Reconstitution of Tonoplast H+-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls in Liposomes

Reconstituted proteoliposomes of tonoplast ATPase are formedon solubilization of tonoplast membranes from mung bean (Vignaradiata L.) with deoxycholate (DOC) in the presence of a mixtureof soybean phospholipids (asolectin), after removal of DOC bypassage through a PD-10 column (Pharmacia). This method is idealbecause of its simplicity and rapidity. Selective insertionof sets of tonoplast H⁺-ATPase polypeptides (68 kDa, 60 kDa,16 kDa and several minor polypeptides) into liposomes usingthis method was confirmed by SDS-PAGE and immuno-blotting withantibodies raised against 68-kDa and 60-kDa polypeptides. Pumping of protons across the membranes of the proteoliposomeswas demonstrated by quinacrine-fluorescence quenching in thepresence of ATP-Mg²⁺. ATP-Mg²⁺ was shown to be the preferredsubstrate in both reconstituted and native tonoplast vesicles,and its optimum concentration was 0.75 to 3.0 mM. Quenchingwas completely abolished by a channel-forming ionophore, gramicidinD, and an inhibitor of tonoplast H⁺-ATPase, KNO₃. Antibodiesto 68-kDa and 60-kDa peptides partially inhibited the pumpingof protons. The rate of pumping of protons increased with thenumber of proteoliposomes, the maximal concentration of whichwas equivalent to 250 µg of protein per reaction mixture.The optimum pH for pumping was 6.5 when inside of proteoliposomeswere loaded pH at 7.2. The rate of pumping of protons was reducedwhen proteoliposomes were made using asolectin and cholesterolat 3 : 1 (w/w), as compared with those made with asolectin alone. The ATPase activity in reconstituted proteoliposomes was inhibitedby KNO₃, with half-maximal inhibition at approximately 7 mM.The enzyme actively hydrolyzed ATP in preference to GTP, CTP,UTP, and ADP, but it did not hydrolyze pNPP or AMP. Antibodiesagainst the 60-kDa polypeptide strongly inhibited ATPase activityas compared to antibodies against the 68-kDa polypeptide. Theresults obtained in this study demonstrate directly that functionaltonoplast H⁺-ATPase can be inserted selectively into liposomes. (Received August 31, 1990; Accepted April 18, 1991)     

Irreversible Denaturation of Maltodextrin Glucosidase Studied by Differential Scanning Calorimetry,Circular Dichroism,and Turbidity Measurements

Thermal denaturation of Escherichia coli maltodextrin glucosidase was studied by differential scanning calorimetry, circular dichroism (230 nm), and UV-absorption measurements (340 nm), which were respectively used to monitor heat absorption, conformational unfolding, and the production of solution turbidity. The denaturation was irreversible, and the thermal transition recorded at scan rates of 0.5–1.5 K/min was significantly scan-rate dependent, indicating that the thermal denaturation was kinetically controlled. The absence of a protein-concentration effect on the thermal transition indicated that the denaturation was rate-limited by a mono-molecular process. From the analysis of the calorimetric thermograms, a one-step irreversible model well represented the thermal denaturation of the protein. The calorimetrically observed thermal transitions showed excellent coincidence with the turbidity transitions monitored by UV-absorption as well as with the unfolding transitions monitored by circular dichroism. The thermal denaturation of the protein was thus rate-limited by conformational unfolding, which was followed by a rapid irreversible formation of aggregates that produced the solution turbidity. It is thus important to note that the absence of the protein-concentration effect on the irreversible thermal denaturation does not necessarily means the absence of protein aggregation itself. The turbidity measurements together with differential scanning calorimetry in the irreversible thermal denaturation of the protein provided a very effective approach for understanding the mechanisms of the irreversible denaturation. The Arrhenius-equation parameters obtained from analysis of the thermal denaturation were compared with those of other proteins that have been reported to show the one-step irreversible thermal denaturation. Maltodextrin glucosidase had sufficiently high kinetic stability with a half-life of 68 days at a physiological temperature (37°C).     

Responsibility of phosphatidylglycerol for biogenesis of the PSI complex

Phosphatidylglycerol (PG) ubiquitous in thylakoid membranes of photosynthetic organisms was previously shown to contribute to accumulation of chlorophyll through analysis of the cdsA⁻ mutant of a cyanobacterium Synechocystis sp. PCC6803 defective in PG synthesis (SNC1). Here, we characterized effects of manipulation of the PG content in thylakoid membranes of Synechocystis sp. PCC6803 on the photosystem complexes to specify roles of PG in biogenesis of thylakoid membranes. SNC1 cells with PG deprivation in vivo, together with the chlorophyll decrease, exhibited a decline not in PSII, but in PSI, at the complex level as well as the subunit levels. On the other hand, the decrease in the PSI complex was accounted for by a remarkable decrease in the PSI trimer with an increase in the monomer. These symptoms of SNC1 cells were complemented in vivo by supplementation of PG. Besides, a reduction in the PG content of thylakoid membranes isolated from the wild type in vitro on treatment with phospholipase A2 (PLA2), similar to the PG-deprivation in SNC1 in vivo, brought about a decrease in the trimer population of PSI with accumulation of the monomer. These results demonstrated that PG contributes to the synthesis and/or stability of the PSI complex for maintenance of the cellular content of chlorophyll, and also to construction of the PSI trimer from the monomer at least through stabilization of the trimerized conformation.     

