The lung lamellar body as a functioning membrane in protein-catalyzed phosphatidylcholine transfer

Lung lamellar bodies and liver mitochondria were used to demonstrate that soluble phospholipid transfer proteins from lung transfer phosphatidylcholine to both of these acceptors. The initial rate of transfer to lung lamellar bodies is about half that of the rate of transfer to the liver mitochondria when both acceptor membranes are present at saturating concentrations. Phosphatidylcholine unilamellar vesicles were used to demonstrate that the fatty acyl composition of the membrane phosphatidylcholine is a significant determinant of the rate of phosphatidylcholine transfer catalyzed by these proteins. The lamellar bodies have a unique phosphatidylcholine composition, and these studies suggest that this is an important factor in determining the lower initial rate of transfer to lamellar bodies. The studies have also characterized two phospholipid transfer proteins in rat lung in terms of isoelectric point. Isoelectric points for the two proteins which transfer phosphatidylcholine were found to be 5.6 ± 0.08 and 6.2 ± 0.03.     

Studies on bovine transferrin; isolation and partial characterization

Bovine serum transferrin (type Tf-A) was isolated by a series of four techniques; (a) precipitation with Rivanol; (b) chromatography of the soluble protein fraction on a column of Sephadex G-150; (c) chromatography of the transferrin containing protein zone on a column of DEAE-Sephadex; and (d) chromatography on a column of DEAE-Sephadex after transferrin was treated with neuraminidase.
It was found that an unidentified protein binds firmly to transferrin, and its removal is only possible after the release of the sialic acid residues with neuraminidase. It is possible that this protein is hemopexin. The occurrence of multiple transferrin components is, in part, dependent on the number of sialic acid residues; possible differences in molecular weight or size seem not to be a factor. The amino acid composition of bovine transferrin, and that of each of three subfractions, resembles that of human transferrin. The calculated mol. wt. of bovine transferrin was found to be 67,000 from sedimentation and viscosity data and 72,400 from sedimentation and diffusion measurements. Sedimentation and viscosity data in concentrated urea suggest that bovine transferrin is composed of two subunits, an observation which is in contrast to data from studies which suggest that human transferrin is composed of a single polypeptide chain.     

The isolation and chemical characterization of phosphorylated enkephalin-containing peptides from bovine adrenal medulla

There is increasing evidence that the opioid peptide precursor, proenkephalin A, and its products undergo extensive post-translational modification, in addition to the cleavage at dibasic amino acid sites. We have used an antiserum directed toward the C terminus of Met-enkephalin Arg6-Phe7 in a radioimmunoassay to monitor the purification to homogeneity of four peptide B variants from bovine adrenal medulla, using gel filtration, anion exchange chromatography, and reverse phase high performance liquid chromatography. Amino acid sequence analysis, together with immunochemical data, confirmed that each comprised the primary sequence, proenkephalin A-(209-239). In addition, three of the four variants were shown to be phosphorylated by alkaline phosphatase digestion, microphosphate analysis, and ethanethiol derivatization coupled with amino acid sequence analysis; these variants were shown to have 1, 2, or 3 phosphate groups per peptide chain, which corresponded to their increasing acidic nature. The phosphorylation sites were clustered together at positions Ser7, Ser13, and Ser15 and were in close association with acidic residues. The clustering of phosphorylated residues is unique among regulatory peptide precursors. This region of proenkephalin A is well conserved, which suggests that it constitutes an important novel functional domain.     

Isolation and characterization of thrombomodulin from bovine lung

Bovine thrombomodulin was isolated from the lung by Triton X extraction, affinity chromatography on diisopropyl phosphate-thrombin-agarose, and gel filtration on Ultrogel AcA-44. The final preparation was purified 6000-fold from the membrane extract with a yield of 21%. It showed apparent Mr of 78,000 and 105,000, before and after reduction, respectively, on polyacrylamide gel electrophoresis in SDS. The activity of the thrombomodulin was stable under the conditions of 1% SDS, 8 M urea, pH 2 and 10, and heat treatment at 60 degrees C for 30 min, but was unstable against treatment with 2-mercaptoethanol. Activation of protein C by thrombin in the presence of the thrombomodulin depended on Ca2+, and an equimolar complex formation between thrombin and thrombomodulin was required for the maximum rate activation. The rate of protein C activation by thrombin was increased 900-fold by thrombomodulin. Thrombomodulin inhibited the thrombin-induced fibrinogen clotting and platelet activation. However, it did not affect the inhibition of thrombin by antithrombin III with or without heparin, a protein C inhibitor or several synthetic inhibitors. These properties of bovine thrombomodulin were similar to those of rabbit thrombomodulin reported earlier.     

The isolation and characterization of the link proteins from proteoglycan aggregates of bovine nasal cartilage.

