Human lymphocyte tubulin: Purification and characterization in normal and leukemic cells

Tubulin has been purified from human blood and tonsil lymphocytes. Using gel filtration, the molecular weight of human lymphocyte tubulin was estimated to be 119 000. The proteins was shown to consist of two subunits, with molecular weights of 61 000 and 58 000 comparable to the α and β polypeptides of human brain tubulin. A partial identity reaction was observed between lymphocyte tubulin and human tubulin when tested by double immunodiffusion against a rabbit anti-human brain tubulin antibody. In the presence of GTP, the purified protein polymerized to form microtubules. Tubulin was localized to the cell's juxtacentriolar region by immunofluorescence and electron microscopy. When assayed by a colchicine-binding assay corrected for time decay, the binding affinity was 1.50 ± 0.86 · 10⁶M^?1 and a level in normal lymphocytes of 1.21 · 10^?2 ± 0.79 g/g of soluble protein was determined. Since chronic lymphocytic leukemia lymphocytes have an anomalous capping behavior as well as an unusual susceptibility to colchicine toxicity, the properties and levels of tubulin were determined in these cells. Similar values were obtained for the level, decay rate, molecular weight, and K_a for colchicine as for normal lymphocytes. Chronic lymphocytic leukemia lymphocyte tubulin polymerized in a normal fashion. It thus appears that a decrease in the quantity or function of tubulin does not account for these anomalies in the chronic lymphocytic leukemia lymphocyte.     

The role of the medium in long-range electron transfer

 The role of the polypeptide matrix in electron transfer processes in proteins has been studied in two distinct systems: first in a protein where the induced ET is artificial, and second as part of the catalytic cycle of an enzyme. Azurins are structurally well-characterized blue single-copper proteins consisting of a rigid β-sheet polypeptide matrix. We have determined rate constants and activation parameters for intramolecular long-range electron transfer between the disulfide radical anions (generated by pulse radiolysis) and the copper(II) centre as a function of driving force and nature of the intervening medium in a large number of wild-type and single-site-mutated proteins. In ascorbate oxidase, for which the three-dimensional structure is equally well characterized, the internal ET from the type-I Cu(I) to the trinuclear Cu(II) centre has been studied. We find that the results correlate well with distance through well-defined pathways using a through-bond electron tunnelling mechanism. Received: 2 January 1997 / Accepted: 6 February 1997     

Isolation, partial chemical and biological characterization of thymone B

A new approach to the study of the molecular arrangements of proteins in membranes is described. Irradiation with visible light of native erythrocytes or washed erythrocyte membranes suspended in buffers containing a) riboflavin, fluorescein or fluorescein coupled to dextran and b) ³H-labelled tryptophan resulted in incorporation of radioactivity into the membrane proteins. Polyacrylamide gel electrophoresis of solubilized membranes followed by radioactivity measurements of the separated membrane proteins revealed that in native erythrocytes the protein components known to be located at the exterior cell surface, Band 3 and the major sialoglycoproteins became specifically labelled, whereas in washed lysed cells all of the major membrane proteins were labelled.     

Purification and characterization of chick intestine brush border membrane Effects of 1α(OH) vitamin D3 treatment

A new technique has been developed for the isolation of membrane vesicles from the vitamin D-deficient and vitamin D-treated chick intestinal brush border membrane. The technique involves removal of nuclei from a low speed pellet by discontinuous sucrose gradient centrifugation. The resulting intact brush borders are then homogenized in 0.5 M Tris and the membrane fragments purified on a glycerol gradient. This preparation represents a 20-fold purification of the brush border marker sucrase. After 1α-hydroxyvitamin D₃ treatment there is a significant increase in membrane phospholipid phosphorous, an alteration in the fatty acid composition of the phosphatidylcholine fraction of membrane phospholipid, and a decrease in sucrase specific activity.     

Electrophoretic profiles of cyanobacterial membrane polypeptides showing heme-dependent peroxidase activity

The photosynthetic membranes of Anacystis nidulans R2 were examined electrophoretically following solubilization with lithium dodecyl sulfate. Electrophoresis yielded six prominent chlorophyll-containing bands. In addition, five polypeptides were observed which possessed heme-dependent peroxidase activity, monitored by incubating gels with 3,3′,5,5′-tetramethylbenzidine plus hydrogen peroxide. One such polypeptide, at 105 kdaltons, was removed by repeated washing of the membranes. Four remaining peroxidase-active polypeptides were observed at 7.2, 13.5, 18.5 and 33 kdaltons. Further examination of these four polypeptides yielded the following results. (1) The mobility of the 33 kdalton polypeptide was altered from 29 to 33 kdaltons upon heating (70°C) during membrane solubilization. (2) All four polypeptides showed stable heme-protein associations in the presence of 8 M urea; however, in the presence of urea, alterations in protein mobility were observed for each poly-peptide and only two (at 13.5 and 33 kdaltons) showed peroxidase activity following heating (70°C) during membrane solubilization. (3) The presence of thiols during membrane solubilization at 0°C was required to observe peroxidase activity at 7.2 kdaltons. These results, when compared to known properties of isolated cytochromes, suggest that the four polypeptides characterized here correspond to the subunits of photosynthetic cytochromes. Electrophoretic assessment of maize mutants lacking cytochrome f and b₆ activity supports this suggestion.     

