The glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Transport mechanism, regulation by ATP and characterization of the glutamine/glutamate antiport

The glutamine/amino acid transporter solubilized from rat renal apical plasma membrane (brush-border membrane) with C₁₂E₈ and reconstituted into liposomes has been previously identified as the ASCT2 transporter. The reconstituted transporter catalyses an antiport reaction in which external glutamine and Na⁺ are cotransported in exchange with internal glutamine (or other amino acids). The glutamine-Na⁺ cotransport occurred with a 1:1 stoichiometry. The concentration of Na⁺ did not influence the Km for glutamine and vice versa. Experimental data obtained by a bi-substrate analysis of the glutamine-Na⁺ cotransport, together with previous report on the glutamine_ex/glutamine_in pseudo bi-reactant analysis, indicated that the transporter catalyses a three-substrate transport reaction with a random simultaneous mechanism. The presence of ATP in the internal compartment of the proteoliposomes led to an increase of the Vmax of the transport and to a decrease of the Km of the transporter for external Na⁺. The reconstituted glutamine/amino acid transporter was inhibited by glutamate; the inhibition was more pronounced at acidic pH. A kinetic analysis revealed that the inhibition was competitive with respect to glutamine. Glutamate was also transported in exchange with glutamine. The external Km of the transporter for glutamate (13.3 mM) was slightly higher than the internal one (8.3 mM). At acidic pH the external but not the internal Km decreased. According with the Km values, glutamate should be transported preferentially from inside to outside in exchange for external glutamine and Na⁺.     

Evidence that late-endosomal SNARE multimerization complex is promoted by transmembrane segments

Assembly of SNARE proteins into quaternary complexes is a critical step in membrane docking and fusion. Here, we have studied the influence of the transmembrane segments on formation of the late endosomal SNARE complex. The complex was assembled in vitro from full-length recombinant SNAREs and from mutants, where the transmembrane segments were either deleted or replaced by oligo-alanine sequences. We show that endobrevin, syntaxin 7, syntaxin 8, and vti1b readily form a complex. This complex forms a dimer as well as multimeric structures. Interestingly, the natural transmembrane segments accelerate the conversion of the quaternary complex to the dimeric form and are essential for multimerization. These in vitro results suggest that the transmembrane segments are responsible for supramolecular assembly of the endosomal SNARE complex.     

Reversible unfolding of the severe acute respiratory syndrome coronavirus main protease in guanidinium chloride

Chemical denaturant sensitivity of the dimeric main protease from severe acute respiratory syndrome (SARS) coronavirus to guanidinium chloride was examined in terms of fluorescence spectroscopy, circular dichroism, analytical ultracentrifuge, and enzyme activity change. The dimeric enzyme dissociated at guanidinium chloride concentration of <0.4 M, at which the enzymatic activity loss showed close correlation with the subunit dissociation. Further increase in guanidinium chloride induced a reversible biphasic unfolding of the enzyme. The unfolding of the C-terminal domain-truncated enzyme, on the other hand, followed a monophasic unfolding curve. Different mutants of the full-length protease (W31 and W207/W218), with tryptophanyl residue(s) mutated to phenylalanine at the C-terminal or N-terminal domain, respectively, were constructed. Unfolding curves of these mutants were monophasic but corresponded to the first and second phases of the protease, respectively. The unfolding intermediate of the protease thus represented a folded C-terminal domain but an unfolded N-terminal domain, which is enzymatically inactive due to loss of regulatory properties. The various enzyme forms were characterized in terms of hydrophobicity and size-and-shape distributions. We provide direct evidence for the functional role of C-terminal domain in stabilization of the catalytic N-terminal domain of SARS coronavirus main protease.     

Analysis of membrane fusion as a two-state sequential process: evaluation of the stalk model

We propose a model that accounts for the time courses of PEG-induced fusion of membrane vesicles of varying lipid compositions and sizes. The model assumes that fusion proceeds from an initial, aggregated vesicle state ((A) membrane contact) through two sequential intermediate states (I(1) and I(2)) and then on to a fusion pore state (FP). Using this model, we interpreted data on the fusion of seven different vesicle systems. We found that the initial aggregated state involved no lipid or content mixing but did produce leakage. The final state (FP) was not leaky. Lipid mixing normally dominated the first intermediate state (I(1)), but content mixing signal was also observed in this state for most systems. The second intermediate state (I(2)) exhibited both lipid and content mixing signals and leakage, and was sometimes the only leaky state. In some systems, the first and second intermediates were indistinguishable and converted directly to the FP state. Having also tested a parallel, two-intermediate model subject to different assumptions about the nature of the intermediates, we conclude that a sequential, two-intermediate model is the simplest model sufficient to describe PEG-mediated fusion in all vesicle systems studied. We conclude as well that a fusion intermediate "state" should not be thought of as a fixed structure (e.g., "stalk" or "transmembrane contact") of uniform properties. Rather, a fusion "state" describes an ensemble of similar structures that can have different mechanical properties. Thus, a "state" can have varying probabilities of having a given functional property such as content mixing, lipid mixing, or leakage. Our data show that the content mixing signal may occur through two processes, one correlated and one not correlated with leakage. Finally, we consider the implications of our results in terms of the "modified stalk" hypothesis for the mechanism of lipid pore formation. We conclude that our results not only support this hypothesis but also provide a means of analyzing fusion time courses so as to test it and gauge the mechanism of action of fusion proteins in the context of the lipidic hypothesis of fusion.     