A preparation containing the link proteins may be obtained from bovine nasal cartilage by extraction with 4 M guanidine hydrochloride and by equilibrium density gradient centrifugations of the extract as commonly employed in the isolation of proteoglycan monomers. In the present paper, protein-rich proteoglycans have been removed from such a preparation to give purified link proteins by chromatography on Sepharose CL-6B in 1% sodium dodecyl sulfate. The individual link proteins, which in order of increasing electrophoretic mobility are termed link proteins 1, 2, and 3, have been separated and isolated in a subsequent preparative gel electrophoresis step. The link proteins present in largest amount, link proteins 1 and 2, have essentially the same amino acid compositions, and following partial digestion with the V8 protease from Staphylococcus aureus and analytical electrophoresis in sodium dodecyl sulfate, their peptide patterns closely resemble each other. Therefore,it is probable that link proteins 1 and 2 are structurally similar. Link protein 1 contains more carbohydrate than link protein 2 (9.5% and 3.0%, respectively) and it is suggested that the major difference between them is in carbohydrate content.     

The isolation and characterization of a colony stimulating factor from human lung.

Serum-free conditioned medium from human lung obtained at autopsy provides a rich source of colony stimulating factor which stimulates granulocytic and macrophagic colony growth in both mouse and human bone marrow. The appearance of the factor is enhanced by endotoxin and inhibited by either puromycin or actinomycin D. Human lung colony stimulating factor is stable at the pH range of 6.5-10 and temperature of 56 degrees C for 30 min. It is resistant to trypsin and neuraminidase but is sensitive to subtilisin, chymotrypsin and periodate. It shows heterogeneity on Sephadex gel filtration with two activity peaks having molecular weight of 200 000 and 40 000, respectively. Upon gel electrophoresis, human lung colony stimulating factor migrates in the alpha-globulin post-albumin region. Using the combination procedures of hydroxyapatite chromatography and preparative polyacrylamide gel electrophoresis a 600-fold purification was achieved with a final specific activity of 6-10(5) units per mg protein. The purified colony stimulating factor is very labile; however, the activity can be stabilized by the addition of gelatin or bovine serum albumin at the concentration of 0.1% and 0.2 mg/ml, respectively.     

Purification and characterization of bovine lung endothelin receptor.

Endothelin receptor was purified from bovine lung by a rapid and simple two-step procedure: 1) solubilization with the detergent 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and digitonin and 2) affinity chromatography using biotinylated endothelin and avidin-agarose. Starting from 3.5 kg of bovine lung, about 200 micrograms of pure receptor were obtained. Microsequencing of tryptic fragments of the purified protein revealed a high sequence similarity with the rat endothelin ETB receptor that has very recently been cloned by expression cloning and shown to be nonselective in terms of the ligand specificity. Purification of the receptor in the presence of low (1 mM) and high (50 mM) concentrations of EDTA yielded, as a major form, 34- and 52-kDa species, respectively, indicating that the lower Mr species (34 kDa) is a proteolytic product of the 52-kDa species. Interestingly, this metal proteinase-mediated limited proteolysis did not affect the ligand binding properties of the receptor.     

Purification and characterization of annexin proteins from bovine lung

Calcium-dependent association with a detergent-extracted particulate fraction was used as the first step in the purification of a group of phospholipid binding proteins. Elution of the detergent-insoluble fraction with excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) resulted in the release of several soluble proteins, termed calcium-activated proteins or CAPs. In the present paper, we describe the simultaneous purification of these CAPs and characterize their interaction with phospholipid, actin, and calmodulin. Partial sequence analysis has identified the majority of the CAPs as members of the annexin family of calcium and phospholipid binding proteins. Two additional CAPs may be novel proteins, one of which appears to be an annexin protein. All CAPs demonstrated Ca2(+)-dependent binding to phosphatidylserine vesicles but did not bind to phosphatidylcholine vesicles. The majority of CAPs exhibited Ca2(+)-dependent binding to F-actin; however, only CAP-III affected the rate of conversion of G-actin to F-actin. The interaction of CAP-III and lipocortin-85 with F-actin resulted in a Ca2(+)-dependent increase in both light scattering and sedimentation of F-actin under comparatively low centrifugal force. In contrast, only lipocortin-85 caused the formation of F-actin bundles. Although all of the CAPs bound to a calmodulin affinity column in a Ca2(+)-dependent manner, attempts to demonstrate binding of CAPs to native calmodulin were unsuccessful. These studies therefore document the similar behavior of the CAPs toward phospholipid and calmodulin but clearly show that F-actin binding or bundling is not a general property of these proteins. The reported purification procedure should allow further comparative studies of these proteins.     

The isolation and characterization of the tropomyosin binding component (TN-T) of bovine cardiac troponin.

The tropomyosin binding component (TN-T) of troponin was purified from bovine cardiac muscle using a combination of ion exchange chromatographies in the presence of urea. Sedimentation equilibrium experiments suggest a molecular weight for cardiac TN-T of 36 300 +/- 2 000, consistent with a value of 37 000 +/- 1 000 determining by polyacrylamide gel electrophoresis. Calculations based upon circular dichroism spectra indicate an apparent alpha-helical content of 43 +/- 3% for TN-T. Polyacrylamide gel electrophoresis and the effects of the calcium binding component (TN-C) upon the solubility of TN-T suggest that the two cardiac troponin components can interact with each other. Cosedimentation analysis of solutions containing cardiac tropomyosin and TN-T provide evidence for complex formation involving these two proteins. The data presented on the physical and chemical properties of TN-T, as well as the interaction studies indicate that the cardiac muscle regulatory system operates in a manner similar to that proposed for skeletal muscle.     

