Large-scale purification of bovine brain lactate dehydrogenase by affinity chromatography on immobilized colchicine

Lactate dehydrogenase (LDH) [EC 1.1.1.27] in a crude extract (40-80% ammonium sulfate fraction) of bovine brain was adsorbed on an immobilized colchicine column and specifically eluted by addition of 1 mM NADH. The purity and subunit composition of the pooled LDH were estimated by two-dimensional gel electrophoresis. With an increase of NaCl concentration from 0 to 2.0 M, ligand saturation of LDH on immobilized colchicine increased from 6.8 to 14%, whereas that on immobilized Cibacron blue F3GA decreased from 2.1 to 0%. In the presence of high NaCl concentration, immobilized colchicine enabled both large- and small-scale purification of LDH by affinity chromatography and resulted in a yield of 117 mg from 1 kg of bovine brain in the presence of 2.5 M NaCl or higher recoveries of 54-96% from various tissues of one rat in the presence of 1.0 M NaCl. These results indicate that immobilized colchicine is an excellent adsorbent for the isolation and purification of LDH by affinity chromatography and has a high LDH-adsorbing capacity dependent upon a high NaCl concentration. Kinetic studies revealed that colchicine apparently competed with cofactor NAD for the active site of LDH and the Ki values of colchicine decreased with an increase of NaCl concentration. The chemical specificity of the colchicine-binding site of LDH was studied by the use of colchicine analogues and it is concluded that both the tropolone moiety (C-ring) and the amido bond in a side chain of colchicine structure are essential to the colchicine-LDH interaction.     

Effect of a light-induced pH gradient on purple-to-blue and purple-to-red transitions of bacteriorhodopsin

Bacteriorhodopsin-containing vesicles that were able to alkalize the extravesicular medium by greater than 1.5 pH units under illumination, i.e., inside-out vesicles, were reconstituted by reverse-phase evaporation with Halobacterium halobium polar lipids or exogenous phospholipids. Acid titration of a dark-adapted sample was accompanied by a color change from purple to blue (pKa = 2.5-4.5 in 0.15 M K2SO4), and alkali titration resulted in the formation of a red species absorbing maximally at 480 nm (pKa = 7 to greater than 9), the pKa values and the extents of these color changes being dependent on the nature of lipid. When a vesicle suspension at neutral or weakly acidic pH was irradiated by continuous light so that a large pH gradient was generated across the membrane, either a purple-to-blue or a purple-to-red transition took place. The light-induced purple-to-red transition was significant in an unbuffered vesicle suspension and correlated with the pH change in the extravesicular medium. The result suggests that the purple-to-red transition is driven from the extravesicular side, i.e., from the C-terminal membrane surface. In the presence of buffer molecules outside, the dominant color change induced in the light was the purple-to-blue transition, which seemed to be due to a large decrease in the intravesicular pH. But an apparently inconsistent result was obtained when the extravesicular medium was acidified by a HCl pulse, which was accompanied by a rapid color change to blue. We arrived at the following explanation: The two bR isomers, one containing all-trans-retinal and the other 13-cis-retinal, respond differently to pH changes in the extravesicular and the intravesicular medium. In this relation, full light adaptation was not achieved when the light-induced purple-to-blue transition was significant; i.e., only the 13-cis isomer is likely to respond to a pH change at the N-terminal membrane surface.     

Ether lysophospholipid-induced production of platelet-activating factor in human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMN) produced considerable amounts of platelet-activating factor (PAF) when exposed to various concentrations of lyso-PAF, especially in the absence of albumin. The amount of produced PAF in the presence of 5 microM lyso-PAF (without albumin) was 1.1 pmol/10 min per 2.5 X 10(6) cells, which was close to the level in the case of opsonized zymosan stimulation. We found that the activity of neither acetyltransferase nor acetylhydrolase was affected markedly by the treatment of cells with lyso-PAF, suggesting that the increased availability of lyso-PAF could be responsible for the induction of PAF synthesis. We also found that PAF synthesis was induced not only by lyso-PAF but also by ether-containing ethanolamine lysophospholipids, 1-alkenyl(alkyl)-sn-glycero-3-phosphoethanolamine (GPE). The addition of 1-alkenyl(alkyl)-GPE caused the degradation of pre-existing 1-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (GPC) and an increased level of lyso-PAF, followed by the formation of PAF. By contrast, 1-acyl-GPC and 1-acyl-GPE failed to induce PAF production. These results suggest a possible key role of the availability of lyso-PAF in triggering the biosynthesis of PAF in human PMN.     

