BBA - General Subjects (v.1524, #1)

Switching osmolyte strategies: response of Methanococcus thermolithotrophicus to changes in external NaCl by Deana D Martin; Rose A Ciulla; Patrice M Robinson; Mary F Roberts (1-10).
Methanococcus thermolithotrophicus, a thermophilic methanogenic archaeon, produces and accumulates β-glutamate and l-α-glutamate as osmolytes when grown in media with <1 M NaCl. When the organism is adapted to grow in >1 M NaCl, a new zwitterionic solute, N ϵ-acetyl-β-lysine, is synthesized and becomes the dominant osmolyte. Several techniques, including in vivo and in vitro NMR spectroscopy, HPLC analyses of ethanol extracts, and potassium atomic absorption, have been used to monitor the immediate response of M. thermolithotrophicus to osmotic stress. There is a temporal hierarchy in the response of intracellular osmolytes. Changes in intracellular K+ occur within the first few minutes of altering the external NaCl. Upon hypoosmotic shock, K+ is released from the cell; relatively small changes occur in the organic osmolyte pool on a longer time scale. Upon hyperosmotic shock, M. thermolithotrophicus immediately internalizes K+, far more than would be needed stoichiometrically to balance the new salt concentration. This is followed by a decrease to a new K+ concentration (over 10–15 min), at which point synthesis and accumulation of primarily l-α-glutamate occur. Once growth of the M. thermolithotrophicus culture begins, typically 30–100 min after the hyperosmotic shock, the intracellular levels of organic anions decrease and the zwitterion (N ϵ-acetyl-β-lysine) begins to represent a larger fraction of the intracellular pool. The observation that N ϵ-acetyl-β-lysine accumulation occurs in osmoadapted cells but not immediately after osmotic shock is consistent with the hypothesis that lysine 2,3-aminomutase, an enzyme involved in N ϵ-acetyl-β-lysine synthesis, is either not present at high levels or has low activity in cells grown and adapted to lower NaCl. That lysine aminomutase specific activity is 8-fold lower in protein extracts from cells adapted to low NaCl compared to those adapted to 1.4 M NaCl supports this hypothesis.
Keywords: Osmoregulation; Methanogen; β-Amino acid; Potassium ion; Osmolyte; Nuclear magnetic resonance spectroscopy; High-performance liquid chromatography;

Antifreeze proteins have been reported to be capable of maintaining the membrane integrity of cold sensitive mammalian cells when exposed to hypothermic temperatures. However the mechanism(s) whereby these proteins exert this protective effect is unknown. The present study used liposomes as a model system to examine the nature of the interactions between four antifreeze (glyco)protein types (AFP I, II, III and AFGP) and albumin, with lipid membranes. Fluorescein isothiocyanate labelling indicated that all of the proteins bound to the three liposome types (dielaidoylphosphatidylcholine (DEPC), dielaidoylphosphatidylethanolamine (DEPE) and dielaidoylphosphatidylglycerol (DEPG)). AFGP was found to be highly effective at preventing leakage from all three liposome compositions as they were cooled through their phase transition temperatures. This was not the case for the other proteins. All four antifreeze types prevented zwitterionic DEPC liposomes from leaking as they were cooled through their phase transition temperature. However, albumin was equally as effective, indicating that this capacity was not unique to antifreeze proteins. All of the proteins, except AFGP, induced the negatively charged DEPG liposomes to leak prior to cooling, and were less effective than AFGP in preventing phase transition leakage from DEPE liposomes. It is proposed that many proteins, including antifreeze proteins, can protect zwitterionic liposomes, such as DEPC, by binding to the lipid bilayer thereby maintaining the ordered structure of the membrane during phase transition. However, when the membrane contains a negatively charged polar group, such as with DEPE and DEPG, proteins, although bound to them, may not be able to maintain sufficient membrane organization to prevent leakage during phase transition or, they may gain entry into the lipid bilayer, disrupt the structure and induce leakage. These results imply that the efficacy of antifreeze proteins in the cold protection of mammalian cells will not only depend on protein structure, but also on the lipid composition of the cell membrane.
Keywords: Antifreeze protein/glycoprotein; Ica; Cell membrane; Liposome; Phospholipid; Structure/function relationship;

