The glyceraldehyde 3-phosphate dehydogenase holoenzyme of Bacillus stearothermophilus possesses precise 222 symmetry: in this respect it differs from the reported structure of the lobster muscle enzyme. Pairs of active sites are linked through a flexible polypeptide loop which probably mediates the structural changes giving rise to cooperative effects. Three additional salt bridges made by each subunit to others would make a major contribution to thermostability of the tetramer.

Abstract. Tat, the transactivation factor of human immunodeflciency virus type 1 (HIV-1), contains the highly conserved tripeptide sequence Arg-Gly-Asp (RGD) that characterizes sites for integrin-mediated cell adhesion. The tat protein was assayed for cell attachment activity by measuring the adhesion of monocytic, T lymphocytic, and skeletal muscle-derived cell lines to tat-coated substratum. All cell lines tested bound to tat in a dose-dependent manner and the tat cell adhesion required the RGD sequence because tat mutants constructed to contain an RGE or KGE tripeptide sequence did not mediate efficient cell adhesion. The tat-mediated cell attachment also required divalent cations and an intact cytoskeleton. In addition, cell adhesion to tat was inhibited in the presence of an RGD-containing peptide GRGDSPK or an antitat mAb that recognizes the RGD epitope. These results strongly suggest that cells are bound to tat through an integrin. Interestingly, myoblast cells bound to tat remained round, whereas the same cells attached through an integrin for a matrix protein typicaUy flatten and spread. The role of this RGD-dependent cellular adhesion of tat in HIV-1 infection remains to be determined.p ROTEINS that interact with integrin cell adhesion receptors frequently contain the amino acid tripeptide RGD sequence (5,20,26,30) within the integrin binding site. RGD sequences are found in fibronectin, vitronectin and collagen and constitute extracellular matrix attachment sites used for integrin-mediated cell adherence during development and differentiation (30). Integrin receptors on leukocytes bind to coagulation proteins (von Willebrand factor, fibrinogen, thrombospondin) and complement components (C3b), and participate in cell-cell adhesion (LFA-1 with I-CAM). These interactions are involved in homeostatic regulation, phagocytosis, cell migration, cell signaling, cellular trafficking, and lymphocyte recognition (11, 23,30,39). In addition, certain bacterial, parasitic, and viral proteins possess RGD sequences which recognize integrin receptors and may contribute to pathogenesis (1, 30, 31).Human immunodeficiency virus type I (HIV-1),~ the etiologic agent of AIDS (6,17,22,28), encodes a gene for a transactivating protein, termed tat, which contains an RGD sequence. HIV-1 tat is an 86-amino acid-long protein, which greatly increases viral gene expression and replication (2,4,13,14,34,35). The tripeptide RGD sequence in tat is 10-Dr. Brake's present address is Biological Research, SmithKline Beecham Animal Health Products, King of Prussia, PA, 19406-0939. Address all correspondence to Dr. C: Debouck. cated in the carboxy-terminal portion of the protein and is highly conserved among HW-1 isolates (Fig. 1) (24). The presence of an RGD sequence within tat raised the intriguing possibility that this tripeptide could constitute a cell attachment site. In this study, purified tat protein was assayed for cell attachment to various cell types. The observed cell adhesion was further characterized using an RGD-containing pep...

No abstract

The membrane attack complex (MAC) of the complement system was localized in both glomeruli and peritubular regions of 22 kidneys manifesting systemic lupus erythematosus (SLE) nephritis. A similar distribution was observed for immune complex markers (IgG, Clq, and C3) and MAC in glomeruli, although the deposits of MAC were more discrete and showed lesser immunofluorescence staining intensity compared with immunoglobulins and complement components. In contrast, peritubular immune complexes were present in only 7 out of 22 kidneys, involved comparatively small clusters of tubules, exhibited weaker immunofluorescence staining than MAC, and failed to correlate with interstitial foci of inflammation. Granular or irregular, linear aggregates of the MAC were observed at the periphery of larger groups of tubules contiguous to areas of interstitial inflammation. Comparable amounts of IgG, Clq, C3, and MAC were present in blood vessel walls in areas of fibrinoid necrosis. These data suggest that the MAC is a direct mediator of tissue injury occurring in renal glomeruli, tubules, and blood vessels. The discordance between immune complexes and MAC localized in the peritubular region, but not in glomeruli or blood vessels, raises the possibility that both immune complexes and nonimmune agents, such as bacterial antigens, may activate the classical or alternative complement pathways and thereby play a role in the pathogenesis of tubulointerstitial lesions of SLE nephritis.