Birth of normal calves resulting from bovine oocytes matured, fertilized, and cultured with cumulus cells in vitro up to the blastocyst stage

Bovine oocytes matured in vitro were fertilized in high proportions (92% of matured oocytes) by sperm capacitated with Ca ionophore A23187. Eight percent of inseminated oocytes that were denuded 96 h after insemination developed to the morula stage when cultured for 6-120 h after insemination with cumulus cells from the original oocytes. Inseminated oocytes denuded 96 h after insemination developed to the blastocyst stage when cultured with or without cumulus cells or in the conditioned medium from 96 h to 168-216 h after insemination (9.0%, 8.1%, and 6.8% of inseminated oocytes respectively). Six frozen-thawed blastocysts were transferred nonsurgically to 3 recipients (2 embryos/recipient). Two of the 3 recipients became pregnant, with one delivering live twins at term. Seven fresh blastocysts were transferred nonsurgically to 6 recipients (1-2 embryos/recipient). Three of the 6 recipients became pregnant, with 2 delivering live calves.     

Hormone and DNA binding activity of a purified human thyroid hormone nuclear receptor expressed in Escherichia coli

Using a T7 expression system, large amounts of the human placental c-erbA protein (h-TR beta 1) were expressed. From 1 liter of Escherichia coli culture, approximately 50-100 micrograms of purified h-TR beta 1 were obtained. Analysis of the binding data indicated that the purified h-TR beta 1 binds to 3,3',5-triiodo-L-thyronine (T3) with a Ka = 2.8 x 10(9) M-1. It binds to 3,3',5-triiodo-L-thyropropionic acid, 3,3',5-triiodo-L-thyroacetic acid, D-T3, L-thyroxine (T4), and 3',5',3-triiodo-L-thyronine with 475, 120, 39, 7, and 0.1%, respectively, of the activity of L-T3. This order of binding activity to T3 analogs is similar to that reported for the T3 nuclear receptor identified in tissues or cultured cells. Furthermore, the purified h-TR beta 1 binds to the T3 response element of the rat growth hormone gene. Thus, the purified h-TR beta 1 is active. To identify the hormone binding domain, the purified h-TR beta 1 was affinity labeled with underivatized [3',5'-125I]T4. A partial digestion by trypsin yielded a 125I-labeled 25-kDa fragment which was identified to be the domain Phe240-Asp456 by amino acid sequencing. Thus, the purified h-TR beta 1 appears suitable for other structural and functional studies.     

Preventive effect of angiotensin I on weight reduction in the adrenal glands of DOCA/salt hypertensive rats

We examined the effect of angiotensin I (AI), without the effect of angiotensin II (AII) converted from AI, on the weight of the adrenal glands, adrenal corticosterone (B) and adrenal aldosterone under conditions where the renin-angiotensin system was suppressed, since a reduction in the size of the adrenal glands is often observed in DOCA/salt hypertensive rats. Sixty male Wistar rats fed on a 1% NaCl solution were divided into 6 groups as follows: a) Salt group: received sesame oil and vehicle, b) Salt + C group: received sesame oil and MK422 (0.14 mg/day), an angiotensin converting enzyme inhibitor (CEI), c) DOCA group: received DOCA (30 mg/week) and vehicle, d) DOCA + A group: received DOCA and AI (0.5 mg/kg/day), e) DOCA + A + C group: received DOCA and AI with MK422, and f) DOCA + C group: received DOCA and MK422. After 4 weeks, the rats were sacrificed to sample their blood and remove their adrenal glands. There was no significant difference in adrenal B among the groups apart from the DOCA + C group. Adrenal aldosterone was lower in the groups of DOCA/salt hypertensive rats than in the Salt group and Salt + C group. Furthermore, the DOCA + A + C group and DOCA + C group had lower adrenal aldosterone levels than the DOCA group and DOCA + A group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Controlling mechanism of ATP regeneration by glycolytic pathway in a yeast: Hansenula jadinii

Summary The regulatory mechanism of ATP regeneration by the glycolytic pathway in Hansenula jadinii cells was investigated by analyzing the initial stage of CDP-choline fermentation. As a result, the on-off of ATP regeneration was found to be determined by the ATP concentration overcoming the inhibitory effect of phosphate buffer on hexokinase activity. The concentration of ATP at the initial stage of fermentation was greatly influenced by the kinds and amounts of glycogen in cells. Based on these results, the regulatory mechanism of ATP regeneration by the glycolytic pathway is discussed in detail.     

Age-dependent modifications of lipogenic enzymes

When 1-, 2- and 9-month-old rats previously adapted to a commercial stock diet were fed a fat-free diet (induction) and also when the process was reversed (repression), the turnovers of lipogenic enzymes in liver were measured by following time courses for the change of the enzyme activities. The magnitudes of the induction of glucose-6-phosphate dehydrogenase, malic enzyme and acetyl-CoA carboxylase were very high in 1-month-old rats and then decreased with aging. In the induction phase, the rates of synthesis of the enzymes were markedly decreased with increasing age as compared with the rate constants of degradation. The age-dependent alterations were not shown so much in the repression phase as in the induction phase. It is suggested that the age-dependent impairment in induction may be due to some alterations in the enzyme-forming system.