Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

A directed nucleotide-sequencing approach for single-stranded vectors based on recloning intermediates of a progressive DNA synthesis reaction

A simple method for site-directed nucleotide sequencing is presented that uses a novel procedure for generating nested 'deletions' within inserts of single-stranded clones. In this method, single-stranded template, sequencing primer, and the Klenow fragment of Escherichia coli DNA polymerase I are used to initiate progressive DNA synthesis of the entire insert of the clone. By time-dependent sampling and pooling of intermediates from the synthesis reaction a series of nested double-stranded DNA subfragments of the insert can be created. Nested subclones are then produced by S1-endonuclease treatment and oriented subcloning methods. First, smaller quantities of template DNA can be used, equivalent to a fraction of a small DNA sequencing prep. Second, it works with single-stranded M13 phage DNA rather than requiring the preparation of double-stranded replicative form DNA as in ExoIII-based methods. Third, the 'deletions' it generates can span areas of simple nucleotide sequence or secondary structure that often halt digestion in the single-stranded exonuclease-based method. Last, the method is adaptable to a larger variety of insert cloning sites than the ExoIII-based method. The main disadvantage of the method is that, due to the lower efficiency of subcloning larger DNA fragments, subclone inserts larger than 3 kb are generated only infrequently.     

A mutation in yeast mitochondrial DNA results in a precise excision of the terminal intron of the cytochrome b gene

The yeast nuclear gene CBP2 was previously proposed to code for a protein necessary for processing of the terminal intron in the cytochrome b pre-mRNA (McGraw, P., and Tzagoloff, A. (1983) J. Biol. Chem. 258, 9459-9468). In the present study we describe a mitochondrial mutation capable of suppressing the respiratory deficiency of cbp2 mutants. The mitochondrial suppressor mutation has been shown to be the result of a precise excision of the last intervening sequence from the cytochrome b gene. Strains with the altered mitochondrial DNA have normal levels of mature cytochrome b mRNA and of cytochrome b and exhibit wild type growth on glycerol. These results confirm that CBP2 codes for a protein specifically required for splicing of the cytochrome b intron and further suggest that absence of the intervening sequence does not noticeably affect the expression of respiratory function in mitochondria.     

Characterization of the binding activities of proteinase-adhesin complexes from Porphyromonas gingivalis.

Adhesins from oral bacteria perform an important function in colonizing target tissues within the dentogingival cavity. In Porphyromonas gingivalis certain of these adhesion proteins exist as a complex with either of two major proteinases referred to as gingipain R (arginine-specific gingipain) and gingipain K (lysine-specific gingipain) (R. N. Pike, W. T. McGraw, J. Potempa, and J. Travis, J. Biol. Chem. 269:406-411, 1994). With specific proteinase inhibitors, it was shown that hemagglutination by either proteinase-adhesin complex could occur independently of proteinase activity. Significantly, low concentrations of fibrinogen, fibronectin, and laminin inhibited hemagglutination, indicating that adherence to these proteins and not the hemagglutination activity was a primary property of the adhesin activity component of complexes. Binding studies with gingipain K and gingipain R suggest that interaction with fibrinogen is a major function of the adhesin domain, with dissociation constants for binding to fibrinogen being 4 and 8.5 nM, respectively. Specific association with fibronectin and laminin was also found. All bound proteins were degraded by the functional proteinase domain, with gingipain R being more active on laminin and fibronectin and gingipain K being more effective in the digestion of fibrinogen. Cumulatively, these data suggest that gingipain R and gingipain K, acting as proteinase-adhesin complexes, progressively attach to, degrade, and detach from target proteins. Since such complexes appear to be present on the surfaces of both vesicles and membranes of P. gingivalis, they may play an important role in the attachment of this bacterium to host cell surfaces.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

Characterization of binding kinetics of A2AR to Gαs protein by surface plasmon resonance

Because of their surface localization, G protein-coupled receptors (GPCRs) are often pharmaceutical targets as they respond to a variety of extracellular stimuli (e.g., light, hormones, small molecules) that may activate or inhibit a downstream signaling response. The adenosine A_2A receptor (A_2AR) is a well-characterized GPCR that is expressed widely throughout the human body, with over 10 crystal structures determined. Truncation of the A_2AR C-terminus is necessary for crystallization as this portion of the receptor is long and unstructured; however, previous work suggests shortening of the A_2AR C-terminus from 412 to 316 amino acids (A_2AΔ316R) ablates downstream signaling, as measured by cAMP production, to below that of constitutive full-length A_2AR levels. As cAMP production is downstream of the first activation event—coupling of G protein to its receptor—investigating that first step in activation is important in understanding how the truncation effects native GPCR function. Here, using purified receptor and Gα_s proteins, we characterize the association of A_2AR and A_2AΔ316R to Gα_s with and without GDP or GTPγs using surface plasmon resonance (SPR). Gα_s affinity for A_2AR was greatest for apo-Gα_s, moderately affected in the presence of GDP and nearly completely ablated by the addition of GTPγs. Truncation of the A_2AR C-terminus (A_2AΔ316R) decreased the affinity of the unliganded receptor for Gα_s by ～20%, suggesting small changes to binding can greatly impact downstream signaling.