Plant responses to H2S and SO2 fumigation. I. Effects on growth, transpiration and sulfur content of spinach

Exposure of spinach (Spinacia oleracea L. cv. Monosa) to 0.25 μl l_?1 H₂S reduced the relative growth rate by 26, 47 and 60% at 15, 18 and 25°C, respectively. Shoot to root ratio decreased in plants fumigated at 18 and 25°C. Growth of spinach was not affected by a 2-week exposure to 0.10 or 0.25 μl l_?1 SO₂. Both H₂S and SO₂ fumigation increased the content of sulfhydryl compounds and sulfate. A 2-week exposure to 0.25 μl l_?1 H₂S resulted in an increase in sulfhydryl and sulfate content of 250 to 450% and 63 to 248% in the shoots, respectively, depending on growth temperature. Exposure to 0.15 and 0.30 μl l_?1 H₂S at 20°C for 2 weeks resulted in a 46% increase in sulfate content of the shoots at 0.30 μl l_?1 and no detectable increase at 0.15 μl l_?1 H₂S; the sulfate content of the roots increased by 195 and 145% at 0.15 and 0.30 μl l_?1 H₂S, respectively. Fumigation with 0.25 μl l_?1 SO₂ at 20°C for 2 weeks resulted in an increase in sulfhydryl content and sulfate content in the shoots of 285% and 300 to 1100%. H₂S fumigation during the 12 h light period or only during the dark period resulted in identical growth reduction and accumulation of sulfhydryl compounds; they were about 50 and 67% of those observed in continuously exposed plants. H₂S- and SO₂-exposed plants showed an increased transpiration rate, which was mainly caused by an increased dark-period transpiration. No effect of H₂S and SO₂ on the water uptake of the plants and the osmotic potential of the leaves was detected. Plants fumigated with 0.25 μl l_?1 H₂S for 2 weeks were smaller and differed morphologically from the control plants by slightly more abaxially curved leaf margins. Cross sections of the leaves showed smaller cells at the margins and smaller and fewer air spaces. The increased transpiration in the H₂S-exposed plants is discussed in relation to the observed morphological changes.     

The effect of pretransplant inoculation with VA mycorrhizal fungi on the subsequent growth of leeks in the field

Summary Leek plants were preinoculated with a mixed inoculum ofGlomus caledonium, Glomus fasciculatum andGlomus sp., and transplanted to Dazomet disinfected and untreated field plots of moderate P deficiency. Successive harvests were made until 99 days after transplanting. Preinoculated leeks attained marketable weights 25 days earlier than uninoculated leeks from untreated soil and their final dry matter yields were 5.7 and 1.5 times as high as those of uninoculated leeks from disinfected and untreated soil, respectively. Phosphorus concentration in preinoculated leeks remained highest for at least 22 and 75 days after transplanting in untreated and disinfected soil, respectively. Preinoculation had a similar, although smaller, influence on Cu and Zn concentrations. Infection levels produced by introduced and indigenous VA endophytes in leeks reached plateaus of 90% and 40%, respectively, 47 days after transplanting. It is concluded that VAM is essential to leeks grown in moderately P deficient soils, and the potential for inoculating seedlings in commercial leek production is discussed.     

Investigations on the mechanism of hypercholesterolemia observed in copper deficiency in rats

The mechanism of hypercholesterolemia effect of Cu²⁺ deficiency was studied in rats. There was increased activity of hepatic hydroxymethylglutaryl-coenzyme A reductase and increased incorporation of labelled acetate into free cholesterol of liver in the Cu²⁺ deficient rats. Incorporation of label into ester cholesterol was however decreased in the liver. Concentration of bile acids in the liver was not significantly altered. Increase in the incorporation of labelled acetate into serum cholesterol and increase in the concentration of cholesterol and apo B in the low density lipoproteins + very low density lipoproteins fractions were observed. Activity of lipoprotein lipase of the extrahepatic tissues decreased in the Cu²⁺ deficient rats.