极端嗜热菌Thermus thermophilus HB8中天冬氨酸转氨酶在大肠杆菌中的表达、纯化及酶学性质研究

为获得具有热稳定性的天冬氨酸转氨酶,从极端嗜热细菌Thermus thermophilus HB8中克隆得到天冬氨酸转氨酶基因aspC,并在大肠杆菌BL21(DE3)和Rosetta(DE3)中进行表达,发现在Rosetta(DE3)中具有较高的表达量。重组酶的最适反应pH是7.0,37 ℃下在pH8～10的缓冲液中保温1 h酶活几乎不改变。重组酶反应的最适温度为75 ℃,酶活稳定的温度范围为25～55℃。重组酶在65℃时半衰期为3.5h,75℃时为2.5h。重组酶的K_m^KG为7.559mmol/L,V_max^KG为0.086mmol/(L·min),K_m^Asp为2.031mmol/L,V_max^Asp为0.024mmol/(L·min)。Ca²⁺、Fe³⁺、Mn²⁺等金属离子对酶活性有微弱抑制作用。     

Molecular analysis of complement component C8β and C9 cDNAs of Japanese flounder, Paralichthys olivaceus

 The amino acid sequences of the human terminal complement components show extensive structural similarity to each other. In this study the C8β and C9 cDNAs of Japanese flounder, Paralichthys olivaceus, were cloned and analyzed. The derived deduced amino acid sequences of the two terminal components were homologous to those of humans, in that the sequences of both species contained LDL receptor, EGF precursor, and two thrombospondin domains. Japanese flounder C9 was found to have a second thrombospondin region in the C-terminus, similar to that reported for rainbow trout and pufferfish. Moreover, these two complement component cDNAs of Japanese flounder had partial similarity to human perforin. These findings show that Japanese flounder C8β and C9 have similar structures, which supports the hypothesis that the terminal complement genes originated from the same ancestral gene. Collectively, these features emphasize the strong similarity among the members of the terminal complement family. Received: 23 March 1999 / Revised: 1 June 1999     

Formation of 9-hydroxy linoleic acid as a product of phospholipid peroxidation in diabetic erythrocyte membranes

The increased production of oxygen-derived free radicals (OFR) and lipid peroxidation may contribute to vascular complications in diabetes. Some lipid peroxidation products have already been reported to be formed via glucose-induced oxidative stress. We have identified 9-hydroxy linoleic acid (9-OH-C18:2) in the red cell membrane phospholipid of diabetic subjects. We hypothesized that 9-OH-C18:2 would be formed in hydroxyl radical reactions to linoleic acid (C18:2) during glucose-induced oxidative stress, and confirmed that the formation of 9-OH-C18:2 was induced by ultraviolet (UV)-C irradiation to the synthetic C18:2. UV-C light generates highly reactive hydroxy radicals. C18:2 is confirmed to be the precursor of 9-OH-C18:2. To estimate the degree of oxidative damage to red cell membrane phospholipids, we developed a selective ion monitoring gas chromatography-mass spectrometric measurement for C18:2 and 9-OH-C18:2, following methanolysis of red cell membrane phospholipids. The relative peak height ratio of C18:2 to 9-OH-C18:2 (9-OH-C18:2/C18:2) was measured in phospholipid extracts of red cell membranes from healthy (n=29, 3.1±1.9%) and diabetic (n=27, 20.9±16.1%) subjects. It was confirmed that 9-OH-C18:2/C18:2 is significantly (P<0.001) elevated in patients with diabetes. The measurement of 9-OH-C18:2/C18:2 in red cell membranes should be useful for assessing oxidative damage to membrane phospholipids in diabetes.     

西藏块菌属的分类研究

首次报道产于西藏的块菌属３种,包括２个新种和１个新记录种。新种是刘氏块菌ＴｕｂｅｒｌｉｕｉＡ．Ｓ．Ｘｕｓｐ．ｎｏｖ．和西藏块菌ＴｕｂｅｒｘｉｚａｎｇｅｎｓｅＡ．Ｓ．Ｘｕｓｐ．ｎｏｖ,新记录种是少孢块菌Ｔｕｂｅｒｏｌｉｇｏｓｐｅｒｍｕｍ（Ｔｕｌ．＆Ｃ．Ｔｕｌ．）Ｔｒａｐｐｅ．标本全部保存于西藏高原生态研究所标本室（ＨＸＺＥ）。