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The effect of the ATPase activity of Manduca sexta V 1 ATPase by the amphipathic detergent lauryldimethylamine oxide (LDAO) and the relationship of these activities to the subunit composition of V 1 were studied. The Vacuolar ATPases (V 1 V O ATPases) define an ubiquitous class of proton pumps, which utilize ATP hydrolysis to maintain an acidic pH inside the vacuole (1). The electrochemical ion gradient created across the vacuolar membrane is used for the accumulation of positively charged substrates such as calcium and basic amino acids (2). In addition to this storage function, the vacuolar compartment has secretory and proteolytic functions (3, 4). The V-ATPases, consisting of at least thirteen distinct subunits (A 3 :B 3 :C:D:E:F:G y :H z :a:d:c:cЈ:cЉ) are morphologically subdivided in two components: a membrane-bound domain, V O , that contains the ion channel, and an extrinsic domain, V 1 , in which ATP hydrolysis takes place (4, 5). The two major subunits A and B, in a stoichiometry of A 3 :B 3 , contain the nucleotide-binding sites and are connected to the V O part by the so-called stalk subunits C-H (6). Seen from the side the structures of the V 1 ATPase, recently identified from Caloramator fervidus (7) and the tobacco hornworm, M. sexta (8,9), revealed a molecule with a single, compact stalk.The V 1 ATPase from M. sexta, which reversibly dissociates from the V O part as an in vivo regulatory mechanism (10), is the object of our studies and comprises the eight subunits A, B, H, C, D, E, G, and F with apparent molecular masses of 67, 56, 54,40,32,28,14, and 16 kDa, respectively (11). Low resolution structural studies of this V 1 complex using small-angle x-ray scattering have shown that the hydrated enzyme is an elongated molecule. The x-ray data define a mushroom-shaped V 1 ATPase, which consists of an ϳ145 Å headpiece, joined by an elongated stalk (8). Image processing of electron micrographs of negatively stained V 1 (9, 12, 13) has revealed that the headpiece consists of a pseudo-hexagonal arrangement of six masses surrounding a seventh mass. These barrel-shaped masses of approximately 30 Å in diameter and 80 Å in length, which consist of the major subunits A and B, are arranged in an alternating manner (9). The hexagonal barrel of subunits A and B encloses a cavity of ϳ32 Å in which part of the central stalk is asymmetrically located. The stalk protrudes from the bottom side of the headpiece forming an angle of ϳ7°with the vertical axis of the molecule. At the upper end of the hexagonal barrel extensions can be observed, assumed to belong to the N termini of subunit A (9, 13). Further insights into the topology of the M. sexta V 1 ATPase were obtained by differential protease sensitivity, release by chaotropic agents (13), and cross-linking studies (13,14). These studies resulted in a model in which the subunits H, C, D, G, and F are exposed in the enzyme, whereas subunit E is shielded in the complex (6,13,14).Here we report an investigation of the structure-function relationship of the V 1 stalk...

The response of V(1) ATPase of the tobacco hornworm Manduca sexta to Mg(2+) and nucleotide binding in the presence of the enhancer methanol has been studied by CuCl(2)-induced disulfide formation, fluorescence spectroscopy, and small-angle X-ray scattering. When the V(1) complex was supplemented with CuCl(2) nucleotide-dependence of A-B-E and A-B-E-D cross-linking products was observed in absence of nucleotides and presence of MgADP+Pi but not when MgAMP.PNP or MgADP were added. A zero-length cross-linking product of subunits D and E was formed, supporting their close proximity in the V(1) complex. The catalytic subunit A was reacted with N-4[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide (CM) and spectral shifts and changes in fluorescence intensity were detected upon addition of MgAMP.PNP, -ATP, -ADP+Pi, or -ADP. Differences in the fluorescence emission of these nucleotide-binding states were monitored using the intrinsic tryptophan fluorescence. The structural composition of the V(1) ATPase from M. sexta and conformational alterations in this enzyme due to Mg(2+) and nucleotide binding are discussed on the basis of these and previous observations.

Obesity can cause insulin resistance and cardiovascular and liver disease. The aim of this study was to analyze changes in laboratory values, body composition, and physical fitness before and after a one-year weight loss program with nutritional education, psychological care, and physical exercise. Twenty-two obese children (16 boys, 6 girls; median age 11.9 [range 7-15] years; BMI SDS +2.4 [1.6-3.1]) participated in the program. Outcome measures included liver enzymes, insulin resistance (HOMA), lipids, body composition, physical strength and endurance. All children had an inverse HOMA/body composition correlation; Group 1 (reduced BMI SDS after one year) had lower triglycerides, liver enzymes and improved body composition and fitness (p < 0.05). Group 2 (unchanged or increased BMI SDS) had worse body composition and increased endurance and strength of trunk extension (p < 0.05). Weight loss reduced risk factors for liver disease and improved insulin sensitivity. Body composition proved useful as a non-invasive indicator for insulin sensitivity.

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