Neutrophils are possibly involved in the pathogenesis of various lung diseases through the release of numerous mediators. In the present study, we studied the regulation of IL-8 gene induction and protein secretion in human blood neutrophils. Northern blot analysis revealed that LPS increased IL-8 mRNA levels in neutrophils, with a maximal fivefold increase by 2 h. IL-8 mRNa levels returned to baseline values within 12 h. In contrast, LPS-stimulated monocytes demonstrated a sustained increase of IL-8 mRNA levels for more than 24 h. TNF-alpha, IL-1 beta, and phorbol myristate acetate also increased IL-8 mRNA levels in neutrophils. Immunohistochemical analysis confirmed that IL-8 was localized within stimulated neutrophils. IL-8 secretion by neutrophils and monocytes was quantified using a specific ELISA for IL-8. Resting neutrophils secreted minimal IL-8 activity. However when cells were stimulated with LPS, TNF-alpha, or IL-1B, neutrophils secreted IL-8. IL-8 secretion was most marked during the first 2 h after stimulation and decreased thereafter. In contrast, monocytes maintained a high rate of IL-8 secretion over 12 h. Although a single monocyte secreted 70-fold more IL-8 than did a single neutrophil after 4 h of incubation, the high abundance of neutrophils in peripheral blood made the neutrophil-secreted IL-8 more significant. During the first 2 h, neutrophils secreted approximately 40% of the IL-8 released by monocytes in the same volume of blood. This ratio decreased to 9% after 12 h. Neutrophil-secreted IL-8 may play an autocrine or paracrine role during the initial stage of inflammation.
Dopamine-,6-hydroxylase (EC 1.14.2.1) has been isolated as a pure protein from bovine-adrenal glands. Although the molecular weight of the native protein is well established (290,000), sodium dodecyl sulfate-gel electrophoresis under dissociating conditions gave a single band with a molecular weight of about 75,000. The enzyme contains about 4% carbohydrate, which consists of residues of mannose, glucosamine, galactose, glucose, fucose, and sialic acid. The pure enzyme also contains about 4 atoms of copper per molecule. It is concluded that dopamine-,6-hydroxylase is a tetrameric glycoprotein.Dopamine-f-hydroxylase (EC 1.14.2.1) was first purified from adrenal glands in 1960 (1). It has been characterized as a copper-containing enzyme with a molecular weight of 290,000. It is estimated that from 3 to 7 mol of copper per mol of enzyme are present (2). Dopamine-f3-hydroxylase has been localized in granulated vesicles of both sympathetic nerve terminals and adrenal medulla chromaffin cells (3). In the chromaffin cells, about half of the dopamine-#-hydroxylase is present in soluble form; the remaining half is membrane bound (4, 5). Only the soluble enzyme is released together with catecholamines upon nerve stimulation (6); the mechanism of release has been suggested to be exocytosis (3). Two laboratories have reported that the soluble enzyme cannot be differentiated from the membrane enzyme by various criteria, such as gel electrophoresis, aminoacid composition, immunological crossreactivity, and size (7,8).Many large proteins consist of subunits. Moreover, proteins involved with membranes or in secretion are often found to be glycoproteins. It therefore seemed of interest to investigate the detailed structure of dopamine-,B-hydroxylase. The present paper is a report of a study of the subunit size and copper and carbohydrate composition of pure bovine-adrenal dopamine-ghydroxylase.METHODS Dopamine-,B-hydroxylase was isolated by a modification of the method of Foldes et al. (8). An additional DEAE-cellulose column with a linear salt concentration gradient of 0-0.4 M NaCl in 10 mM phosphate buffer (pH 7.2) was used after the final G-200 Sephadex chromatography of the above procedure. The fractions with the highest specific activity were concentrated by Diaflo filtration and stored in dry-ice in a freezer. This preparation was stable for several weeks under these storage conditions.Polyacrylamide gel electrophoresis was performed with the system [5% acrylamide (pH 8.1)] described by Clarke (9). (10% acrylamide) were prepared, and the samples were run as described by Weber and Osborn (10) for high molecular weight proteins. Gels were stained for protein-bound carbohydrates with the periodic acid-Schiff reagent (11). The periodate cleavage of ,B-hydroxyphenylethyl amines has been used as the basis of a dopamine-,3-hydroxylase assay by several investigators (1, 12, 13). The following modification of this method was used. The octopamine formed from tyramine was converted to p-hydroxybenzaldehyde by sodium periodat...
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