Complex subcellular distributions of enzymatic markers in intestinal epithelial cells

Summary Current procedures for isolating intestinal epithelial cell surface and intracellular membranes are based on the assumption that each organelle is marked by some unique constitutent. This assumption seemed inconsistent with the dynamic picture of subcellular organization emerging from studies of membrane turnover and recycling. Therefore, we have designed an alternative fractionation which is independent ofa priori marker assignments. We subjected mucosal homogenates to a sequence of separations based on sedimentation coefficient, equilibrium density, and partitioning in aqueous polymer twophase systems. The resulting distributions of protein and enzymatic markers define a total of 17 physically and biochemically distinct membrane populations. Among these are: basal-lateral membranes, with Na,K-ATPase enriched 21-fold; brush-border membranes, with alkaline phosphatase enriched as much as 38-fold; two populations apparently derived from the endoplasmic reticulum; a series of five populations believed to have been derived from the Golgi complex; and a series of five acid phosphatase-rich populations which we cannot identify unequivocally. Each of the five enzymatic markers we have followed is associated with a multiplicity of membrane populations. Basallateral, endoplasmic reticulum, and Golgi membranes contain alkaline phosphatase at the same specific activity as the initial homogenate. Similarly, Na,K-ATPase appears to be associated branes at specific activities two-to seven-fold that of the initial homogenate.     

The nitrogen cycle in lodgepole pine forests,southeastern Wyoming

Storage and flux of nitrogen were studied in several contrasting lodgepole pine (Pinus contorta spp.latifolia) forests in southeastern Wyoming. The mineral soil contained most of the N in these ecosystems (range of 315–860 g · m^–2), with aboveground detritus (37.5–48.8g · m^–2) and living biomass (19.5–24.0 g · m^–2) storing much smaller amounts. About 60–70% of the total N in vegetation was aboveground, and N concentrations in plant tissues were unusually low (foliage = 0.7% N), as were N input via wet precipitation (0.25 g · m^–2 · yr^–1), and biological fixation of atmospheric N (<0.03 g · m^–2 · yr^–1, except locally in some stands at low elevations where symbiotic fixation by the leguminous herbLupinus argenteus probably exceeded 0.1 g · m^–2 · yr^–1).Because of low concentrations in litterfall and limited opportunity for leaching, N accumulated in decaying leaves for 6–7 yr following leaf fall. This process represented an annual flux of about 0.5g · m^–2 to the 01 horizon. Only 20% of this flux was provided by throughfall, with the remaining 0.4g · m^–2 · yr^–1 apparently added from layers below. Low mineralization and small amounts of N uptake from the 02 are likely because of minimal rooting in the forest floor (as defined herein) and negligible mineral N (< 0.05 mg · L^–1) in 02 leachate. A critical transport process was solubilization of organic N, mostly fulvic acids. Most of the organic N from the forest floor was retained within the major tree rooting zone (0–40 cm), and mineralization of soil organic N provided NH₄ for tree uptake. Nitrate was at trace levels in soil solutions, and a long lag in nitrification was always observed under disturbed conditions. Total root nitrogen uptake was calculated to be 1.25 gN · m^–2 · yr^–1 with estimated root turnover of 0.37-gN · m^–2 · yr^–1, and the soil horizons appeared to be nearly in balance with respect to N. The high demand for mineralized N and the precipitation of fulvic acid in the mineral soil resulted in minimal deep leaching in most stands (< 0.02 g · m^–2 · yr^–1). These forests provide an extreme example of nitrogen behavior in dry, infertile forests.     