Limited proteolysis of bovine alpha-lactalbumin: isolation and characterization of protein domains

The partly folded states of alpha-lactalbumin (alpha-LA) exposed to acid solution at pH 2.0 (A-state) or at neutral pH upon EDTA-mediated removal of the single protein-bound calcium ion (apo form) have been probed by limited proteolysis experiments. These states are nowadays commonly considered to be molten globules and thus protein-folding intermediates. Pepsin was used for proteolysis at acid pH, while proteinase K and chymotrypsin at neutral pH. The expectations were that these proteolytic probes would detect sites and/or chain regions in the partly folded states of alpha-LA sufficiently dynamic, or even unfolded, capable of binding and adaptation to the specific stereochemistry of the protease's active site. A time-course analysis of the proteolytic events revealed that the fast, initial proteolytic cuts of the 123-residue chain of alpha-LA in its A-state or apo form by the three proteases occur at the same chain region 39-54, the actual site(s) of cleavage depending upon the protease employed. This region in native alpha-LA encompasses the beta-sheets of the protein. Subsequent cleavages occur mostly at chain regions 31-35 and 95-105. Four fragment species of alpha-LA have been isolated by reverse-phase high-performance liquid chromatography, and their conformational properties examined by circular dichroism and fluorescence emission spectroscopy. The single chain fragment 53-103, containing all the binding sites for calcium in native alpha-LA and cross-linked by two disulfide bridges, maintains in aqueous buffer and in the presence of calcium ions a folded structure characterized by the same content of alpha-helix of the corresponding chain segment in native alpha-LA. Evidence for some structure was also obtained for the two-chain species 1-40 and 104-123, as well as 1-31 and 105-123, both systems being covalently linked by two disulfide bonds. In contrast, the protein species given by fragment 1-34 connected to fragment 54-123 or 57-123 via four disulfide bridges adopts in solution a folded structure with the helical content expected for a native-like conformation. Of interest, the proteolytic fragment species herewith isolated correspond to the structural domains and subdomains of alpha-LA that can be identified by computational analysis of the three-dimensional structure of native alpha-LA (Siddiqui AS, Barton GI, 1995, Protein Sci 4:872-884). The fast, initial cleavages at the level of the beta-sheet region of native alpha-LA indicate that this region is highly mobile or even unfolded in the alpha-LA molten globule(s), while the rest of the protein chain maintains sufficient structure and rigidity to prevent extensive proteolysis. The subsequent cleavages at chain segment 95-105 indicate that also this region is somewhat mobile in the A-state or apo form of the protein. It is concluded that the overall domain topology of native alpha-LA is maintained in acid or at neutral pH upon calcium depletion. Moreover, the molecular properties of the partly folded states of alpha-LA deduced here from proteolysis experiments do correlate with those derived from previous NMR and other physicochemical measurements.     

The isolation and characterization of the ATPase inhibitory protein (TN-I) from bovine cardiac muscle.

The inhibitory component of the troponin complex (TN-I) was purified from bovine cardiac muscle, using a combination of ion exchange and molecular exclusion chromatographies in the presence of urea. It has the ability to inhibit the Mg2+-activated APTase (EC 3.6.1.3) of a synthetic cardiac actomyosin preparation and this inhibition is reversed by the addition of cardiac calcium binding component of troponin (TN-C). Conventional sedimentation equilibrium experiments suggest a molecular weight for cardiac TN-I of 22 900 +/- 500. However, sodium dodecyl sulfate (SDS) gels indicate a molecular weight of 27 000 +/- 1000. The mobility of TN-I on SDS gels may be anomalous due to the high proportion of basic amino acid residues in the protein. Cardiac TN-I and TN-C interact to form a tight complex, even in the presence of 6 M urea. The results of this study invite direct comparison with results published for rabbit skeletal TN-I.     

Localization, isolation and characterization of Mn-superoxide dismutase in bovine adrenocortical cells

Cyanide-insensitive superoxide dismutase activity was present in both cytosol (26%) and mitochondrial (64%) fractions of bovine adrenal cells. The cyanide-insensitive superoxide dismutase was isolated from the mitochondrial fraction. It contained 2.2 g atoms of manganese per mol of enzyme. The enzyme had a molecular weight of 82,000 and a subunit molecular weight of 22,000. The isoelectric point, amino acid composition, and spectra of visible and ultraviolet regions were similar to those of the Mn-superoxide dismutase from the other mitochondria.     

The isolation and characterization of individual phenol-soluble nuclear proteins from various reproductive cells during bovine spermatogenesis

The metabolic turnover of phenol-soluble nuclear proteins during bovine spermatogenesis is described. Several methods for the isolation and purification of two proteins, one from early developing sperm cells, and one from mature sperm cells, are also discussed. These methods involve ways in which single proteins can be extracted from polyacrylamide gels and ultimately suspended in an aqueous phase. Preliminary evidence is presented which suggests that these two proteins are chemically distinct and may be functionally related to sperm maturation.