ABSTRACT

Soluble guanylyl cyclase was purified from bovine lung by an immunoaffinity chromatographic method using IgG fractions of antisera against a synthetic peptide of the C-terminus of the 70-kDa subunit of the enzyme. After anion-exchange chromatography, the enzyme was bound to an immunoaffinity column and was eluted with the synthetic peptide. This method allowed the convenient isolation of 2 mg of apparently homogeneous enzyme from 40 g cytosolic proteins. The enzyme had an apparent molecular mass of about 150 kDa and consisted of two subunits (70 kDa and 73 kDa) as determined by gel permeation fast protein liquid chromatography and SDS/PAGE. The basal activities determined in the presence of Mg2+ and Mn2+ were 10-20 nmol.min-1.mg-1 and 80-100 nmol.min-1.mg-1, respectively. The enzyme exhibited an ultraviolet-visible absorption spectrum typical for hemoproteins, with a Soret band at 430 nm. The purified enzyme was stimulated by NO-containing compounds. Maximal enzyme activities measured in the presence of sodium nitroprusside were 1.2-2.4 mumol.min-1.mg-1 (half-maximal effect of sodium nitroprusside at 1.3-1.9 microM) and 0.9-1.8 mumol.min-1.mg-1 (half-maximal effect at 0.28-0.41 microM sodium nitroprusside) in the presence of Mg2+ and Mn2+, respectively. The method developed for the large-scale purification of soluble guanylyl cyclase by immunoaffinity chromatography, using synthetic peptides for the elution of the enzyme, appears to be superior to previously described methods. As antibodies against synthetic peptides corresponding to deduced amino acid sequences of the respective protein are easily obtained, the described method may be suitable for a convenient large-scale purification of various proteins.

No abstract

The primary structure of the larger subunit of the soluble guanylyl cyclase from bovine lung, which catalyzes the formation of cyclic GMP from GTP, has been determined. Two clones, isolated from two bovine libraries yielded a total of 3261 bp with a coding region of 2073 bp. The open reading frame encodes a protein of 691 amino acids and a molecular mass of 77 500. The deduced amino acid sequence reveals regions which are, to a large extent, homologous to the sequence of the smaller subunit of the enzyme as well as to the sequences of other gyanylyl and adenylyl cyclases.

Members of the fatty-acid-binding protein (FABP) family are thought to play an important role in fatty acid transport within the cytosol and thus to be involved in lipid metabolism. As previous data on the occurrence of distinct FABP types in total lung are contradictory, we determined the expression of FABP types in three isolated cell types of rat lung, which are characterised by active lipid metabolism. Alveolar type-II cells synthesise, store and secrete pulmonary surfactant, a phospholipid-rich surfacetension-lowering agent, whereas lung fibroblasts, localised adjacent to the alveolar type-II cells, are assumed to provide neutral lipid substrate to alveolar type-II cells around birth, and alveolar macrophages are known to degrade complex lipids. Initial screening by reverse transcriptase PCR revealed the occurrence of heart (H-), epidermal (E-) and liver FABP in rat lung, the latter being not detectable in the three cell types studied. Cells were analysed by northern and western blotting, then quantitatively by sandwich ELISA, for which recombinant rat E-FABP was prepared. E-FABP mRNA was found in all three cell types, and E-FABP was detected in the following amounts: 240.9Ϯ19.0 ng/mg cytosolic protein in alveolar type-II cells; 172.3Ϯ0.7 ng/mg protein for lung fibroblasts ; and 36.9 Ϯ3.5 ng/mg protein for alveolar macrophages. This indicates a basic function of E-FABP in cellular lipid metabolism. In contrast, H-FABP probably is involved in the metabolism of neutral lipids because H-FABP mRNA was found only in lung fibroblasts with a corresponding protein level of 315.5Ϯ6.9 ng/mg. Small amounts of H-FABP protein were present in alveolar type-II cells and alveolar macrophages.Keywords : fatty-acid-binding protein; alveolar type-II cell; lung fibroblast ; alveolar macrophage; ELISA.In rat lung, alveolar type-II cells synthesise, store and secrete thought to be involved in a facilitated on-demand supply of surfactant substrate [9]. pulmonary surfactant, a phospholipid · protein complex that preParticipation of alveolar macrophages in surfactant metabovents the collapse of the alveoli by reducing surface tension at lism was suggested from in vivo and in vitro experiments [10Ϫ the air-liquid interface. This surfactant is highly enriched in 12]. Theoretically, alveolar macrophages could account for the phospholipids, particularly dipalmitoyl phosphatidylcholine.entire catabolism of alveolar surfactant in rat lung [11]. Fatty Fatty acids required for the biosynthesis of phospholipids are acids derived from phosphatidylcholine degradation are reused either synthesised de novo within this type-II cell [1Ϫ3] or taken for triacylglycerol synthesis [13]. up from exogenous sources [4Ϫ6]. Uptake and intracellular It is obvious from the above that fatty acids are needed for transfer of fatty acids by these alveolar type-II cells are not well lung surfactant homeostasis, in which the three cell types menunderstood [7], but the high turnover of phospholipids requires tioned appear to be involved. As a consequence, fatty ac...