Localization of chitinolytic enzymes in blood of turbot, Scophthalmus maximus, and their possible roles in defence

The intracellular localizations ofchitinase and β-N-acetylglucosaminidase were detected in turbot blood smears, using a novel method employing fluorogenic substrates. The two enzymes showed different distributions, with chitinase being more generally distributed and N-acetylglucosaminidase being strongly associated with distinct intracellular bodies, probably lysosomes. The fluorogenic substrates were used to analyse soluble and membrane fractions of homogenates of red and white blood cells prepared on Percoll gradients. In the leucocytes, the chitinase and N-acetylglucosaminidase activities were mostly in the soluble fraction. In the erythrocytes the activities were lower, at about one-hundredth and one-tenth specific activities, respectively, and were distributed between soluble and membrane-bound fractions at about 2 : 1 and 3 : 1, respectively. In contrast, lysozyme had a soluble distribution in leucocytes and was not detected in erythrocytes. Plasma was rich in chitinase and lysozyme activities but had no detectable N-acetylglucosaminidase. Two possible roles for the chitinolytic enzymes are discussed: defence against pathogens and processing of glycoproteins or glucosaminoglycans. Evidence for a defence role for the chitinase and lysozyme is provided by demonstrating that they had inhibitory activity against the chitinous fungus Mucor mucedo .     

The binding of palmitoyl-CoA to bovine serum albumin

Bovine serum albumin (BSA) is routinely utilized in vitro to prevent the adverse detergent effects of long-chain acyl-CoA esters (i.e., palmitoyl-CoA) in enzyme assays. Determination of substrate saturation kinetics in the presence of albumin would only be valid if the relationship between bound and free substrate concentrations was known. To elucidate the relationship between bound and free palmitoyl-CoA concentrations in the presence of BSA, several different techniques including equilibrium dialysis, equilibrium partitioning, fluorescence polarization and direct fluorescence enhancement were investigated. Direct fluorescence enhancement using a custom synthesized fluorescent probe, 16-(9-anthroyloxy)palmitoyl-CoA (AP-CoA), was the best approach to this question. Measurement of the relationship between mol of palmitoyl-CoA bound per mol of BSA (nu) versus -log[free palmitoyl-CoA] revealed that the binding of palmitoyl-CoA to BSA, like palmitate was nonlinear, suggesting the presence of more than one class of acyl-CoA binding sites. Computer analyses of the binding data gave a best fit to the 2,4 two-class Scatchard model, suggesting the presence of two high-affinity primary binding sites (k1 = (1.55 +/- 0.46) x 10(-6) M-1) and four lower affinity secondary binding sites (k2 = (1.90 +/- 0.09) x 10(-8) M-1). Further analyses using the six parameter stoichiometric (stepwise) ligand binding model supports the existence of six binding sites with the higher affinities associated with the binding of the first mole of palmitoyl-CoA and weaker binding occurring after the first two sites are occupied. The association constants from this model of multiple binding diminish sequentially (i.e., K1 greater than K2 greater than K3 greater than...greater than or equal to K6), suggesting that each mol of long-chain acyl-CoA binds to BSA with decreasing affinities.     

Evolution of alpha 2-macroglobulin. The purification and characterization of a protein homologous with human alpha 2-macroglobulin from plaice (Pleuronectes platessa L.) plasma.

A papain-binding protein (PB-protein) was purified to homogeneity from the plasma of plaice (Pleuronectes platessa L.). PB-protein inhibited the activity of trypsin and pancreatic elastase (serine proteinases), thermolysin (a metalloproteinase) and papain (a cysteine proteinase). Presaturation of PB-protein with trypsin prevented the subsequent inhibition of thermolysin, and vice versa. Only catalytically active endopeptidases were bound by PB-protein. The catalytic activity of trypsin bound by PB-protein was inhibited by 95% against an insoluble protein substrate, but only by 38% against a low-molecular-weight synthetic substrate. The remaining activity of the bound trypsin was partially protected against further inhibition by soya-bean trypsin inhibitor. Trypsin bound by PB-protein showed a decrease of 67% in its reactivity with antibodies. The inhibitory activity of PB-protein was inactivated at pH 8.0 by methylamine (0.2M) or dithiothreitol (1 mM). The inhibition of proteinases by plaice PB-protein shows the distinctive characteristics of inhibition by human alpha 2-macroglobulin, and it is concluded that the plaice protein is a homologue of the human macroglobulin.     

Influence of Protein Synthesis on NO(3) Reduction, NH(4) Accumulation, and Amide Synthesis in Suspension Cultures of Paul's Scarlet Rose

Changes in the concentrations of NH₄⁺ and amides during the growth of suspension cultures of rose (Rosa cv. Paul's Scarlet) cells were examined. When cells were grown in medium possessing only NO₃⁻ as a nitrogen source, the concentrations of NH₄⁺ and amides increased to 4.0 × 10⁻¹ and 5.9 micromoles per gram fresh weight, respectively. The amounts of both constituents declined during the later stages of growth. When a trace amount of NH₄⁺ was added to the NO₃⁻ base starting medium, the concentration of NH₄⁺ in the cells was increased to 7.0 × 10⁻¹ micromoles per gram fresh weight.     

Schistosoma mansoni: Electrophoretic characterization of strains selected for different levels of infectivity to snails

Individual adult Schistosoma mansoni from strains selected for high or low infectivity to specific strains of the snail intermediate host, Biomphalaria glabrata, were subjected to enzyme electrophoresis on starch gels. Fourteen enzyme systems were analyzed in an attempt to find electrophoretic markers associated with genes for infectivity to snails. The S. mansoni strains were selected from different isolates from Puerto Rico in several strains of B. glabrata. Of an estimated 18 loci, 3 were polymorphic and the remainder monomorphic. For 1 of the 3 polymorphic enzyme loci, lactate dehydrogenase (Ldh, EC 1.1.1.27), phenotype frequencies were correlated with infectivity to snails. In schistosome strains of low infectivity, frequencies of the Ldh-N phenotype ranged between 0.56 and 0.69, while in strains of high infectivity, Ldh-N frequencies were typically 0.91 to 1.00. Whether the correlation is accidental or due to some form of association, such as chromosomal linkage, between the locus responsible for variation in lactate dehydrogenase and a gene for infectivity to snails remains to be determined.     

Dynamic light scattering from collagen solutions. II. Photon correlation study of the depolarized light.

The depolarized forward-scattered light from solutions of rat tail collagen has been studied by photon correlation spectroscopy. The measured autocorrelation function is seen to decay on two widely different time scales. The decay time for the fast component is consistent with the rotational diffusion of rodlike collagen monomers. The slowly decaying autocorrelation component is attributed to large nonspecific aggregates of collagen. A substantial fraction of the collagen is in this aggregated form. Extrapolation of the faster decay times to zero concentration yields a value of θ = 1082 ± 30 sec^?1 for the rotational diffusion coefficient of the collagen monomer.     

P blood group regulation of glycosphingolipid levels in human erythrocytes.

The neutral glycosphingolipid content of normal human erythrocytes was analyzed by a new method which utilizes high performance liquid chromatography. This rapid and accurate technique permits the quantitation of each of the major neutral glycolipids from individual blood samples. A correlation between the P blood group and the relative quantities of neutral glycosphingolipids is demonstrated. Erythrocytes from P1 individuals are shown to contain more globotriaosylceramide and less lactosylceramide than do erythrocytes from P2 individuals. The results of these experiments suggest the existence of a new phenotype in the P blood group system, and have further implications regarding the biosynthesis of the P blood group glycosphingolipids.