crude extract (starting material) when the activity was assessed by direct cell counts.     

Metabolism of 3-phosphoglyceroyl phosphate in phosphoenolpyruvate-enriched human erythrocytes

The concentration of 3-phosphoglyceroyl phosphate in erythrocytes was increased by more than 100-fold when red cells were incubated with extracellular phosphoenolpyruvate at 37 degrees C. Since these elevated levels were maintained for 60 min, the metabolism of 3-phosphoglyceroyl phosphate and related compounds could be investigated in phosphoenolpyruvate-treated erythrocytes. 2,3-Bisphosphoglycerate synthesis was not affected by intracellular pH when the 3-phosphoglyceroyl phosphate level was constant but did vary with 3-phosphoglyceroyl phosphate concentration. On the other hand, the relationship between the rate of 2,3-bisphosphoglycerate synthesis and 3-phosphoglyceroyl phosphate concentration was not straightforward. At relatively low concentrations of 3-phosphoglyceroyl phosphate, the observed rate of 2,3-bisphosphoglycerate synthesis agreed with a rate calculated from a formula incorporating kinetic parameters of purified 2,3-bisphosphoglycerate synthase (Rose, Z.B. (1973) Arch. Biochem. Biophys. 158, 903-910). However, at high concentrations of 3-phosphoglyceroyl phosphate, the observed rate of 2,3-bisphosphoglycerate synthesis was lower than the calculated value. The concentration of glucose 1,6-bisphosphate did not increase even when 3-phosphoglyceroyl phosphate was elevated to 200 microM. Elevated levels of intracellular 2,3-bisphosphoglycerate did not inhibit glycolytic activity in these erythrocytes. These results suggest that incubation of erythrocytes with phosphoenolpyruvate is a useful technique to investigate the effect of metabolic perturbations at the intermediate stages of glycolysis.     

Calcium-activated protease in the giant algaChara australis

Summary There is a protease, which is activated by Ca²⁺ (about 100 M), works at neutral pH and exists in the cytoplasm inChara australis. This protease may correspond to calpain, the calcium-activated neutral protease, which has been studied in animal cells. This is the first report showing the existence of a calcium-activated protease in plant cells.     

Exposure of Leaves of Cucumis sativus L. to Low Temperatures in the Light Causes Uncoupling of Thylakoids II. Non-Destructive Measurements with Intact Leaves

To examine the effects of chilling of leaves of cucumber (Cucumissativus L.) in moderate light on the coupling state of thylakoidsin situ, changes in fluorescence, changes in light scatteringand flash-induced changes in absorbance at 518 nm were examinedin intact leaves. After chilling of leaves at 5?C in the lightfor 5 h, the non-photochemical quenching of fluorescence, ameasure of energisation of thylakoids, was largely suppressed.The treatment also caused a suppression of light-induced changesin the light scattering by leaves, which depends on the formationof a pH gradient across thylakoid membranes. When thylakoidswere prepared by very gentle methods from the leaves chilledin the light, through a step of preparation of intact chloro-plasts,the transport of electrons from H₂O to ferricyanide was uncoupled,being insensitive to an uncoupler, methylamine. These data provide consistent evidence that the thylakoids areuncoupled in situ by the chilling of leaves in the light and,as a consequence of the uncoupling, the energisation of themembranes is suppressed. However, the decay of the flash-inducedchange in absorbance at 518 nm in leaves was not markedly acceleratedby the treatment. The thylakoids isolated from leaves chilledin the light, which were in the uncoupled state, also did notshow a rapid decay, unless an efficient uncoupler such as gramicidinwas added. These results suggest that even a considerable uncouplingof thylakoids, brought about by chilling of leaves in the light,is not sufficient to cause a marked acceleration of the decayof the flash-induced change in absorbance at 518 nm. Therefore,analysis at 518 nm is not always a sensitive method for assessingthe coupling state of thylakoids. (Received July 1, 1991; Accepted October 4, 1991)     

Triterpenes from Periandra dulcis roots

Three oleanane triterpenes were isolated from the roots of Periandra dulcis,and identified as 3β-hydroxy-25-al-olean-18-en-30-oic acid (periandric acid I), 3β-hydroxy-25-al-olean-12-en-30-oic acid (periandric acid II) and 3-oxo-25-hydroxy-olean-12-en-30-oic acid. The former two compounds (periandric acids I and II) were identical with the aglycones obtained by hydrolysis of periandrin I and II, respectively and the latter one was a new triterpene.     

Photoreception in pineal organs of larval and adult lampreys,Lampetra japonica

A comparative study of the larval and adult pineal organs, which are sensitive to incident light, was carried out in the river lamprey Lampetra japonica, using intracellular recording from the pineal photoreceptors. The tissue overlying the larval pineal organ is transparent, whereas that over the adult pineal is translucent. The optical density of this oval pineal window in the adult lamprey was 1.2. In order to elucidate the early development of the larval pineal, the ratio r of the diameter (micron) of the pineal to the body-length (cm) was measured. The value of r was 62.5 in a small larva of 2.8 cm, 29.7 in a larger one of 14.3 cm, and 9.3 in an adult of 54 cm body-length. The intracellular response to light of the larval pineal was a hyperpolarization, showing fundamentally the same pattern as that of the adult pineal. It was possible to record a typical response even from the pineal of the smallest larva, 2.8 cm in body length, used in this study. The intensity-amplitude relationship was analysed after Naka-Rushton's hyperbolic equation. The value of sigma of isolated larval pineals was 0.88 log unit higher than that of adults. The value of n was larger in larvae, suggesting a sensitive reaction to changing photic stimulus. The spectral sensitivity was compared. The peak was at 505 nm in the larva, but 525 nm in the adult. A change of visual pigment in the pineal during metamorphosis is suggested.     

Ammonia Regulation of Intermediary Metabolism in Photosynthesizing and Respiring Chlorella pyrenoidosa: Comparative Effects of Methylamine

The natural ¹³C abundance (¹³C value) of the field-grown leguminousplants (soybean, kidney bean, pea, azuki bean, mung bean, peanutand cowpea) was investigated by mass spectrometry with a precisionbetter than %0.2 for ¹³C. Among organs of premature plants,the leaves had the most negative values, and the nodules generallyhad the least negative values, and other organs, fruits, stemsand roots, showed intermediate values. In the soybeans so farinvestigated, the grains of nodulating plants exhibited higher¹³C values than nonnodulating lines. The ¹³C values of the grainsvaried depending on the species: peanuts showed the most negativevalues. Possible causes underlying these variations are discussed. (Received March 2, 1983; Accepted May 27, 1983)     

Localization and Properties of Enzymes Involved with Electron Transport Activity in Mycobacteria

A study of the subcellular localization of the nicotinamide adenine dinucleotide (NADH)-3-(4, 3-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) oxido-reductase systems in Mycobacterium was presented. Evidence based on starch gel electrophoresis and different responses of the subcellular fractions to heat inactivation suggested the existence of more than one enzyme responsible for the NADH-MTT oxido-reductase activity. One type of activity was found in the membrane-mesosome fraction which contained labile and electrophoretically non-migrating enzymes. Another type of activity was also detected in the soluble fraction which on starch gel electrophoresis exhibited 4 bands of activity, two of which showed heat resistance.