Bacterial peptidoglycan binds to tubulin by Roman Dziarski; Mark M. Rasenick; Dipika Gupta (17-26).
A search for cellular binding proteins for peptidoglycan (PGN), a CD14- and TLR2-dependent macrophage activator from Gram-positive bacteria, using PGN-affinity chromatography and N-terminal micro-sequencing, revealed that tubulin was a major PGN-binding protein in mouse macrophages. Tubulin also co-eluted with PGN from anti-PGN vancomycin affinity column and bound to PGN coupled to agarose. Tubulin-PGN binding was preferential under the conditions that promote tubulin polymerization, required macromolecular PGN, was competitively inhibited by soluble PGN and tubulin, did not require microtubule-associated proteins, and had an affinity of 100–150 nM. By contrast, binding of tubulin to lipopolysaccharide (LPS) had 2–3 times lower affinity, faster kinetics of binding, and showed positive cooperativity. PGN enhanced tubulin polymerization in the presence of 4 M glycerol, but in the absence of glycerol, both PGN and LPS decreased microtubule polymerization. These results indicate that tubulin is a major PGN-binding protein and that PGN modulates tubulin polymerization.
Keywords: Lipopolysaccharide; Microtubule; Peptidoglycan; Tubulin;

Purification and characterization of α-galactosidase from a thermophilic fungus Thermomyces lanuginosus by Vladimı&#x0301;r Puchart; Mária Vršanská; Mahalingeshwara K. Bhat; Peter Biely (27-37).
An extracellular α-galactosidase was purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of a mannanolytic strain of the thermophilic fungus Thermomyces lanuginosus. Molecular mass of the enzyme is 57 kDa. The pure enzyme which has a glycoprotein nature, afforded several forms on IEF, indicating its microheterogeneity. Isoelectric point of the major form was 5.2. Enzyme is the most active against aryl α-d-galactosides but efficiently hydrolyzed α-glycosidically linked non-reducing terminal galactopyranosyl residues occurring in natural substrates such as melibiose, raffinose, stachyose, and fragments of galactomannan. In addition, the enzyme is able to catalyze efficient degalactosylation of polymeric galactomannans leading to precipitation of the polymers. Stereochemical course of hydrolysis of two substrates, 4-nitrophenyl α-galactopyranoside and galactosyl1mannotriose, followed by 1H NMR spectroscopy, pointed out the α-anomer of d-galactose was the primary product of hydrolysis from which the β-anomer was formed by mutarotation. Hence the enzyme is a retaining glycosyl hydrolase. In accord with its retaining character the enzyme catalyzed transgalactosylation from 4-nitrophenyl α-galactopyranoside as a glycosyl donor. Amino acid sequence alignment of N-terminal and two internal sequences suggested that the enzyme is a member of family 27 of glycosyl hydrolases.
Keywords: α-Galactosidase; Retaining glycosyl hydrolase; Galactomannan; Thermophilic fungus; Thermomyces lanuginosus;

