Renaturation of citrate synthase: influence of denaturant and folding assistants.

Citrate synthase (CS), which has been denatured in either guanidine hydrochloride (GdnHCl) or urea can be assisted in its renaturation in a variety of ways. The addition of each of the assistants--bovine serum albumin (BSA), oxaloacetate (OAA), and glycerol--promotes renaturation. In combination, the effect of these substances is additive with respect to the yield of folded CS. The report of Buchner et al. (Buchner, J., Schmidt, M., Fuchs, M., Jaenicke, R., Rudolph, R., Schmid, F.X., & Kiefhaber, T., 1991, Biochemistry 30, 1586-1591) that refolding of CS is facilitated by the GroE system (an Escherichia coli chaperonin [cpn] that is composed of GroEL [cpn60] and GroES [cpn10]) has been confirmed. However, we observed substantially higher yield of reactivated CS, 82%, and almost no reactivation in the absence of GroES, < 5%, whereas Buchner et al. reported 28% and 16%, respectively. In addition, we find that GroE-assisted refolding is more efficient for CS denatured in GdnHCl than for CS denatured in urea. This result is discussed in light of the known difference in the denatured states generated in GdnHCl and urea. Because GroEL inhibits the BSA/glycerol/OAA-assisted refolding, this system will be useful in future studies on the mechanism of GroE-facilitated refolding.     

Carnitine acetyltransferase activity in oleaginous yeasts

Abstract The highest activities of carnitine acetyltransferase (CAT) were found in non-oleaginous yeasts ( Candida utilis and Saccharomyces cerevisiae ); lower activities, ranging from 50% down to 3% of the highest values, were found in various strains of oleaginous yeasts ( Candida curvata, Lipomyces starkeyi, Rhodosporidium toruloides and Trichosporon cutaneum ). Supply of acetyl units into the cytosol of the latter, but not of the former yeasts, was therefore necessarily reliant on the action of ATP: citrate lyase (ACL), which was present in all oleaginous yeasts. There was no correlation, however, between the amount of lipid in the oleaginous yeasts and the specific activities of either CAT or ACL. Activity of CAT was increased up to 30-fold by growing yeasts on a triacylglycerol.     

Negatively Charged Lipids Are Essential for Functional and Structural Switch of Human 2-Cys Peroxiredoxin II

The function of ubiquitous 2-Cys peroxiredoxins (Prxs) can be converted alternatively from peroxidases to molecular chaperones. This conversion has been reported to occur by the formation of high-molecular-weight (HMW) complexes upon overoxidation of or ATP/ADP binding to 2-Cys Prxs, but its mechanism is not well understood. Here, we show that upon binding to phosphatidylserine or phosphatidylglycerol dimeric human 2-Cys PrxII (hPrxII) is assembled to trefoil-shaped small oligomers (possibly hexamers) with full chaperone and null peroxidase activities. Spherical HMW complexes are formed, only when phosphatidylserine or phosphatidylglycerol is bound to overoxidized or ATP/ADP-bound hPrxII. The spherical HMW complexes are lipid vesicles covered with trefoil-shaped oligomers arranged in a hexagonal lattice pattern. Thus, these lipids with a net negative charge, which can be supplied by increased membrane trafficking under oxidative stress, are essential for the structural and functional switch of hPrxII and possibly most 2-Cys Prxs.     

Coupling of the polyamine and iron metabolism pathways in the regulation of proliferation: Mechanistic links to alterations in key polyamine biosynthetic and catabolic enzymes

Many biological processes result from the coupling of metabolic pathways. Considering this, proliferation depends on adequate iron and polyamines, and although iron-depletion impairs proliferation, the metabolic link between iron and polyamine metabolism has never been thoroughly investigated. This is important to decipher, as many disease states demonstrate co-dysregulation of iron and polyamine metabolism. Herein, for the first time, we demonstrate that cellular iron levels robustly regulate 13 polyamine pathway proteins. Seven of these were regulated in a conserved manner by iron-depletion across different cell-types, with four proteins being down-regulated (i.e., acireductone dioxygenase 1 [ADI1], methionine adenosyltransferase 2α [MAT2α], Antizyme and polyamine oxidase [PAOX]) and three proteins being up-regulated (i.e., S-adenosyl methionine decarboxylase [AMD1], Antizyme inhibitor 1 [AZIN1] and spermidine/spermine-N¹-acetyltransferase 1 [SAT1]). Depletion of iron also markedly decreased polyamine pools (i.e., spermidine and/or spermine, but not putrescine). Accordingly, iron-depletion also decreased S-adenosylmethionine that is essential for spermidine/spermine biosynthesis. Iron-depletion additionally reduced ³H-spermidine uptake in direct agreement with the lowered levels of the polyamine importer, SLC22A16. Regarding mechanism, the “reprogramming” of polyamine metabolism by iron-depletion is consistent with the down-regulation of ADI1 and MAT2α, and the up-regulation of SAT1. Moreover, changes in ADI1 (biosynthetic) and SAT1 (catabolic) partially depended on the iron-regulated changes in c-Myc and/or p53. The ability of iron chelators to inhibit proliferation was rescuable by putrescine and spermidine, and under some conditions by spermine. Collectively, iron and polyamine metabolism are intimately coupled, which has significant ramifications for understanding the integrated role of iron and polyamine metabolism in proliferation.     

Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line

A serum-free medium, WCM5, has been developed for the large scale propagation of CHO (Chinese hamster ovary) cells which express recombinant protein using dihydrofolate reductase as a selectable marker. WCM5 was prepared by supplementing Iscoves medium without lecithin, albumin or transferrin with a number of components which were shown to benefit growth. WCM5 medium contained 5 mg l^–1 human recombinant insulin (Nucellin) but was otherwise protein-free. CHO 3D11^* cells which had been engineered to express a humanised antibody, CAMPATH^*-1H, were routinely grown using serum-containing medium. From a seeding density of 10⁵ cells ml^–1, cells grown in static culture with serum reached a maximal cell density of 6.5×10⁵ cells ml^–1 after 6 days in culture and produced a maximal antibody concentration of 69 mg l^–1 after 11 days in culture. CHO 3D11^* cells grown with serum were washed in serum-free medium then cultured in WCM5 medium. Following a period of adaptation the cell growth and product yield was superior to that achieved with serum-containing medium. CHO cells producing CAMPATH-1H grown in an 8000 l stirred bioreactor seeded with 2×10⁵ cells ml^–1 reached a maximal viable cell density of 2.16×10⁶ cells ml^–1 after 108 h in culture and a maximal antibody concentration of 131.1 mg l^–1 after 122 h in culture.Abbreviations CHO Chinese hamster ovary - dhfr dihydrofolate reductase - dhfr dihydrofolate reductase deficient - MTX methotrexate - H hypoxanthine - T thymidine - T/V trypsin versene - F12 Hams F12 medium - NEAA non essential amino acids     

Estrogenic and antiestrogenic properties of clomiphene citrate in laboratory mice

The estrogen agonistic and antagonistic properties of clomiphene citrate were investigated in the mice. Clomiphene citrate was tested at various doses of 0.1, 1.0, 10 and 100 μg for three consecutive days in immature and mature bilaterally ovariectomized mice. Clomiphene citrate showed uterotrophic activity in both immature and ovariectomized conditions. The lower doses of 0.1 and 1.0 μg were ineffective to show any uterotrophic stimulation. Clomiphene citrate at 10 μg dose produced 305.56% increase in uterine weight i.e., 27.70 ± 0.24 vs 6.83 ± 0.06 in immature and 182.27% i.e., 42.68 ± 1.12 vs 15.12 ± 0.57 in ovariectomized mice. Clomiphene citrate at 100 μg dose showed significant uterotrophic effect e.g., 435.57% i.e., 36.58 ±0.34 vs 6.83 ± 0.06 in immature and 586% i.e., 103.80 ± 0.60 in ovariectomized mice. When clomiphene citrate was administered in combination with 0.32 μg of estradiol 17-β it caused significant antagonistic effect (decrease in uterine weight) at 10 and 100 μg respectively. Clomiphene citrate at 10 μg dose produced 32% i.e., 28.93 ± 0.43 vs 38.04 ± 2.68 in immature and 35% i.e., 59.64±1.44 vs 83.34 ±0.25 in ovariectomized mice respectively. Histological observation clearly showed that clomiphene citrate at 10 and 100 μg doses did not cause any differential hypertrophy of the epithelial layer. Similar doses in combination with estradiol produced significant antagonistic effect on uterine weight and luminal epithelial cell height.     

Ligand effects on aluminium sorption by calcium pectate

Although soluble aluminium (Al) has long been recognised as an important limitation to plant growth on acid soils, the biochemical basis of Al toxicity has not been elucidated. Aluminium accumulation in the cell wall may be important, especially the reaction of Al with calcium (Ca) pectate. A study was conducted to investigate the effects of six ligands, citrate, malate, galacturonate, fluoride, sulfate and chloride, on the sorption of Al by Ca pectate prepared from two sources of pectin that differed in degree of methyl esterification (DE). The sorption of Al by Ca pectate increased linearly with increase in Al added from 25 to 100 µM (or 50 to 200 µM in the case of Al₂(SO₄)₃). There was a significant reduction in Al sorption in the presence of those ligands that form strong complexes with Al, especially citrate and, to a lesser extent, malate and fluoride. There was little difference in Al sorption by Ca pectate prepared from pectin of differing DE. Calcium in the supernatant solution increased linearly by 1.5 nmol for each 1 nmol increase in Al sorbed. The results support the hypothesis that strong complexes of Al with organic and inorganic ligands reduce Al sorption by Ca pectate in the cell wall.     

The absorption of sugars and organic acids by the daughter sporocysts of Microphallus similis (Jäg.)

The kinetics of uptake, accumulation against a concentration gradient, competitive inhibition and metabolic inhibition suggest that d-glucose enters the daughter sporocysts of Microphallus similis by active transport.Studies of competitive inhibition suggest that galactose, fructose, d-fucose, α-methyl-d-glucoside, d-mannose and 3-O-methylglucose are actively absorbed at the same locus as d-glucose. Glucosamine, d-ribose and l-fucose, however, may be actively transported at a different locus or may be taken up by simple diffusion.Acetate, pyruvate, citrate and succinate are absorbed by simple diffusion.     

Changes in mitochondrial ultrastructure induced by gramicidin plus protamine

The changes in isolated rat liver mitochondria induced by protamine or gramicidin and by protamine plus gramicidin have been investigated electron microscopically. The results are as follows:

1.

1. Inhibition of succinate (plus rotenone) oxidation induced by protamine caused mitochondrial shrinkage with collapse of outer and inner lamina of mitochondrial membrane and of cristae.