Total Membrane Fraction Research Articles

Mechanism of Assembly of the Outer Membrane of Salmonella typhimurium: SITE OF SYNTHESIS OF LIPOPOLYSACCHARIDE

The site of synthesis of lipopolysaccharide has been investigated in cytoplasmic and outer membrane fractions isolated by isopycnic sucrose gradient centrifugation of the total membrane fraction from lysozyme-EDTA spheroplasts. Evidence that synthesis of O-antigen occurs exclusively in cytoplasmic membrane was obtained by pulse-chase experiments in vivo and by assay of biosynthetic enzymes in isolated membrane fractions. O-Antigen chains pulse-labeled in vivo with [14C]mannose appeared initially in cytoplasmic membrane, but were rapidly transferred to outer membrane during a subsequent chase with nonradioactive mannose. In accord, the specific activities of enzymes of O-antigen synthesis in isolated cytoplasmic membrane fractions were 15- to 30-fold greater than in outer membrane. The cytoplasmic membrane fractions were also enriched for glycosyltransferase activities involved in biosynthesis of the core region of lipopolysaccharide. However, unequivocal localization of core transferase enzymes in the isolated membranes was not possible since these activities were also found with soluble fraction, and secondary binding of soluble enzyme to both cytoplasmic and outer membrane was shown to occur during the isolation procedure. Evidence that the cytoplasmic membrane is the site of synthesis of the core region was derived from pulse-chase experiments in a mutant in which [14C]galactose is incorporated exclusively into the core portion of the polysaccharide. Over 90% of the [14C]galactose incorporated into lipopolysaccharide during a 1-min pulse was recovered in the cytoplasmic membrane fraction. The mechanism of translocation of lipopolysaccharide to the outer membrane is unknown. Evidence suggesting that the process is not readily reversible was obtained in experiments in mutants with conditional defects in lipopolysaccharide synthesis. Incomplete lipopolysaccharides incorporated into outer membrane during growth under nonpermissive conditions could not be completed following subsequent shift to permissive growth conditions.

Enzymes of phospholipid metabolism: Localization in the cytoplasmic and outer membrane of the cell envelope of Escherichia coli and Salmonella typhimurium

The phospholipid biosynthetic enzymes of the cell envelope of Escherichia coli and Salmonella typhimurium had highest specific activities in the cytoplasmic membrane fraction and lowest specific activities in the outer membrane fraction obtained by isopycnic sucrose density gradient centrifugation of the total membrane fraction of lysozyme-EDTA spheroplasts. This finding suggests that the site of phospholipid synthesis is the cytoplasmic membrane. Several degradative enzymes of phospholipid metabolism had highest specific activities in the outer membrane fraction and lowest specific activities in the cytoplasmic membrane. These data suggest an intracellular separation of the degradative and biosynthetic enzymes of phospholipid metabolism.

The Relief of Postoperative Pain

Targeting peripheral neuropathic pain at its origin may prevent the development of hypersensitivity. Recently we showed this can be mediated by opioid receptors at the injured nerve trunk. Here, we searched for the most relevant peripheral site to block transient receptor potential vanilloid 1 (TRPV1), and investigated analgesic interactions between TRPV1 and opioids in neuropathy. In a chronic constriction injury (CCI) of the sciatic nerve in mice, we assessed the effects of μ-, δ- and κ-opioid receptor agonists and TRPV1 antagonist (SB366791) injected at the CCI site or into the injured nerve-innervated paw on spontaneous paw lifting, heat and mechanical sensitivity. We also examined TRPV1 expression in total membrane and plasma membrane fractions from nerves and paws. We found that opioids and SB366791 co-injected in per se nonanalgesic doses at the CCI site or into the paw diminished heat and mechanical sensitivity. SB366791 alone dose-dependently alleviated heat and mechanical sensitivity. TRPV1 blockade in the paw was more effective than at the CCI site. None of the treatments diminished spontaneous paw lifting. TRPV1 expression analysis suggests that the levels of functional TRPV1 do not critically determine the TRPV1 antagonist-mediated analgesia. Together, the identification of the primary action site in damaged nerves is crucial for effective pain control. Contrary to opioids, the TRPV1 blockade in the injured nerve peripheral terminals, rather than at the nerve trunk, appears promising against heat pain. Opioid/TRPV1 antagonist combinations at both locations partially reduced neuropathy-triggered heat and mechanical pain.