Identification of proteins associated with apolipoprotein A-I-containing lipoproteins purified by selected-affinity immunosorption.

The isolation of apolipoprotein A-I-containing lipoproteins [Lp(A-I)] by selected-affinity immunosorption minimizes the loss of associated proteins that occurs during the isolation of high-density lipoproteins (HDL) by sequential ultracentrifugation. We have used two-dimensional gel electrophoretic analysis to separate the proteins associated with Lp(A-I). Using a combination of amino acid sequencing of transblotted proteins and Western blotting with specific antisera, we have identified a number of associated proteins. The positions of the apolipoproteins (apo) A-I, A-II, A-IV, C-III, D, and E were located on the gels. Lecithin-cholesterol acyltransferase and cholesteryl ester transfer protein were identified in association with Lp(A-I) to a greater extent than found associated with HDL after centrifugation. In addition to those proteins previously identified in association with HDL, we detected a number of plasma proteins associated with Lp(A-I), namely, fibrinogen, haptoglobin, proline-rich protein (C4b-binding protein), and apolipoprotein J (SP40,40 sulfated glycoprotein). The co-isolation of these proteins with Lp(A-I) does not appear to be an artifact in that they have very low affinity for a sham column containing covalently bound preimmune goat IgG in place of the anti-apoA-I IgG. These findings suggest that in addition to apolipoproteins that exist largely in association with lipoproteins there is another class of proteins which exist in lipoprotein-associated form and in the dispersed state. Detection and identification of these lipoprotein-associated proteins may aid in the mechanistic determination of a number of observed functions attributed to HDL.

The molecular biology of human renin and its gene.

The molecular biology of renin, prorenin, and the renin gene have been studied. A tissue-specific pattern of expression was found in rat and human tissues. In the human placenta, the transfected and endogenous renin promoters are active, and renin mRNA levels and transfected promoter activity are increased by a calcium ionophore plus cAMP. Cultured pituitary AtT-20 cells transfected with a preprorenin expression vector mimick renal renin release by converting prorenin to renin and releasing renin in response to 8Br-cAMP. Studies with mutant renin genes suggest that the body of renin directs renin to the regulated secretory pathway, and renin glycosylation affects its trafficking. Chinese hamster ovary cells were used to produce recombinant prorenin. Infused prorenin was not converted to renin in monkeys. Renin crystals were used to determine its three-dimensional structure. Renin resembles other aspartyl proteases in the active site and core, but it differs in other regions that probably explain renin's unique substrate specificity. Based on structural and mutational analysis, a model for human prorenin was built that suggests lysine -2 of the prosegment interacts with active site aspartate residues, and that the prosegment inactivation of renin is stabilized by binding of an amino terminal beta strand into a groove on renin.

Structure of recombinant human renin, a target for cardiovascular-active drugs, at 2.5 A resolution.

The x-ray crystal structure of recombinant human renin has been determined. Molecular dynamics techniques that included crystallographic data as a restraint were used to improve an initial model based on porcine pepsinogen. The present agreement factor for data from 8.0 to 2.5 angstroms (A) is 0.236. Some of the surface loops are poorly determined, and these disordered regions border a 30 A wide solvent channel. Comparison of renin with other aspartyl proteinases shows that, although the structural cores and active sites are highly conserved, surface residues, some of which are critical for specificity, vary greatly (up to 10A). Knowledge of the actual structure, as opposed to the use of models based on related enzymes, should facilitate the design of renin inhibitors.

Semi-preparative purification of recombinant human renin and prorenin.

Chinese hamster ovary (CHO) cells, transfected with a vector containing cDNA coding for preprorenin, have been shown to secrete authentic prorenin into the culture supernatant. Purification of the expressed prorenin and purification of active renin, generated by solid-phase trypsin treatment of the conditioned media, have been achieved by conventional chromatographic methods. Scale-up of the initial steps of these procedures is described, including the use of radial-flow columns and automation with fast protein liquid chromatography valves and pumps. This semi-preparative scheme has allowed hundreds of milligrams of both proteins to be isolated.

Characterization of recombinant human prorenin and renin.

A cell line that secretes substantial quantities of recombinant human prorenin was prepared by transfecting Chinese hamster ovary cells with a gene encoding preprorenin. The prorenin was purified to homogeneity and was found to have a single amino terminus, reflecting cleavage after a typical 23 amino acid signal sequence. The purified inactive prorenin was not a substrate for active renin and was not capable of self-activation. Prorenin could be converted to renin by addition of exogenous protease, and deglycosylation of the prorenin did not alter the sensitivity to protease activation. The enzymatic activity of deglycosylated renin was kinetically identical to that of the native protein. Multimilligram quantities of recombinant human renin and prorenin were purified, providing suitable material for studies directed toward greater understanding of the function of these proteins and for structural studies such as x-ray diffraction for use in design of renin inhibitors.

Hypothalamic factor inhibits the (Na,K)ATPase from the extracellular surface. Mechanism of inhibition.