The molecular basis of the membranolytic activity of the membrane attack complex (MAC) of complement was investigated. By using density gradient equilibrium ultracentrifugation, the binding of egg yolk lecithin to the isolated MAC and to its intermediate complexes and precursor proteins was measured. No stable phospholipid-protein complexes were formed with the MAC precursor components C5b-6, C7, C8, and C9. Stable complexes of phospholipid and protein were formed by CMb-7, C5b-8, CAb-9, and the MAC (C5b-9 dimer) and they exhibited densities of 1.2164, 1.184, 1.2055, and 1.2275 g/ml, respectively. The molar phospholipid/protein ratios for the four complexes were determined to be: CMb-7, 399:1; C5b-8, 841:1; CMb-9, 9181; and C5b-9 dimer, 1460:1. Electron microscopy of the isolated phospholipid-protein complexes revealed no lipid bilayer structures. The magnitude of the phospholipid binding capacity of the MAC is consistent with the interpretation that the MAC forms phospholipid-protein mixed in micelles in lipid bilayers and biological membranes and thus causes formation of hydrophilic lipid channels.

The membranolytic C5b-9 complement membrane attach complex (MAC) is assembled after activation of either the classic or the alternative complement pathway. The quaternary configuration of the MAC macromolecule presents neoantigenic determinants not present on precursor molecules. Consequently, antibodies specific for these neoantigen(s) do not detect nonspecifically bound native complement precursors of MAC. By means of antibodies rendered specific for MAC neoantigen(s), MAC was localized by the immunoperoxidase reaction in cryostat sections of human muscle. In 66 biopsy specimens containing necrotic muscle fibers (Duchenne dystrophy, 13; other dystrophies, 15; inflammatory myopathies, 31; miscellaneous myopathies, 7) all of the necrotic fibers reacted for MAC neoantigen(s). C3 and C9 were also consistently localized in necrotic fibers, but localization of C1q, C4, and IgG was variable and often did not exceed background staining. None of the nonnecrotic fibers reacted for immunoglobulin or complement. Detection of MAC neoantigen(s) in necrotic fibers in a wide variety of muscle disease unambiguously shows that (1) the lytic complement pathway is consistently activated and participates in muscles fiber necrosis in vivo, and (2) complement reaction products are generated than can stimulate cellular infiltration and phagocytosis of the necrotic fiber. The findings also suggest that cell necrosis in general may involve participation of complement.

The membrane attack complex (MAC) of complement was extracted from the membranes of cells lysed by human complement and its properties were compared with those of the fluid phase complex SC5b-9. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunochemical analysis, the two isolated complexes had identical subunit compositions, except that the MAC lacked the S-protein. The sedimentation coefficient and molecular weight of the extracted and isolated MAC were, respectively, 33.5 S and 1.7 x 10(6) daltons, compared to 23 S and 1.0 x 10(6) dalton for SC5b-9. Because the molecular weight of the MAC is approximately two times greater than that of C5b-0 (800,000 daltons), the MAC is considered the dimer of C5b-9. Under specified conditions, the 33.5 S dimer could be converted to the 23 S monomer without dissociation of subunits. The MAC had the electron microscopic appearance and dimensions that are characteristic for the complement produced ultrastructural membrane lesions. SC5b-9 had a different ultrastructure that is dissimilar to the morphology of the lesions. The isolated MAC could be reincorporated into phospholipid bilayers and assumed on the surface of the resultant lipid vesicles the orientation and appearance of typical complement lesions.

The membrane attack complex (MAC) 1 of complement has been shown to be a dimer of C5b-9 (1) and the ring-shaped ultrastructural membrane lesion induced by complement (2) was identified as the membrane-bound MAC (1, 3). Electron microscopic examination of the isolated MAC has revealed a 50-× 200-A ring structure with 100-,~-long perpendicular attachments. The latter structures may represent stalklike appendages (1) or the wall of a hollow cylinder (3). Although the precise geometry of the MAC remains uncertain at present, it appeared feasible to gain insight into the process of MAC assembly by a comparative study of the morphology and particle size of the intermediate complexes C5b-6, C5b-7, C5b-8, and C5b-9. For this purpose recombinants of the intermediate complexes and phospholipids were prepared using dioleoyl lecithin single bilayer vesicles and purified human C5b-6, C7, C8, and C9. The results show that each intermediate complex has a characteristic morphology which is referable to the overall morphology of the fully assembled MAC and that MAC assembly constitutes an intricate process involving both protein association and dissociation reactions. It is proposed that production of a functional membrane lesion by the MAC is the result of phospholipid reorganization caused by highly complex protein-protein and protein-phospholipid interactions. Materials and MethodsChemicals. Dioleoyl lecithin (DOL) was purchased from Avanti Bioehemicals, Inc. (Birmingham, Ala.), sodium deoxycholate (DOC) was obtained from Sigma Chemical Co. (St. Louis, Mo.). All other chemicals were of the best grade commercially available. ComplementProteins. C5b-6 (4), C7 (5), C8 (6), and C9 (7, 8) were purified according to published procedures. The proteins were radiolabeled by the method of McConahey and Dixon (9).OystaUization of C5b-6. C5b-6 at concentrations between 1 and 2.5 mg/ml dissolved in veronal (3.3 mM)-buffered saline (0.15 M), pH 7.4, containing 0.15 mM CaClz and 0.5 mM MgCI~ (VB) was held at 4°C for 72 h. The resulting paracrystaIs were sedimented in a