Albumin modulates S1P delivery from red blood cells in perfused microvessels: mechanism of the protein effect.

Removal of plasma proteins from perfusates increases vascular permeability. The common interpretation of the action of albumin is that it forms part of the permeability barrier by electrostatic binding to the endothelial glycocalyx. We tested the alternate hypothesis that removal of perfusate albumin in rat venular microvessels decreased the availability of sphingosine-1-phosphate (S1P), which is normally carried in plasma bound to albumin and lipoproteins and is required to maintain stable baseline endothelial barriers (Am J Physiol Heart Circ Physiol 303: H825-H834, 2012). Red blood cells (RBCs) are a primary source of S1P in the normal circulation. We compared apparent albumin permeability coefficients [solute permeability (Ps)] measured using perfusates containing albumin (10 mg/ml, control) and conditioned by 20-min exposure to rat RBCs with Ps when test perfusates were in RBC-conditioned protein-free Ringer solution. The control perfusate S1P concentration (439 ± 46 nM) was near the normal plasma value at 37 °C and established a stable baseline Ps (0.9 ± 0.4 × 10(-6) cm/s). Ringer solution perfusate contained 52 ± 8 nM S1P and increased Ps more than 10-fold (16.1 ± 3.9 × 10(-6) cm/s). Consistent with albumin-dependent transport of S1P from RBCs, S1P concentrations in RBC-conditioned solutions decreased as albumin concentration, hematocrit, and temperature decreased. Protein-free Ringer solution perfusates that used liposomes instead of RBCs as flow markers failed to maintain normal permeability, reproducing the "albumin effect" in these mammalian microvessels. We conclude that the albumin effect depends on the action of albumin to facilitate the release and transport of S1P from RBCs that normally provide a significant amount of S1P to the endothelium.

An optical and microPET assessment of thermally-sensitive liposome biodistribution in the Met-1 tumor model: Importance of formulation.

The design of delivery vehicles that are stable in circulation but can be activated by exogenous energy sources is challenging. Our goals are to validate new imaging methods for the assessment of particle stability, to engineer stable and activatable particles and to assess accumulation of a hydrophilic model drug in an orthotopic tumor. Here, liposomes were injected into the tail vein of FVB mice containing bilateral Met-1 tumors and imaged in vivo using microPET and optical imaging techniques. Cryo-electron microscopy was applied to assess particle shape prior to injection, ex vivo fluorescence images of dissected tissues were acquired, excised tissue was further processed with a cell-digest preparation and assayed for fluorescence. We find that for a stable particle, in vivo tumor images of a hydrophilic model drug were highly correlated with PET images of the particle shell and ex vivo fluorescence images of processed tissue, R(2)=0.95 and R(2)=0.99 respectively. We demonstrate that the accumulation of a hydrophilic model drug is increased by up to 177 fold by liposomal encapsulation, as compared to accumulation of the drug at 24 hours.

Steady-state kinetics of cabbage histidinol dehydrogenase.

Cabbage histidinol dehydrogenase (HDH) oxidizes L-histidinol to L-histidine through two sequential NAD(+)-linked reactions via an alkaline-labile, L-histidinaldehyde intermediate. The kinetic mechanism of the overall reaction as well as the partial reactions involved in the overall catalysis were investigated at pH 7.2 using L-histidinaldehyde as a substrate. Product inhibition patterns conformed to a Bi Uni Uni Bi Ping Pong mechanism as reported for the HDH from Salmonella typhimurium. Thus, the reaction scheme is ordered with the binding of histidinol first and NAD+ second, and histidine is the last product to be released. The intermediate, L-histidinaldehyde, could be a substrate for both the oxidation and the reduction reactions to produce histidine and histidinol, respectively. L-Histidine was not enzymatically reduced in the presence of NADH, indicating that the reaction to oxidize histidinaldehyde is apparently irreversible. L-Histidinaldehyde exhibited a three times greater binding rate constant than histidinol with a considerably small dissociation constant. These results were in agreement with the observation that histidinaldehyde was not released during the overall reaction. The rate of the reduction of histidinaldehyde to histidinol was almost same as that of the overall oxidation reaction. The overall oxidation from histidinol to histidine proceeded about three times slower than the partial oxidation from histidinaldehyde to histidine, suggesting that the first-half forward reaction is the rate-determining step in the total reaction of cabbage HDH.

Site-directed mutagenesis shows that the conserved cysteine residues of histidinol dehydrogenase are not essential for catalysis.