Induction of apoptosis by cigarette smoke via ROS-dependent endoplasmic reticulum stress and CCAAT/enhancer-binding protein-homologous protein (CHOP)

In this report, we investigated a role of endoplasmic reticulum (ER) stress in cigarette smoke (CS)-induced apoptosis of human bronchial epithelial cells (hBEC). Exposure of hBEC to CS or CS extract (CSE) caused expression of endogenous ER stress markers GRP78 and CHOP and induction of apoptosis evidenced by nuclear condensation, membrane blebbing, and activation of caspase-3 and caspase-4. In vivo exposure of mice to CS also caused induction of GRP78 and CHOP in the lung. Attenuation of ER stress by overexpression of ER chaperone GRP78 or ORP150 significantly attenuated CSE-triggered apoptosis. Exposure of hBEC to CSE caused generation of reactive oxygen species, and treatment with antioxidants inhibited CSE-induced apoptosis. Interestingly, antioxidants including a scavenger of O(2)(*-) blunted induction of CHOP by CSE without affecting the level of GRP78, and dominant-negative inhibition of CHOP abolished CSE-induced apoptosis. Furthermore, a generator of O(2)(*-) selectively induced CHOP and apoptosis in hBEC. Our results revealed that: (1) CS induces ER stress in vitro and in vivo, (2) ER stress mediates CS-triggered apoptosis downstream of oxidative stress, (3) CS-initiated apoptosis is caused through oxidative stress-dependent induction of CHOP, (4) O(2)(*-) may play a dominant role in this process, and (5) oxidative stress-independent induction of GRP78 counterbalances the proapoptotic action of CHOP.     

Staphylococcal enterotoxin induces emesis through increasing serotonin release in intestine and it is downregulated by cannabinoid receptor 1

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are the most recognizable bacterial superantigenic toxins causing food poisoning in humans throughout the world. However, it remains unclear how SEs induce emesis and its emetic signal pathway. We investigated a mechanism of SEA-induced emesis using a small emetic animal model, house musk shrew. SEA-induced emesis in the animals was inhibited by a 5-hydroxytryptamine (5-HT) synthesis inhibitor and a 5-HT(3) receptor antagonist. SEA could increase 5-HT release in the small intestine. Pre-treatment with 5,7-dihydroxytryptamine (5,7-DHT) markedly inhibited SEA-induced emesis. SEA-induced emesis was also abolished by surgical vagotomy. Furthermore, cannabinoid (CB) receptor agonists inhibited SEA-induced emesis, and the action was reversed by a CB1 antagonist. Both 5-HT release and CB1 receptor expression were found in the mucosal and myenteric plexus of the intestine. Moreover, a CB1 receptor agonist significantly decreased the 5-HT release in the intestine. These results demonstrate that SEA induces 5-HT release in intestine, rather than in brain, and that the 5-HT(3) receptors on vagal afferent neurons are essential for SEA-stimulated emesis. In addition, SEA-induced emesis is downregulated by the CB system through decreasing 5-HT release in intestine.     

Ceruloplasmin Protects Against Rotenone-Induced Oxidative Stress and Neurotoxicity

To clarify the neuroprotective property of ceruloplasmin and the pathogenesis of aceruloplasminemia, we generated ceruloplasmin-deficient (CP ^−/−) mice on the C57BL/10 genetic background and further treated them with a mitochondrial complex I inhibitor, rotenone. There was no iron accumulation in the brains of CP ^−/− mice at least up to 60 weeks of age. Without rotenone treatment, CP ^−/− mice showed slight motor dysfunction compared with CP ^+/+ mice, but there were no detectable differences in the levels of oxidative stress markers between these two groups. A low dose of rotenone did not affect the mitochondrial complex I activity in our mice, however, it caused a significant change in motor behavior, neuropathology, or the levels of oxidative stress markers in CP ^−/− mice, but not in CP ^+/+ mice. Our data support that ceruloplasmin protects against rotenone-induced oxidative stress and neurotoxicity, probably through its antioxidant properties independently of its function of iron metabolism.     

Involvement of ASK1 in Ca2+-induced p38 MAP kinase activation

The mammalian mitogen-activated protein (MAP) kinase kinase kinase apoptosis signal-regulating kinase 1 (ASK1) is a pivotal component in cytokine- and stress-induced apoptosis. It also regulates cell differentiation and survival through p38 MAP kinase activation. Here we show that Ca²⁺ signalling regulates the ASK1–p38 MAP kinase cascade. Ca²⁺ influx evoked by membrane depolarization in primary neurons and synaptosomes induced activation of p38, which was impaired in those derived from ASK1-deficient mice. Ca²⁺/calmodulin-dependent protein kinase type II (CaMKII) activated ASK1 by phosphorylation. Moreover, p38 activation induced by the expression of constitutively active CaMKII required endogenous ASK1. Thus, ASK1 is a critical intermediate of Ca²⁺ signalling between CaMKII and p38 MAP kinase.