Infrared spectroscopic studies on interactions of water and carbohydrates with a biological membrane

Infrared spectroscopy was used to investigate the changes in bands assigned to phospholipids and proteins in dehydrated and rehydrated sarcoplasmic reticulum. The changes in CH₂ and CH₃ stretching bands, amide bands, and phosphate stretching bands are similar to shifts in frequency seen for those bands in phospholipid and protein preparations during thermotropic phase transitions and hydration. IR studies on dry trehalose-sarcoplasmic reticulum mixtures show similar results; with increasing trehalose concentration in the dry mixtures, amide and phosphate bands shift to frequencies characteristic of hydrated samples. Changes in bands assigned to OH deformations in the trehalose suggest that the interaction between the carbohydrate and membrane is by means of hydrogen bonding between these OH groups and membrane components.     

Enolase isozymes from Ricinus communis: Partial purification and characterization of the isozymes

The plastid and cytosolic isozymes of enolase from developing endosperm of castor oil seeds, Ricinus communis L. cv. Baker 296, were separated and partially purified. Each purified isozyme had a specific activity of approximately 200 μmol min^?1 mg protein. The isozymes have similar pH optima for the forward reaction, but different optima for the reverse reaction. The divalent metal specificity is the same for both isozymes. In addition to differences in charge, the isozymes can be distinguished by their different kinetic constants, thermostability and sensitivity to fluoride inhibition. Antibodies against yeast enolase isozyme I cross-react with Ricinus plastid enolase but not with the cytosolic isozyme.     

In Vitro Inhibition of the Plastid and Cytosolic Isozymess of 6-Phosphogluconate Dehydrogenase from Developing Endosperm of Ricinus communis by Fructose 2,6-bisphosphate

Activities of the cytosolic and plastid isozymes of 6-phosphogluconate dehydrogenase from developing endosperm of Ricinus communis L. seeds were inhibited in vitro by hexosebisphosphates. Inhibition constants for glucose 1,6-bisphosphate were 221 and 209 micromolar for the cytosolic and plastid isozymes, respectively, and corresponding values for fructose 2,6-bisphosphate were 10.5 and 8.6 micromolar. In each case inhibition was of a mixed noncompetitive nature relative to 6-phosphogluconate. While the levels and distribution of fructose 2,6-bisphosphate in castor oil seed endosperm cells are not yet known, the levels reported to occur in leaf cytosol would be high enough to significantly inhibit carbon flux through the pentosephosphate pathway due to inhibition of 6-phosphogluconate dehydrogenase activity.     

Formation and regeneration ofHalobacterium spheroplasts

Spheroplasts ofHalobacterium cutirubrum were formed upon suspension of cell pellets in 0.1M MES buffer, pH 7.0, containing 0.5M sucrose, 0.25M NaCl, and 0.01M MgCl₂. The spheroplasts regenerated into rod-shaped bacteria when plated on a complex medium containing 15% (wt/vol) sucrose, undergoing several divisions as spherical bodies before the rod shape developed. The frequency of regeneration was approximately 5% of the total spheroplasts plated. The yield of regenerants was increased significantly (to approximately 35%) when bovine serum albumin was present in the spheroplasting buffer and dilution media. The conditions for spheroplast formation and regeneration inH. cutirubrum were also found effective forHalobacterium salinarium but not forHalobacterium halobium.NRCC Paper no. 23080.     

Energetic basis of development of salt-tolerant transport in a moderately halophilic bacterium,Vibrio costicola

Active transport of -aminoisobutyric acid (AIB) in Vibrio costicola utilizes a system with affinity for glycine, alanine and, to some extent, methionine. AIB transport was more tolerant of high salt concentrations (3–4 M NaCl) in cells grown in the presence of 1.0 M NaCl than in those grown in the presence of 0.5 M NaCl. The former cells could also maintain much higher ATP contents than the latter in high salt concentrations.Transport kinetic studies performed with bacteria grown in 1.0 M NaCl revealed three effects of the Na⁺ ion: the first effect is to increase the apparent affinity (K _t) of the transport system for AIB at Na⁺ concentrations <0.2 M, the second to increase the maximum velocity (V _max) of transport (Na⁺ concentrations between 0.2 and 1.0 M), and the third to decrease the V _max without affectig K _t (Na⁺ concentrations >1.0 M). Cells grown in the presence of 0.5 M or 1.0 M NaCl had similar affinity for AIV. Thus, the differences in salt response of transport in these cells do not seem due to differences in AIB binding. Large, transport-inhibitory concentrations of NaCl resulted in efflux of AIB from cells preloaded in 0.5 M or 1.0 M NaCl, with most dramatic efflux occurring from the cells whose AIB transport was more salt-sensitive. Our results suggest that the degree to which high salt concentrations affect the transmembrane electrochemical energy source used for transport and ATP synthesis is an important determinant of salt tolerance.Abbreviations AIB -aminoisobutyric acid - pmf proton motive force     

Changes in the metabolism of the proteoglycans from sheep articular cartilage in response to mechanical stress