Immobilization of enzymes in porous supports: Effects of support-enzyme solution contacting

Proteins have been immobilized in porous support particles held in a fixed-bed reactor through which protein solution is continuously circulated. Changing the recirculation flow rate alters the observed immobilization kinetics and the maximum enzyme loading which can be achieved for glucose oxidase and glucoamylase on carbodiimide-treated activated carbon and for glucoamylase immobilized on CNBr-Sepharose 4B. Direct microscopic examination of FITC-labelled protein in sectioned Sepharose particles and indirect activity-loading studies with activated carbon-enzyme conjugates all indicate that immobilized enzyme is increasingly localized near the outer surface of the support particles at larger recirculation flow rates. Restricted diffusion of enzymes may be implicated in this phenomenon. These contacting effects may be significant considerations in the scaleup of processes for protein impregnation in porous supports, since apparent activity and stability of the final preparation depend on internal protein distribution.     

Seasonal changes in photosynthetic activity of snow tussocks (Chionochloa spp.) along an altitudinal gradient in Otago,New Zealand

Summary Photosynthetic characteristics were studied in Chionochloa rigida, an altitudinally widespread snow tussock and a closely related high-altitude species C. macra. Along a gradient from near sea level to 1600 m there were no consistent differences in maximum photosynthetic capacity which averaged 4.5 mol CO₂ m^-2s^-1. The photosynthetic temperature optimum ranged between 15 and 18°C and there was only a limited capacity for seasonal adjustment. Net photosynthesis was light-saturated at about 500 mol photons m^-2s^-1. In winter, the photosynthetic capacity decreased significantly with increasing altitude of origin of the snow tussocks. A transplant experiment indicated this was partly genetically controlled.     

Neuroendocrine and behavioral effects of m-chlorophenylpiperazine administration in rhesus monkeys

The effects of m-chlorophenylpiperazine (mCPP), a serotonin receptor agonist, on the release of plasma prolactin (PRL), growth hormone (GH), and cortisol in the rhesus monkey were studied. mCPP was administered intravenously at doses of 0.5, 1.5, and 3.0 mg/kg. GH and cortisol were increased significantly at all doses whike PRL was significantly increased only following administration of 3.0 mg/kg mCPP. mCPP administration also produced behavioral alterations in each monkey, including sedation, penile erection, and defecation. PRL, GH and behavioral responses to mCPP were completely blocked by pretreatment with the serotonin anatgonist metergoline (MTG). However, pretreatment with MTG failed to entirely antagonize the cortisol response to mCPP. These data suggest that mCPP has prominent neuroendocrine and behavioral effects which are mediated, in part, by serotonergic mechanisms.     

Analysis of the kinetics of phospholipid activation of cobra venom phospholipase A2

A kinetic analysis of the "dual phospholipid model" for cobra venom phospholipase A2 (Hendrickson, H. S., and Dennis, E. A. (1984) J. Biol. Chem. 259, 5734-5739) was applied to the activation of phospholipase A2-catalyzed hydrolysis of a thiol ester analog of phosphatidylethanolamine (thio - PE) in Triton X - 100/phospholipid mixed micelles by various phosphorylcholine-containing activators. Activation of thio-PE hydrolysis by didecanoylphosphatidylcholine (PC) was found to be a function of the surface concentration of activator rather than bulk concentration. Its presence did not affect the initial binding of enzyme to phospholipid in the micelle surface as determined kinetically. After initial binding of enzyme to the surface, the activation appears to be due to enzyme-lipid binding in the surface. Activation does not appear to affect the affinity of the enzyme for phospholipid substrate, but rather affects the catalytic efficiency of the enzyme as characterized by the value of Vmax. The monomeric phospholipid dibutyryl-PC, when used as an activator at 57 mM (bulk concentration), also showed effects of surface dilution with Triton X-100, which would not be expected unless the lipid is incorporated into the micelles to some extent at these high concentrations. A thiol ester analog of phosphatidylcholine, thio-PC, was less effective than didecanoyl-PC as an activator, but appeared to be more effective than decylphosphorylcholine. A conformational change of the enzyme upon binding of the activator, after enzyme is bound to substrate at the interface, is discussed as a possible mechanism for this activation.