We have characterized the effect of a stable small molecule isolated from bovine hypothalamus (Haupert, G. T., and Sancho, J. M. (1979) Proc. Natl. Acad. Sci. 76, 4658-4660) on mammalian (Na,K)ATPase. This hypothalamus-derived inhibitory factor, HIF, has been shown to inhibit ATPase activity of purified dog kidney enzyme reversibly with high affinity (Haupert, G. T., Carilli, C. T., and Cantley, L. C. (1984) Am. J. Physiol. 247, F919-F924). In this report it is shown that HIF inhibits the ouabain sensitive component of 86Rb+ uptake into human red blood cells. HIF also inhibited (Na,K)ATPase activity of unsealed red cell membranes but not that of sealed inside-out vesicles, indicating that HIF is impermeant to red cell membranes and inhibits the (Na,K)ATPase from the extracellular side. In unsealed human red cell membranes, concentrations of HIF which caused 70% inhibition of the (Na,K)ATPase did not inhibit ATP hydrolysis by plasma membrane (Ca2+)ATPase or (Mg2+)ATPase. However, at a similar concentration, HIF was shown to inhibit rabbit muscle sarcoplasmic reticulum (Ca2+)ATPase. HIF also inhibited p-nitrophenylphosphatase activity of unmodified or fluorescein-5'-iso-thiocyanate labeled dog kidney (Na,K)ATPase. As judged by fluorescein fluorescence of the modified enzyme, HIF stabilized the low fluorescent "E2" conformation of the enzyme similar to that stabilized by ouabain. However, unlike ouabain, HIF blocked covalent phosphorylation of dog kidney (Na,K)ATPase by inorganic phosphate. These studies show that HIF is an inhibitor of (Na,K)ATPase which acts from the extracellular side of the membrane by a mechanism similar to but not identical to that of cardiac glycosides.

Hypothalamic sodium-transport inhibitor is a high-affinity reversible inhibitor of Na+-K+-ATPase.

Bovine hypothalamus contains a stable, low molecular weight substance with ouabain-like properties. To further study its mechanism of action and potential physiological importance we examined its effects on purified Na+-K+-ATPase in a kinetic coupled-enzyme assay. Under optimal conditions up to 95% of Na+-K+-ATPase activity could be inhibited by the factor. Mg2+ is required for maximal inhibitory activity, but ligand requirements for optimal activity are otherwise distinct from those of both ouabain and vanadate. Inhibition is reversed by high concentrations of sodium chloride plus EDTA. Kinetic analysis yielded a Ki = 1.4 nM. The hypothalamic factor is a high-affinity reversible inhibitor of Na+-K+-ATPase, being at least as potent as the cardiac glycoside ouabain and may be a circulating inhibitor of sodium transport, which appears to be associated with experimental volume-expanded hypertension and human essential hypertension.

The amino acid sequence of a fluorescein-labeled peptide from the active site of (Na,K)-ATPase.

(Na,K)-ATPase in an active-transport protein that couples the energy obtained from the hydrolysis of ATP to the transport of Na+ and K+ across animal cell membranes. In order to investigate the enzymatic mechanism of this activity, a peptide derived from the ATP-binding site of (Na,K)-ATPase has been purified and its amino acid sequence has been determined. The peptide was identified by the covalent incorporation of a fluorescent probe, fluorescein 5'-isothiocyanate, into the active site before trypsin digestion of the protein. The labeling of (Na,K)-ATPase by fluorescein 5'-isothiocyanate was associated with the irreversible inhibition of enzymatic activity, and both the labeling of the tryptic peptide and inhibition of activity were prevented when the reaction was performed in the presence of ATP. An apparent KD of 5.7 microM was calculated when the reaction between (Na,K)-ATPase and fluorescein 5'-isothiocyanate was performed under pseudo first-order conditions. The amino acid sequence of the active-site peptide, His-Leu-Leu-Val-Met-Lys-Gly-Ala-Pro-Glu-Arg, is similar to the sequence of a fluorescein-labeled peptide derived from the active site of the sarcoplasmic reticulum Ca2+-transport ATPase (Mitchinson, C., Wilderspin, A. F., Trinaman, B. J., and Green, N. M. (1982) FEBS Lett. 146, 87-92).

The active site structure of Na+- and K+-stimulated ATPase. Location of a specific fluorescein isothiocyanate reactive site.

Fluorescein 5'-isothiocyanate (FITC) has been shown to specifically inactivate the Na+- and K+-stimulated adenosine triphosphatase ((Na,K)-ATPase) at low concentrations (Karlish, S. J. D. (1979) Na+,K+ATPase Structure and Kinetics 115-128). The site of modification of purified dog kidney (Na,K)-ATPase by FITC has been investigated by enzymatic cleavage and fluorescence resonance energy transfer. The binding of FITC, which occurs at a stoichiometry of approximately one site per ATP binding site, causes an ATP-protectable inactivation of ATPase activity suggesting that it is reacting at the ATP hydrolysis site. The FITC reaction site apparently is located near the center of the COOH-terminal 77,000-dalton peptide fragment obtained by chymotryptic cleavage of the alpha subunit. Addition of ouabain to the native enzyme in the presence of chymotrypsin enhances cleavage at this site and releases the fluorescein moiety from the membrane. It is further shown that the distance from the FITC reaction site to the ouabain binding site, as judged by fluorescence resonance energy transfer from anthroyl ouabain to FITC, is approximately 74 A. These results demonstrate that ouabain inhibits the (Na,K)-ATPase by causing a protein conformational change which extends an unusually large distance across the membrane.