The effect of altered mechanical stress on the metabolism of sheep articular cartilage has been investigated. A simple experimental model involving the immobilisation of a single sheep foreleg was used to study the effect of increased or decreased functional demand on the chemical composition of, and the incorporation of labelled acetate into, the proteoglycans of sheep articular cartilage. By immobilisation of one of the sheep forelegs, mechanical stress is removed from that particular joint, while increased stress is placed on the other foreleg. The load distribution about the two hind legs remains essentially the same. After a 4-week immobilisation period there was a significant increase in the hexuronic acid content of the cartilage from the loadbearing ankle joint, and a corresponding decrease in the hexuronic acid content of the non-loadbearing joint cartilage. Hexosamine analyses of the cartilage from each joint showed that the major chemical occurred in the chondroitin sulphate fraction. From analyses of the extracted and isolated proteoglycans from each experimental joint it was evident that there was a significant decrease in the molecular weight of the proteoglycan from the non-loadbearing joint. In vitro studies showed increased incorporation of labelled acetate into the chondroitin sulphate fraction from the loadbearing joint but a corresponding decreased incorporation into the non-loadbearing immobilised joint cartilage. These results suggest that the changes observed in the chemical composition of the cartilage from the loadbearing and non-loadbearing joints may be accounted for in part by changes in the biosynthesis of the cartilage proteoglycan in response to altered functional demand.     

The modulation of membrane fluidity by hydrogenation processes. III. The hydrogenation of biomembranes of spinach chloroplasts and a study of the effect of this on photosynthetic electron transport

A method is reported for the in situ modification of the lipids of isolated spinach chloroplast membranes. The technique is based on a direct hydrogenation of the lipid double bonds in the presence of the catalyst, chlorotris(triphenylphosphine)rhodium (I). The pattern of hydrogenation achieved suggests that the catalyst distributes amongst all of the membranes. The polyunsaturated lipids within the membranes are hydrogenated at a faster rate and at an earlier stage than are the monoenoic lipids.Whilst addition of the catalyst to the chloroplast causes an initial 10–20% decrease in Hill activity, saturation of up to 40% of the double bonds present can be accomplished without causing further significant alterations in photosynthetic electron transport processes or marked morphological changes of the chloroplast structure as observed in the electron microscope.     

Isozymes of the glycolytic and pentose-phosphate pathways in storage tissues of different oilseeds

Isozymes of hexose-phosphate isomerase (HPI; EC 5.3.1.9), pyruvate kinase (PK; EC 2.7.1.40) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) have been detected in the developing cotyledons of soybean (Glycine max (L.) Merr.), safflower (Carthamnus tinctorius L.) and sunflower (Helianthus annuus L.). In each seed there are two isozymes each of PK and HPI. The isozyme patterns of 6PGDH are more complex: soybean has two forms of the enzyme, safflower three, and sunflower six. In each tissue, at least 25% of the activity of each of the three enzymes is in the plastids. This supports the proposal that the glycolytic and pentose-phosphate pathways are operating in the plastids and that the plastids are the site of long-chain fatty-acid biosynthesis in developing oilseeds.Abbreviations HPI hexose-phosphate isomerase - 6PGDH 6-phosphogluconate dehydrogenase - PK pyruvate kinase     

Species differences in biotransformation of 17α-cyanomethylestradiol 3-methyl ether

Following oral administration of 9,11- ³H-17α-cyano-methylestra-1,3,5(10)-triene-3,17-diol 3-methyl ether, urinary metabolites were studied in man, baboon, beagle dog, minipig and rat. The metabolite pattern revealed remarkable species differences, especially in quantitative respects. 17α-Cyanomethylestra-1,3,5(10)-triene-3,17-diol, 17α-cyanomethylestra-1,3,5(10)-triene-2,3,17-triol 2-methyl ether, 17α-hydroxymethylestra-1,3,5(10)-triene-3,17-diol and 17α-cyanomethylestra-1,3,5(10)-triene-3,16$\text{6}\text{5}$,17-triol were isolated as principal metabolites. In rat bile, a metabolite was tentatively identified as aγ-lactone of a 17α-carbozymethyl-16α-hydroxy compound.     

Protease-nexin: A cellular component that links thrombin and plasminogen activator and mediates their binding to cells

This report identifies a component of normal human fibroblasts that forms a covalent linkage with thrombin and urokinase (urinary plasminogen activator) and mediates most of the specific cellular binding of these proteases. This component, here named protease-nexin (PN), is both associated with the cell surface and released into the culture medium. In several ways PN resembles antithrombin III (AT3), a prominent inhibitor of thrombin in serum: PN links thrombin, probably via an ester bond; PN does not link thrombin blocked at its catalytic site serine; PN has a high-affinity heparin-binding site; and heparin greatly accelerates the rate of linkage between soluble PN and thrombin. Despite these similarities, PN and AT3 are distinct; they differ in size and are not immunologically cross-reactive. Whereas AT3 regulates the proteolytic activity of thrombin in serum, PN may regulate the activity of serine proteases at and near the cell surface.