Ultrastructure of the sodium pump: Comparison of thin sectioning, negative staining, and freeze-fracture of purified, membrane-bound (Na+, K+)-ATPase

Purified (Na+, K+)-ATPase was studied by electron microscopy after thin sectioning, negative staining, and freeze-fracturing, particular emphasis being paid to the dimensions and frequencies of substructures in the membranes. Ultrathin sections show exclusively flat or cup-shaped membrane fragments which are triple-layered along much of their length and have diameters of 0.1-0.6 μm. Negative staining revealed a distinct substructure of particles with diameters between 30 and 50 A and with a frequency of 12,500 +/- 2,400 (SD) per μm(2). Comparisons with sizes of the protein components suggest that each surface particle contains as its major component one large catalytic chain with mol wt close to 100,000 and that two surface particles unite to form the unit of (Na+,K+)-ATPase which binds one molecule of ATP or ouabain. The further observations that the surface particles protrude from the membrane surface and are observed on both membrane surfaces in different patterns and degrees of clustering suggest that protein units span the membrane and are capable of lateral mobility. Freeze-fracturing shows intramembranous particles with diameters of 90-110 A and distributed on both concave and convex fracture faces with a frequency of 3,410 +/- 370 per μm(2) and 390 +/- 170 per μm(2), respectively. The larger diameters and three to fourfold smaller frequency of the intramembranous particles as compared to the surface particles seen after negative staining may reflect technical differences between methods, but it is more likely that the intramembranous particle is an oliogomer composed of two or even more of the protein units which form the surface particles.     

Occurrence of 3-hydroxyretinol in the freshwater fish Bagarius bagarius and Wallago attu. Isolation and synthesis.

1. Four different types of alpha-mannosidase activity were shown to occur in several tissues from the rat. There is the Zn2+-dependent enzyme, active at acidic pH, and three enzymes that are active near to neutral pH. 2. The 'neutral' enzymes are activated by Fe2+, Co2+ or Mn2+. 3. Optimum activities for these three enzymes are shown at pH values of 5.2, 6.5 and 7.3. The activity at pH6.5 is the only one evident without metal-ion activation, but activity is enhanced by all three metal ions. The activity at pH 5.2 is seen only in the presence of Fe2+ or Co2+, and the activity at pH7.3 is seen only in the presence of Co2+ or Mn2+ and in a non-chelating buffer medium. 4. The pH6.5-active enzyme is inactivated by EDTA, but activity is restored by excess of metal ion. 5. The enzymes differ markedly in their stability. The pH6.5-active enzyme is very labile and the pH7.3-active enzyme is the most stable. 6. Tissue preparations vary widely in their activity at pH6.5, but where activity is low it can be increased by incubation with one of the activating metal cations. 7. All the enzymes active at neutral pH are inhibited by heavy-metal ions and stabilized to some extent by thiol groups.     

Instantaneous benthic response to different organic matter quality: In situ experiments in the Benguela Upwelling System

The benthic community in continental slope and deep-sea sediments of the Benguela Upwelling System was supplied with ¹³C-labelled organic matter (OM) of two different qualities using a benthic chamber lander. Freeze-dried cultures of Skeletonema costatum served as 'fresh' OM. 'Altered' OM of the same material had been additionally dialysed to remove low-molecular weight compounds. In order to investigate the benthic response pattern, mineralization of labelled OM, uptake by macrofauna and incorporation into bacteria were followed over 18-36 h. Total oxygen uptake was not affected beyond natural variation by the OM addition. Mineralization dominated the ¹³C-labelled phytodetritus processing, constituting 71-95% of the total processed OM. Bacterial incorporation of phytodetrital carbon exceeded macrofaunal uptake at all stations. Stations situated in a major centre of OM deposition showed phytodetritus processing rates on average twice as high as outside the depocentre. Phytodetritus processing was 1.5, 2.5 and 4.3 times higher for fresh than for altered OM at 605, 1019 and 1335 m water depth, respectively. Our observations clearly indicate the importance of OM quality on mineralization rates.     

Phytochrome mediates germination responses to multiple seasonal cues

We identified a new role of phytochrome in mediating germination responses to seasonal cues and thereby identified for the first time a gene involved in maternal environmental effects on germination. We examined the germination responses of a mutant, hy2-1, which is deficient in the phytochrome chromophore. The background genotype, Landsberg erecta (Ler), lacked dormancy in most treatments, while hy2-1 required cold stratification for germination in a manner that resembled a more dormant ecotype, Columbia (Col). Unlike Col, hy2-1 was not induced into dormancy by warm stratification. Therefore, the down-regulation of phytochrome-mediated germination pathways results in sensitivity to cold, but we found no evidence that reduced phytochrome activity enables the warm-induction of dormancy. Cool temperatures during seed maturation induced dormancy. The hy2-1 mutants did not overcome this dormancy, indicating that phytochrome-mediated pathways are required to break cold-induced dormancy. Ler did not respond to post-stratification temperature, but hy2-1 did respond, suggesting phytochrome pathways are involved in germination responses to temperature. In summary, phytochromes mediate dormancy and germination responses to seasonal cues experienced both during seed maturation and after dispersal. Phytochromes therefore appear to be involved in mediating seasonal germination timing, a trait of great ecological importance and one that is under strong natural selection.     

Structure-based kinetic models of modular signaling protein function: focus on Shp2

We present here a computational, rule-based model to study the function of the SH2 domain-containing protein tyrosine phosphatase, Shp2, in intracellular signal transduction. The two SH2 domains of Shp2 differentially regulate the enzymatic activity by a well-characterized mechanism, but they also affect the targeting of Shp2 to signaling receptors in cells. Our kinetic model integrates these potentially competing effects by considering the intra- and intermolecular interactions of the Shp2 SH2 domains and catalytic site as well as the effect of Shp2 phosphorylation. Even for the isolated Shp2/receptor system, which may seem simple by certain standards, we find that the network of possible binding and phosphorylation states is composed of over 1000 members. To our knowledge, this is the first kinetic model to fully consider the modular, multifunctional structure of a signaling protein, and the computational approach should be generally applicable to other complex intermolecular interactions.     

Quantification of retinyl-β-glucuronides in rat urine by reversed-phase high-performance liquid chromatography with ultraviolet detection

A method is presented for the quantitation of the glucuronide conjugates of 4-oxo-all-trans-, 4-oxo-13-cis-, 13-cis-, 9-cis- and all-trans-retinoic acids in rat urine utilizing solid-phase extraction and gradient reversed-phase HPLC. The range of the R.S.D. (relative standard deviation) for both the inter- and intra-assay precision was 1.45,2–11.60%. The recovery of all retinoyl-β-glucuronides from rat urine ranged between 89 and 99%. The limit of detection was 0.01 μg/ml using 5 ml of rat urine. This method was applied to quantitate the amount of retinoyl-β-glucuronides produced in urine after the single and multiple oral administrations of 13-cis-, 9-cis- and all-trans-retinoic acids to rats.     

The structure and stereochemistry of lantanilic acid,the β,β-dimethylacryloyl ester of lantaninilic acid,isolated from Lantana camara

The structure of lantanilic acid, a new triterpene isolated from the leaves of Lantana camara, has been determined as the β,β-dimethylacryloyl ester of lantaninilic acid.