Preparation of immobilized enzyme with high activity using affinity tag based on proteins A and G

Affinity tag AG consisting of immunoglobulin G (lgG)-binding domains of protein A from Staphylococcus aureus (EDABC) and those of protein G from Streptococcus strain G148 (C2C3) were used to facilitate immobilization of beta-galactosidase (betagal) from Escherichia coli. Poly(methylmethacrylate/N-isopropylacrylamide/methacrylic acid) [P(MMA/NIPAM/MAA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] latex particles, which show thermosensitivity, were used as support materals to prepare affinity adsorbents. Human gamma-globulin (HgammaGb), whose major fraction is lgG, was used as an affinity ligand and was covalently immobilized onto the both latex particles by the carbodiimide method under various conditions. A fusion protein, AGbetagal, was immobilized at pH 7.3 by the specific binding of affinity tag to these affinity adsorbents. The amount of adsorbed AGbetagal per unit amount of immobilized HgammaGb, namely, efficiency of ligand utilization, was strongly affected by the type of latex particles and pH value for HgammaGb immobilization. The efficiency of ligand utilization was maximum in the affinity adsorbents prepared at pH 6.0 to 7.0, and that in the HgammaGb-P(MMA/NIPAM/MAA) latex particles was high. This result could be explained by the conformation and orientation of immobilized HgammaGb molecules. Immobilized AGbetagal retained approximately 75% of its activity in solution and the binding is stable enough to allow repeated use. These results clearly demonstrate that combination of the affinity tag AG and the affinity adsorbents, based on the thermosensitive latex particles, offers a simple and widely applicable method for preparation of immobilized enzyme with high activity. (c) 1995 John Wiley & Sons, Inc.     

Cell Swelling Activates Phospholipase A2 in Ehrlich Ascites Tumor Cells

Ehrlich ascites tumor cells, loaded with ³H-labeled arachidonic acid and ¹⁴C-labeled stearic acid for two hours, were washed and transferred to either isotonic or hypotonic media containing BSA to scavenge the labeled fatty acids released from the cells. During the first two minutes of hypo-osmotic exposure the rate of ³H-labeled arachidonic acid release is 3.3 times higher than that observed at normal osmolality. Cell swelling also causes an increase in the production of ¹⁴C-stearic acid-labeled lysophosphatidylcholine. This indicates that a phospholipase A₂ is activated by cell swelling in the Ehrlich cells. Within the same time frame there is no swelling-induced increase in ¹⁴C-labeled stearic acid release nor in the synthesis of phosphatidyl ¹⁴C-butanol in the presence of ¹⁴C-butanol. Furthermore, U7312, an inhibitor of phospholipase C, does not affect the swelling induced release of ¹⁴C-labeled arachidonic acid. Taken together these results exclude involvement of phospholipase A₁, C and D in the swelling-induced liberation of arachidonic acid. The swelling-induced release of ³H-labeled arachidonic acid from Ehrlich cells as well as the volume regulatory response are inhibited after preincubation with GDP_βS or with AACOCF₃, an inhibitor of the 85 kDa, cytosolic phospholipase A₂. Based on these results we propose that cell swelling activates a phospholipase A₂—perhaps the cytosolic 85 kDa type—by a partly G-protein coupled process, and that this activation is essential for the subsequent volume regulatory response. Received: 23 July 1996/Revised: 17 June 1997     

Acetyl coenzyme A biosynthesis in the chloroplast

Acetyl coenzyme A (CoA) biosynthesis in spinach chloroplasts has been investigated by following the incorporation of bicarbonate and acetate into fatty acids under a variety of conditions. Both substrates were readily incorporated into fatty acids in a light-dependent manner by intact photosynthesising chloroplasts, but when the concentrations of these substrates were adjusted to those found in vivo, i.e. 200 M acetate, 10 M bicarbonate, then acetate was found to supply carbon atoms for fatty acids biosynthesis via acetyl CoA at forty times the rate of bicarbonate. It is proposed that extra-chloroplastic free acetate is the pricipal substrate for chloroplasts acetyl CoA biosynthesis in spinach.Abbreviations ACP acyl carrierprotein - CoASH coenzyme A     

A spectroscopic study of metal ion and ligand binding to β-lactamase II

β-Lactamase II has two metal-binding sites. The electronic spectra of Cd(II)- and Co(II)-substituted β-lactamase II have been investigated. It is suggested that a thiol ligand is involved in metal binding at the first site. The stoichiometric dissociation constants for Co(II) binding to β-lactamase II were estimated to be 0.13 and 2.66 mM (pH 6.0, 4°C, 1 M NaCl) by equilibrium dialysis. Competition between Zn(II) and Co(II) for the first metal binding site suggests a value of 0.7 μM (pH 6.0, 30°C, 1 M NaCl) for the dissociation constant o Zn(II).The electronic spectra of the Co(II) enzyme lead to the suggestion that the coordination geometries around the metal ions in the first and second sites are related to those of a distorted tetrahedron and octahedron, respectively.     

Effects of weaning age on mortality of colony-born Macaca nemestrina

In an effort to determine empirically the proper age for separation of pigtailed macaque (Macaca nemestrina ) infants from their dams in a large domestic breeding colony, we conducted a retrospective survey of 1,592 infants. Survivorship was highest for infants not separated from their dams at all and for those separated within the first four months of life. Survivorship was poor for animals separated during or after the fifth month. Thus, not weaning or weaning early are the most acceptable management strategies in terms of mortality risk. This result is paradoxical with regard to nutritional considerations, but appears to be related to a sensitive period in social development. The relative advantages and disadvantages of various weaning ages are discussed.     

Computer simulation of engulfment and other movements of embryonic tissues

Using a model proposed earlier by Goel et al. and a set of plausible motility rules, a computer simulation of engulfment of two or more intact embryonic tissues is successfully carried out. The same motility rules are used to simulate the rounding up of a tissue, centralization of one tissue within another tissue (a phenomenon not yet observed), and phase inversion, a process which may have relevance to differentiation. The finnal structures bear a good resemblance to those observed experimentally. The software, in conjunction with an appropriate hardware configuration, allows a visual display of the dynamics of cellular movement. These simulations indicate that the range of inter-cellular interactions controlling these tissue rearrangements extends only one or two cell diameters.     

Action of alkyl cations and the natural ATPase inhibitor from mitochondria on soluble mitochondrial ATPase

The effect of the natural ATPase inhibitor and octylguanidine on the ATPase activity of soluble oligomycin-insensitive mitochondrial F₁ were compared. Both compounds induced a maximal inhibition of 60–80% in various preparations of F₁ studied. The inhibition was of the uncompetitive type with respect to MgATP, and the action of the compounds was partially additive. The data suggest that octylguanidine reproduces the action of the natural ATPase inhibitor. Alkylammonium salts also affect the ATPase activity in a similar form. F₁ bound to Sepharose-hexylammonium is largely inactive, whilst free hexylammonium at higher concentrations induces only a partial inhibition of the activity. This suggests that the degree of immobilization of F₁ is related to the magnitude of inhibition of ATPase activity induced by alkyl cations. The binding of F₁ to Sepharose-hexylammonium is prevented by high concentrations of Na⁺ or K⁺.