Acoustic field assisted enhanced demixing of aqueous two-phase systems by N.D Srinivas; R.S Barhate; K.S.M.S Raghavarao; Paul Todd (38-44).
Aqueous two-phase extraction has been recognized as a versatile downstream processing technique for the recovery of biomolecules. A major deterrent to its industrial exploitation is the slow demixing of the two aqueous phases after extraction, due to their similar physical properties. A method to decrease the demixing times of these systems, employing a travelling acoustic wave field, is reported. The effects of phase composition and microbial cells on demixing in a polyethylene glycol/potassium phosphate two-phase system are studied in detail. As phase composition increased, demixing time decreased gradually. Phase volume ratio was found to have a significant effect on demixing time at low phase compositions. However, at intermediate and high phase compositions, only a small effect on demixing time was observed. The effect of phase composition and volume ratio on demixing behavior was explained based on the droplet size of the dispersed phase, which is the resultant effect of the physical properties of the phases. At all the phase compositions studied, the acoustically assisted process decreased the demixing time by 17–60% when compared to demixing under gravity alone. Increasing the cell concentration increased the demixing time markedly in case of yeast cells. However, it remained practically constant in the case of Lactobacillus casei cells. Application of an acoustic field reduced the demixing times up to 60% and 40% in the case of yeast and L. casei cells, respectively. Visual observations indicated that ultrasonication caused mild circulation currents in the phase dispersion enhancing droplet–droplet interaction, which in turn enhanced the rate of coalescence, eventually resulting in an enhanced demixing rate.
Keywords: Aqueous two-phase extraction; Coalescence; Demixing time; Microbial cell; Acoustics;

Uracil salvage pathway in PC12 cells by Laura Mascia; Gino Turchi; Valentina Bemi; Piero Luigi Ipata (45-50).
The salvage anabolism of uracil to pyrimidine ribonucleosides and ribonucleotides was investigated in PC12 cells. Pyrimidine base phosphoribosyl transferase is absent in PC12 cells. As a consequence any uracil or cytosine salvage must be a 5-phosphoribosyl 1-pyrophosphate-independent process. When PC12 cell extracts were incubated with ribose 1-phosphate, ATP and uracil they can readily catalyze the synthesis of uracil nucleotides, through a salvage pathway in which the ribose moiety of ribose 1-phosphate is transferred to uracil via uridine phosphorylase (acting anabolically), with subsequent uridine phosphorylation. This pathway is similar to that previously described by us in rat liver and brain extracts (Cappiello et al., Biochim. Biophys. Acta 1425 (1998) 273; Mascia et al., Biochim. Biophys. Acta 1472 (1999) 93). We show using intact PC12 cells that they can readily take up uracil from the external medium. The analysis of intracellular metabolites reveals that uracil taken up is salvaged into uracil nucleotides, with uridine as an intermediate. We propose that the ribose 1-phosphate-dependent uracil salvage shown by our in vitro studies, using tissues or cellular extracts, might also be operative in intact cells. Our results must be taken into consideration for the comprehension of novel chemotherapeutics’ influence on pyrimidine neuronal metabolism.
Keywords: α-d-Ribose 1-phosphate; Pyrimidine salvage pathway; PC12 cell;

Detection of immunoreactive napsin A in human urine by Vesna Schauer-Vukasinovic; Hanno Langen; Thomas Giller (51-56).
Human napsin A is an aspartic proteinase highly expressed in kidney and lung. To elucidate whether napsin A is excreted in the urine we have performed an immunochemical study using anti-napsin A polyclonal antibody. As a result an immunoreactive band at approx. 38 kDa was detected which corresponds to the molecular mass of recombinant active human napsin A. A deglycosylation study showed that excreted napsin A is N-glycosylated on apparently all of the three potential glycosylation sites. Immunoreactive napsin A was also observed in urine from patients with a transplanted kidney whose kidney function appeared half to fully normal. On the other hand, no or very low immunostaining was detected in samples from patients with diseased kidneys. The urinary excretion pattern correlates well with the enzymatic activity of napsin A. These data show that human napsin A is excreted as functional proteinase in the urine. Furthermore, immunochemical studies suggest a relation between urinary excretion of napsin A and renal function. More specifically, lack of urinary excretion of napsin A could potentially serve as a tool for the detection of kidney dysfunction.
Keywords: Human napsin A; Urine; Human kidney; Napsin antibody; Aspartic proteinase;

