Clustering of lipid-bound annexin V may explain its anticoagulant effect.

In 1985 we isolated a new vascular anticoagulant protein VAC alpha, now called annexin V, with a high binding affinity (Kd less than 10(-10) M) for phospholipids. Its anticoagulant effect was attributed to displacement of coagulation factors from the phospholipid membrane. The present study demonstrates that the inhibition of prothrombinase activity by annexin V strongly depends on the curvature of the membrane surface and on the calcium concentration. Half-maximal inhibition of prothrombinase on and binding of annexin V to small vesicles, composed of 20% phosphatidylserine and 80% phosphatidylcholine, requires 2-3 mM calcium. With large vesicles and planar bilayers considerably less calcium is required for inhibition of prothrombinase and for lipid binding. Half-maximal binding of annexin V to large vesicles and to planar bilayers occurs at 0.7 and 0.2 mM calcium, respectively. This seemingly confirms the displacement model. The displacement of coagulation factors, however, proved to be incomplete, with residual surface concentrations of factors Xa, Va, and prothrombin sufficient for effective production of thrombin. Cryoelectron microscopy revealed that annexin V binding to large vesicles caused planar facets, indicating the formation of large sheets of clustered annexin V. Apparently, the formation of these two-dimensional arrays is promoted by calcium and hampered by high surface curvature. It is speculated that the complete inhibition (greater than 99%) of prothrombinase activity by annexin V is caused by the reduced lateral mobility of prothrombin and factor Xa in rigid sheets of annexin V covering the membrane.     

Inhibition and killing of fungi by the polyamine oxidase-polyamine system

Both components of the polyamine oxidase (PAO)-polyamine system are known to be present in phagocytes and have thus been postulated to contribute to the antimicrobial activity of these cells. Therefore, the effects of the PAO-polyamine system on three medically important opportunistic fungi were examined. Yeasts of Cryptococcus neoformans, but not Candida albicans blastoconidia or Aspergillus fumigatus conidia, were efficiently killed by the system. Two putative end products of the system, hydrogen peroxide and acrolein, both killed C. neoformans at concentrations attainable with the whole system. However, catalase failed to inhibit activity of the whole system, making hydrogen peroxide an unlikely mediator of killing. Although C. albicans blastoconidia and A. fumigatus conidia were not killed by the PAO-polyamine system, germ tube formation by the former, and hyphal growth by the latter, were markedly inhibited. These data establish that the PAO-polyamine system possesses antifungal activity.     

Comparison of the catalytic oxidation of cysteine and o-dianisidine by cupric ion and ceruloplasmin

Several features of the catalytic oxidation of cysteine by ceruloplasmin and nonenzymic Cu(II) at pH 7 have been compared. The oxidation of cysteine by ceruloplasmin has several properties in common with the Cu(II) catalyzed oxidation of cysteine: pH maxima, thiol specificity, lack of inhibition by anions, and high sensitivity to inhibition by copper complexing reagents. These two catalysts differed in their molecular activity, in their ability to oxidize penicillamine and thioglycolate, and in that H₂O₂ was produced as a primary product only during Cu(II) oxidation. The oxidation of cysteine by ceruloplasmin was compared also with the ceruloplasmin catalyzed oxidation of o-dianisidine, a classical pH 5.5 substrate. The mechanism of the oxidation of cysteine by ceruloplasmin at pH 7 differed from that of o-dianisidine oxidation because the latter substrate was inhibited by anions but not by copper complexing agents. Spectral and other data suggest that during the ceruloplasmin reaction with cysteine there is a one electron transfer from cysteine to ceruloplasmin resulting in the specific reduction of type lb Cu(II).     

Parallel activities of fatty acid methyl esters and analogous phorbol diesters toward mouse lymphocytes

Linear saturated fatty acid methyl esters were comitogenic with lectins for mouse lymphocytes, the degree of comitogenicity being strongly dependent on the length of the acyl group, and maximal for methyl tetradecanoate. Lesser effects were found for analogs with 10, 12 or 16 acyl carbon atoms, whereas those with fewer than 10 or more than 16 were inactive. Analogous structure-function relationships have been described for various membrane-active and tumor-promoting phorbol diesters, where there is a similar dependence on ester acyl group length for many activities. The fatty acid esters may therefore represent simple model compounds for studying mechanistic aspects of phorbol diester activity.     

Mineralization of Detrital Lignocelluloses by Salt Marsh Sediment Microflora

Specifically radiolabeled ¹⁴C-(cellulose)-lignocellulose and ¹⁴C-(lignin)-lignocellulose were isolated from labeled cuttings of Spartina alterniflora (cordgrass) and Pinus elliottii (slash pine). These were used to estimate the rates of mineralization to CO₂ of lignocelluloses of estuarine and terrestrial origin in salt marsh estuarine sediments. The lignin moiety of pine lignocellulose was mineralized 10 to 14 times more slowly than that of Spartina lignocellulose, depending on the source of inoculum. Average values for percent mineralization after 835 h of incubation were 1.4 and 13.9%, respectively. For Spartina lignocellulose, mineralization of the cellulose moiety was three times faster than that of the lignin moiety. Average values for percent mineralization after 720 h of incubation were 32.1 and 10.6%, respectively. Lignocellulose and lignin contents of live pine and Spartina plants were analyzed and found to be 60.7 and 20.9%, respectively, for pine and 75.6 and 15.1%, respectively, for Spartina.     

Evidence for Cometabolism in Sewage

A procedure was developed to demonstrate cometabolism in models of natural ecosystems. The procedure involves showing the formation of metabolic products in high yield and the lack of incorporation of substrate carbon into cellular constituents. Samples of four ¹⁴C-labeled herbicides (trifluralin, profluralin, fluchloralin, and nitrofen) were incubated with sewage aerobically and under discontinuous anaerobiosis for 88 days, and fresh sewage was added at intervals. Products were formed from each of the herbicides in nonsterile, but not in sterile, sewage. The yield of recovered products reached 87% for profluralin and more than 90% for fluchloralin and trifluralin, and the number of products ranged from 6 for nitrofen to 12 for fluchloralin. Concentrating the sewage microflora 40-fold greatly enhanced the rate of conversion. None of the radioactivity from the herbicide entered the nucleoside pool of the sewage microflora. The lack of incorporation of substrate carbon into cells and the almost stoichiometric conversion of the substrate to organic products indicate that members of the microbial community were cometabolizing the test compounds.     

Differential precipitation with concanavalin a as a method for the purification of glycoproteins: Human alkaline phosphatase

The purification of a glycoprotein from a mixture by first precipitating with a lectin and fractional solution of the precipitate could be applicable as an important preparative procedure. Another possibility could be fractional precipitation with the lectin by varying the concentration of Ca²⁺ and Mn²⁺ required for binding. These principles have been tested in relation to the preparation of human liver alkaline phosphatase. Both approaches have led to the purification of alkaline phosphatase from single human livers with a high recovery (28 and 34%) and purification factors of 16 × 10³ and 13 × 10³ over the initial aqueous butanl-ol extract. The specific activities for the preparations were 1610 and 1500 units/mg of protein, respectively; a unit is defined as the amount of enzyme catalyzing the hydrolysis of 1.0 μmol of p-nitrophenyl phosphate/min at 35°C in 0.1 m 2-amino-2-methylpropan-1-ol/HCl buffer, pH 10.5, containing 10 mmp-nitrophenyl phosphate, 0.1 mm MgCl₂, and 1 μm ZnSO₄.