High purity preparations of higher plant vacuolar H+-ATPase reveal additional subunits. Revised subunit composition

A fast protein liquid chromatography procedure for purification of the V-type H+-ATPase from higher plant vacuolar membrane to yield near-homogeneous enzyme with a specific activity of 20-25 mumol/mg.min is described. When precautions are taken to ensure the quantitative recovery of protein before sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the preparation is found to be constituted of seven major polypeptides of 100, 67, 55, 52, 44, 32, and 16 kDa, respectively, and two minor components of 42 and 29 kDa. The 52-, 44-, and 32-kDa polypeptides do not cross-react with antisera raised to the 67- and 55-kDa subunits of the enzyme, and two independent sample preparation procedures yield the same apparent subunit composition. The additional polypeptides are not breakdown products or aggregates of the previously identified subunits of the ATPase. The ATPase of tonoplast vesicles is subject to MgATP-dependent cold inactivation, and the conditions for inactivation are identical to those for the bovine chromaffin granule H+-ATPase (Moriyama, Y., and Nelson, N. (1989) J. Biol. Chem. 264, 3577-3582). Cold inactivation is accompanied by the detachment of five major polypeptides of 67, 55, 52, 44, and 32 kDa from the membrane, and all five components co-migrate with the corresponding polypeptides of the purified ATPase upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 100- and 16-kDa polypeptides of the ATPase are not removed from the membrane during cold inactivation, but the latter can be purified to homogeneity by chloroform:methanol extraction of the fast protein liquid chromatography-purified enzyme. It is concluded that the tonoplast H+-ATPase is constituted of 6-7 major polypeptides organized into a peripheral sector comprising the 67-, 55-, 52-, 44-, and 32-kDa components and an integral sector consisting of the 100- and 16-kDa polypeptides. The V-type H+-ATPase from animal endomembranes and higher plant vacuolar membranes therefore have remarkably similar subunit compositions and gross topographies.     

THE DISTRIBUTION AND DIFFERENTIATION OF LYMPH-BORNE IMMUNOBLASTS AFTER INTRAVENOUS INJECTION INTO SYNGENEIC RECIPIENTS

Thoracic duct lymph from inbred, hooded rats was collected 3–5 days after antigenic stimulation of the caudal lymph nodes. During this period the lymph contained 10–15% of large, basophilic lymphoid blast cells (immunoblasts). By incubating the lymph cells at 38.C with radioactive DNA precursors, either ³H-thymidine or ¹²⁵I-deoxyuridine, the immunoblasts became labelled but the small lymphocytes did not. The lymph cells were then washed and injected intravenously into syngeneic recipients which were killed after various intervals up to 24 hr so that the radioactivity of their organs could be assayed by scintillation counting and autoradiography.
The main finding was that in animals killed after 4 or more hours the small gut always contained most of the recoverable activity and autoradiographs showed that this was because the injected cells had infiltrated the lamina propria in large numbers. Earlier, many of the injected cells were retained temporarily in the lungs, liver and spleen but many of them soon left those organs and entered the lamina propria of the small gut.
An electron microscope study of autoradiographs showed that 24 hr after injection the cells which entered the lamina propria of the gut had differentiated into plasma cells so that they displayed abundant, lamellar endoplasmic reticulum.     

How does the extracellular matrix direct gene expression?

Based on the existing literature, a model is presented that postulates a “dynamic reciprocity” between the extracellular matrix (ECM) on the one hand and the cytoskeleton and the nuclear matrix on the other hand. The ECM is postulated to exert physical and chemical influences on the geometry and the biochemistry of the cell via transmembrane receptors so as to alter the pattern of gene expression by changing the association of the cytoskeleton with the mRNA and the interaction of the chromatin with the nuclear matrix. This, in turn, would affect the ECM, which would affect the cell, which…     

The effect of needle age on the acceptability of Sitka spruce needles to the aphid,Elatobium abietinum (Walker)

Summary A comparison of the feeding behaviour of E. abietinum on current year needles and previous year needles of P. sitchensis revealed that during the summer months the aphids preferentially settled on previous year needles, this response not being evident in winter. Aphids on current year needles in summer took a much longer period of time to commence sap uptake than in winter, intake ceasing following a very short feeding period. On previous year needles sap uptake in December commenced after a shorter feeding period than in June. Analysis of total and soluble nitrogen levels in Sitka spruce needles showed that current year needles had initially higher levels during shoot elongation in May and early June, but that previous year needles had higher levels for most of the remainder of the year. Quantitative analyses of amino acids revealed that in current year needles the levels were generally lower than in previous year needles. Less marked proportional differences were observed between previous year needles in May and in July/August when the needles were unsuitable. Addition of amino acids in solution into cut current year shoots resulted in increased longevity on shoots containing introduced iso-leucine, histidine and methionine and revealed a general imbalance of the amino acids.     

Movement of tropomyosin during regulation of vertebrate skeletal muscle: a simple physical model.

Using data obtained from (a) X-ray diffraction patterns of vertebrate skeletal muscle (b) three dimensional reconstructions from electron micrographs of in vitro aggregates of the thin filament proteins in the switched “on” and “off” positions (c) the analysis of the sequence of tropomyosin, a simple model can be proposed which may explain the geometrical arrangement of actin, tropomyosin and troponin during regulation. The “cooperative” behaviour exhibited by the thin filament can also be explained in terms of this arrangement.