Quinacrine has been used for decades and the beneficial effects of this drug are as numerous as its toxic effects. Since endothelial cells (EC) are in many cases the first cells coming in contact with drugs, the effect of quinacrine on certain aspects of EC biology were studied. The presented data demonstrate that quinacrine can have a marked impact on the integrity on EC monolayer without grossly interfering with cell viability. The described impact of quinacrine on EC might explain, at least in part, the toxic effects of this drug observed in the past. Furthermore, quinacrine profoundly effects gene regulation in EC. Quinacrine binds to DNA in a sequence-specific manner. While NF-κB–DNA interactions are not effected, AP-1–DNA binding is blocked by quinacrine. Such differential effects are presumably due to intercalation of quinacrine into the AP-1 consensus element. Preincubation of oligonucleotides resembling this sequence blocked the subsequent binding of nuclear extract containing AP-1 protein(s). Taken together, these data suggest that quinacrine interferes with EC physiology and alters the repertoire of EC to respond to stimuli. Furthermore, the differential effects of quinacrine might be exploited to study and gain additional insight in the involvement of AP-1 and NF-κB in gene regulation.
Keywords: Endothelial cell; Quinacrine; Nuclear factor κB; Activation protein-1;

Scanning electrochemical microscopy (SECM) has been used in the induced transfer (SECMIT) mode to image the permeability of a probe cation, methyl viologen (MV2+), in samples of articular cartilage. An ultramicroelectrode (UME), scanned just above the surface of a sample, is used to amperometrically detect the probe solute. The resulting depletion of MV2+ in solution induces the transfer of this cation from the sample into the solution for detection at the UME. The current provides quantitative information on local permeability, provided that the sample-UME distance is known. It is shown that the necessary topographical information can be obtained using the amperometric response for the oxidation of Ru(CN)4− 6, which does not permeate into the cartilage matrix. This procedure was validated by marking samples in situ, after electrochemical imaging, with subsequent examination by ex situ interferometry and optical microscopy. Wide variations in the permeability of MV2+ have been detected by SECMIT. These observations represent the first demonstration of the inhomogeneous permeability of a cation in cartilage on a micrometre scale. The permeability maps show similar features to the proteoglycan distribution, identified by toluidine blue staining, and it is likely that proteoglycans are the main determinant of MV2+ permeability in articular cartilage.
Keywords: Scanning electrochemical microscopy; Ultramicroelectrode; Diffusion; Permeability; Cartilage; Proteoglycan;

The adhesive properties of recombinant soluble L-selectin are modulated by its glycosylation by Claudia B. Fieger; Sonja Emig-Vollmer; Thomas Petri; Michael Gräfe; Martin Gohlke; Nils Debus; Wolfhard Semmler; Rudolf Tauber; Barbara Volz (75-85).
The leukocyte adhesion molecule L-selectin, which mediates the initial steps of leukocyte attachment to vascular endothelium, is intensely glycosylated. Different glycoforms of L-selectin are expressed on different leukocyte subsets and differences in L-selectin glycosylation appear to be correlated with the leukocyte’s ability to attach to different endothelial targets. In the present study we addressed the question whether glycosylation of L-selectin influences L-selectin–ligand interactions. To obtain different glycoforms of L-selectin, recombinant proteins were expressed both in the baby hamster kidney (BHK) cell line and in the human myelogenous cell line K562, resulting in sL-sel[BHK] or sL-sel[K562], respectively. The glycosylation characteristics of the purified proteins were determined. The most striking differences in glycosylation were seen in the terminal sialylation. Each of the two proteins carried sialic acids in the α2-3 position, while α2-6-bound sialic acids were found exclusively on sL-sel[K562]. To investigate their adhesive properties, both recombinant sL-selectins were used in cell adhesion assays and interactions with the ligands present on various hematopoietic cell lines or activated human cardiac microvascular endothelial cells were examined. The binding capacity of sL-sel[K562] was about 1.6 fold higher compared to sL-sel[BHK] under static as well as under flow conditions. These findings indicate that the terminal sialylation pattern of L-selectin modulates its binding characteristics.
Keywords: Recombinant L-selectin; Glycosylation; Sialic acid; Leukocyte endothelial interaction; Flow chamber;