A particulate arachidonate lipoxygenase in human blood platelets

The microsomal fraction of human platelets contains a lipoxygenase activity in addition to the thromboxane-synthesizing activity. The enzymatic activity was stimulated by tryptophan, but inhibited by catecholamine, methemoglobin, and hydroquinones.     

Examining Polyglutamine Peptide Length: A Connection between Collapsed Conformations and Increased Aggregation

Abnormally expanded polyglutamine domains in proteins are associated with several neurodegenerative diseases, of which the best known is Huntington's. Expansion of the polyglutamine domain facilitates aggregation of the affected protein, and several studies directly link aggregation to neurotoxicity. The age of onset of disease is inversely correlated with the length of the polyglutamine domain; this correlation motivates an examination of the role of the length of the domain on aggregation. In this investigation, peptides containing 8 to 24 glutamines were synthesized, and their conformational and aggregation properties were examined. All peptides lacked secondary structure. Fluorescence resonance energy transfer studies revealed that the peptides became increasingly collapsed as the number of glutamine residues increased. The effective persistence length was estimated to decrease from ∼ 11 to ∼ 7 Å as the number of glutamines increased from 8 to 24. A comparison of our data with theoretical results suggests that phosphate-buffered saline is a good solvent for Q8 and Q12, a theta solvent for Q16, and a poor solvent for Q20 and Q24. By dynamic light scattering, we observed that Q16, Q20, and Q24, but not Q8 or Q12, immediately formed soluble aggregates upon dilution into phosphate-buffered saline at 37 °C. Thus, Q16 stands at the transition point between good and poor solvent and between stable and aggregation-prone peptide. Examination of aggregates by transmission electron microscopy, along with kinetic assays for sedimentation, provided evidence indicating that soluble aggregates mature into sedimentable aggregates. Together, the data support a mechanism of aggregation in which monomer collapse is accompanied by formation of soluble oligomers; these soluble species lack regular secondary structure but appear morphologically similar to the sedimentable aggregates into which they eventually mature.     

Radioimmunoassay of prostaglandin F2α using sephadex G-75 gel chromatography

The development of a “bound—free” separation technique and its application to the radioimmunoassay of prostaglandin F_2α is described. The method is simple, rapid, free of non-specific binding and could be performed either at 4°C or at room temperature. A total of 100 tubes could be subjected to “bound—free” separation in 30 min at 4°C. The bound fraction is collected directly into scintillation vials. The total column length was 9.5 cm, of which the bed volume was 2.5 ml. The PGF_2α radioimmunoassay incubation volume of 0.3 ml when bedded in required 1.4 ml of elution buffer to elute the antibody-bound fraction. The free fraction was washed out with 4.0 ml of buffer and the columns were ready for further use. A standard curve of high sensitivity (5 pg) and good reproducibility (CV %: intra-assay = 6.54; inter-assay = 9.68) was obtained.     

Biosynthesis of thromboxane B2: assay, isolation, and properties of the enzyme system in human platelets.

The microsomal fraction of human platelets catalyzed the conversion of arachidonic acid to an unstable platelet-aggregating factor and a hydrolyzed product on the thin-layer chromatography (TLC). This product was isolated on TLC, purified by silica gel column chromatography and identified by combined gas chromatography-mass spectrometry as the hemiacetal derivative of 8-(1-hydroxy-3-oxopropyl)-9, 12L-dihydroxy-5, 10-heptadecatrienoic acid (thromboxane B2). The enzymatic activity was dependent upon methemoglobin and tryptophan as cofactors. Reduced glutathione had no effect either alone or in combination with other cofactors. Methemoglobin could be replaced by hematin or hemin; and tryptophan by 3-indolacetic acid or catecholamines. The apparent requirement for methemoglobin is due to the reductive activity of ferriprotoporphyrin IX. The reaction, however, catalyzed by the ferriprotoporphyrin IX in the thromboxane synthesizing system is different from that described for the decomposition of lipid peroxides. Certain transition metals and hydrogen donors, such as hydroquinone and ascorbate, which have been shown to stimulate the catalytic activity of ferriproroporphyrin IX in the decomposition of 15-hydroperoxy-prostaglandin E1 are inhibitors of thromboxane B2 formation. This enzyme preparation also transformed eicosa-8. 11, 14-trienoic acid to an unknown product on TLC. The enzyme system was rapidly inactivated upon incubation in the reaction mixture.     

1,25-Dihydroxyvitamin D receptors in an established bone cell line. Correlation with biochemical responses

A stable cell line derived from mouse bone (cell line MMB-1) has been used for studies of the cellular receptor for 1,25-dihydroxyvitamin D3 in osteoblasts. Previous studies have demonstrated that collagen synthesis in the MMB-1 cell line is specifically inhibited by 1,25-dihydroxyvitamin D3 as well as by other bone-regulating hormones. Incubation of cell homogenates with [3H]1,25-dihydroxyvitamin D3 indicated the presence of a specific receptor which was located primarily in the chromatin fraction. Optimum conditions for the receptor assay required the inclusion of 500 kallikrein-inactivating units of Trasylol/ml and 10 mM NaMoO4. Under these conditions the receptors were stable for 2 h at 23 degrees C and for 24 h at 4 degrees C. Cellular content of receptors was dependent upon the state of confluency of the cells: fully confluent cells contained minimal concentrations of receptors. In cultures of 70-80% confluency, the 1,25-dihydroxyvitamin D3 receptors demonstrated linear Scatchard plots with Kd = 0.4 nM. Peak receptor activity was found at 3.7 S in linear sucrose gradient fractions of cell homogenates. The synthesis of collagen by MMB-1 cells was inhibited by 1,25-dihydroxyvitamin D3 in direct proportion to the concentration of cellular receptors at varying levels of culture confluence. The data indicate that MMB-1 cells contain cytoplasmic/nuclear receptors for 1,25-dihydroxyvitamin D3 which are similar to the receptors found in other target tissues for this hormone and suggest that these receptors are mediators of the effects of 1,25-dihydroxyvitamin D3 on collagen synthesis.