Histidinol dehydrogenase (HDH) catalyzes two sequential oxidation reactions to produce histidine from histidinol via histidinaldehyde. In HDH proteins so far reported, two Cys residues are conserved. From the results of the studies on Salmonella typhimurium HDH, it has been proposed that one of these two conserved Cys residues is involved in the thiohemiacetal formation at the aldehyde oxidation step [Grubmeyer and Gray (1986) Biochemistry 25, 4778-4784]. To clarify the reaction mechanism, we investigated the role of the conserved Cys residues by site-directed mutagenesis in cabbage HDH. Thus, Cys-112, that corresponds to the catalytic Cys residue of the Salmonella enzyme, and the other conserved one, Cys-149, were replaced with Ala, Ser, or Phe. All the Cys-112 mutant HDHs catalyzed both the alcohol dehydrogenase and aldehyde dehydrogenase reactions, producing 1 mol of L-histidine during the reduction of 2 mol of NAD+, as did the wild type HDH. Site-directed mutagenesis at Cys-149 did not cause significant changes in the catalytic properties, either. These observations, together with the results of detailed comparison of the catalytic properties of mutant HDHs, clearly indicate that neither Cys-112 nor Cys-149 is involved in the reaction, and ruled out the involvement of thiohemiacetal formation in the histidinol dehydrogenase reaction.

Incorporation and activation of a membrane-bound enzyme in bilayers of liposomes.

The effects of lipid composition and fluidity of lipid bilayers on incorporation and activation of membrane-bound D-fructose dehydrogenase are described in this study. The incorporation of the enzyme into bilayers of small unilamellar vesicles (SUV) made of several phospholipids resulted in enzyme activation with magnitudes higher than that observed in the presence of Triton X-100, indicating that this higher activation is due to lipid-protein interaction. The activity was highest in the presence of SUV formed by the addition of 10% DL-alpha-dipalmitoylphosphatidylethanolamine to L-alpha-dimyristoylphosphatidylcholine, which resulted in eightfold higher activation compared with that of the enzyme in its free state. This activation did not appear to be due to the degree of incorporation of the enzyme, indicating that incorporation is distinct from the activation event. Thus, it is probably the lipid environment that leads to higher activation of the enzyme. A break in the Arrhenius plot of the activity of the membrane-bound enzyme at temperatures close to the phase transition of the phospholipid implies that changes in the physical state of the lipid bilayer influence the enzyme activity. Furthermore, immobilization of D-fructose dehydrogenase, previously adsorbed to SUV, on urethane prepolymer also resulted in about eightfold higher activation than that of the free enzyme.

Reaction characteristics and stability of a membrane-bound enzyme reconstituted in bilayers of liposomes.

The effects ensuing from the interaction between membrane-bound sarcosine dehydrogenase and the surrounding lipids as well as the effects of membrane fluidity were described in this study. A 25-fold activation was observed upon the reconstitution of the enzyme in bilayers of SUVs made of DMPC. The considerable decrease in K(m) and increase in V(max) suggest the induction of favorable conformational changes in both the substrate-binding site and the catalytic site of the enzyme due to the lipid-protein interaction. In SUVs of negatively charged phospholipids, the enzyme retained its initial activity over 1 month. The break point in the Arrhenius plot of the activity of reconstituted enzyme was found at temperatures close to the gel-liquid crystalline transition point of the phospholipid showing that the activity is sensitive to the physical state of membrane phospholipids. Further, immobilization of the reconstituted enzyme by use of ENT prepolymer resulted in a high activity, whereas no remarkable activity was detected with the immobilized enzyme without reconstitution.

Effects of arachidonic acid concentration on prostaglandin biosynthesis and feasibility of semibatch processes.

The reaction characteristics of prostaglandin E2 biosynthesis by PGH-synthase and PGE2 isomerase and the substrate dependency of this biosynthesis were studied. The activity of PG-synthases was blocked by the inhibitory action of one or more byproducts, probably resulting from the action of PGH-synthase. This inhibitory action then appeared to be partly reversible, indicating that the substrate and the inhibitor compete for the catalytic sites. According to these findings, the feasibility of a successful semibatch biosynthesis was investigated. A combination of the substrate concentration reducing procedure and the semibatch process resulted in an about 3.5-fold higher increase in the total amount of PGE2 formed in comparison with the batch results obtained at the substrate concentration of 1.0 mg/cm3. Since the cost of enzyme is a governing factor in this biosynthesis, development of semibatch biosynthesis of PGE2 becomes a matter of economic importance.