Three high molecular weight protease inhibitors of rat plasma. Isolation, characterization, and acute phase changes.

Rat blood plasma contains three high molecular weight thiol ester-containing proteinase inhibitors, alpha 1-macroglobulin (alpha 1M), alpha 1-inhibitor III (alpha 1I3), and alpha 2-macroglobulin (alpha 2M). Rat serums have been analyzed using a two-dimensional gel electrophoretic technique which optimizes recovery of high molecular weight proteins. alpha 1M, and (alpha beta)4-tetramer in native solution, separated in the second sodium dodecyl sulfate-containing electrophoretic dimension as a disulfide-linked (alpha beta)2-dimer with an approximate Mr of 360 kDa. alpha 1I3 separated in the gels as a single 190-kDa polypeptide. It is also a monomer in native solution by ultracentrifugation criteria. Native rat alpha 2M is a tetramer, but it separates in the gels as a disulfide-linked dimer with an Mr of approximately 360 kDa. The kinetics of changes in concentration of these proteins during the induction of polyarthritis was also measured by quantitative immunoelectrophoresis. In rats with adjuvant-induced polyarthritis, the concentration of alpha 1I3 dramatically decreases and alpha 2M appears and continues to increase in a biphasic manner for 2 weeks. The alpha 1M concentration remains relatively constant. All three macroglobulins were purified utilizing modern rapid chromatographic techniques, and parallel comparisons of their native physicochemical properties were carried out. The N-terminal sequence of the alpha-chain of rat alpha 1M was also shown to share sequence homology with that of alpha 2M. In agreement, Esnard et al. (Esnard, F., Gutman, N., El Moujahed, A., and Gauthier, F. (1985) FEBS Lett. 182, 125-129) recently reported that alpha 1I3 also contains a thiol ester bond, as do alpha 1M and alpha 2M, since it reacts covalently with [14C]methylamine and is cleaved autolytically at 80 degrees C. We have examined negatively stained preparations of native, trypsin-treated, and methylamine-treated human alpha 2M, rat alpha 2M, and rat alpha 1M in the electron microscope. Trypsin appears to convert globular ring-shaped native molecules to rectangular box-like structures, in agreement with the conclusions of a recent report on human alpha 2M (Tapon-Bretaudiere, J., Bros, A., Couture-Tosi, E., and Delain, E. (1985) EMBO J. 4, 85-89).

Vacuolar localization of proteases and degradation of chloroplasts in mesophyll protoplasts from senescing primary wheat leaves.

Mesophyll protoplasts isolated from primary leaves of wheat seedlings were used to follow the localization of proteases and the breakdown of chloroplasts during dark-induced senescence. Protoplasts were readily obtained from leaf tissue, even after 80% of the chlorophyll and protein had been lost. Intact chloroplasts and vacuoles could be isolated from the protoplasts at all stages of senescence. All the proteolytic activity associated with the degradation of ribulose bisphosphate carboxylase in the protoplasts could be accounted for by that localized within the vacuole. Moreover, this localization was retained late into senescence. Protoplasts isolated during leaf senescence first showed a decline in photosynthesis, then a decline in ribulose bisphosphate carboxylase activity, followed by a decline in chloroplast number. There was a close correlation between the decline in chloroplast number and the loss of chlorophyll and soluble protein per protoplast, suggesting a sequential degradation of chloroplasts during senescence. Ultrastructural studies indicated a movement of chloroplasts in toward the center of the protoplasts during senescence. Thus, within senescing protoplasts, chloroplasts appeared either to move into invaginations of the vacuole or to be taken up into the vacuole.

Osmoregulation in Cotton in Response to Water Stress : I. ALTERATIONS IN PHOTOSYNTHESIS, LEAF CONDUCTANCE, TRANSLOCATION, AND ULTRASTRUCTURE.

Cotton plants subjected to a series of water deficits exhibited stress adaptation in the form of osmoregulation when plants were subjected to a subsequent drying cycle. After adaptation, the leaf water potential coinciding with zero turgor was considerably lower than in plants that had never experienced a water stress. The relationship between leaf turgor and leaf water potential depended on leaf age.Nonstomatal factors severely limited photosynthesis in adapted plants at high leaf water potential. Nonetheless, adapted plants maintained photosynthesis to a much lower leaf water potential than did control plants, in part because of increased stomatal conductance at low leaf water potentials. Furthermore, adapted plants continued to translocate recently derived photosynthate to lower leaf water potentials, compared with control plants.Stress preconditioning modified cellular ultrastructure. Chloroplasts of fully turgid adapted leaves contained extremely large starch granules, seemed swollen, and had some breakdown of thylakoid membrane structure. In addition, cells of adapted leaves appeared to have smaller vacuoles and greater nonosmotic cell volume than did control plants.

Binding characteristics of n(2)-fixing bacteria to cereal roots.

The attachment of Rhizobium japonicum 61A89 and Rhizobium spp. 32H1 to the roots of wheat and rice seedlings is analyzed in terms of an equilibrium model. A Langmuir adsorption isotherm describes the binding. Strain 61A89 binds to a greater extent than does strain 32H1, and the equilibrium constants for each strain binding to wheat are strongly temperature dependent. Both time-dependent dissociation and association, predicted by an equilibrium model, have been found. The dissociation rate constant for 32H1 is approximately twice that of 61A89, and each is weakly temperature dependent. The rate equation for the binding of exponentially growing 61A89 to wheat roots has been solved as a function of time. Theory and experiment both indicate that the binding at very short times is much less than the equilibrium values. The binding of Azotobacter vinelandii 12837 to wheat roots has also been measured. Root-associated Azotobacter fixes nitrogen, whereas under aerobic growth conditions, root-associated 61A89 and 32H1 do not. The effect of metabolic inhibitors and antibiotics on the binding of Rhizobia and Azotobacter was examined.

Octabromobiphemyl-induced ultrastructural changes in rat liver.

Single oral administration of octabromobiphenyls at 1,000 mg/kg, or two consecutive doses of 3,000 mg/kg, to rats produced liver enlargement. Light microscopic examination revealed hepatocellular hyperplasia, margination of basophillic cytoplasm, and foamy cytoplasmic alteration on the third day posttreatment. Laminated cytoplasmic inclusions developed seven days after treatment and subsequently disappeared about one week later. Under electron microscopy, the cytoplasmic margination corresponded to peripherally displaced granular reticulum and the foamy cytoplasm was recognizable as proliferating agranular reticulum with depletion of particualte glycogen. The cytoplasmic inclusions were identified as myelin configurations enclosing lipid bodies, membrane-bound vacuoles with whorled figures, and vesicular agranular reticulum. In the early developmental stages, the myelin figures were studded with ribosomes and associated with the granular reticulum.