Antibodies to steroid receptor deoxyribonucleic acid binding domains and their reactivity with the human glucocorticoid receptor

Functional properties of the DNA-binding domain of the human glucocorticoid receptor were investigated using high titer polyclonal antibodies produced against single synthetic peptides or a mixture of peptides whose sequences were derived from the DNA-binding domain of steroid receptor proteins. Three of seven antisera recognized both native and denatured forms of the glucocorticoid receptor, although considerably lower antisera dilutions were required for antibody binding to native receptor. Activation of the glucocorticoid receptor to its DNA-binding form was required for antibody recognition of the native receptor. Antisera to the second finger region of the DNA-binding domain caused a portion of the activated 4S glucocorticoid receptor to sediment as 7 or 9S in sucrose gradients containing 0.4 M KCl, but did not alter the sedimentation of the nontransformed 8S receptor. Specificity of the glucocorticoid receptor-antibody interaction was demonstrated by loss of reactivity after preabsorption with peptide antigens. Antisera that interacted specifically with the glucocorticoid receptor inhibited DNA binding of the activated receptor by as much as 80%. Thus, antibody probes directed against DNA-binding domain sequences provide immunological evidence that glucocorticoid receptor activation exposes the DNA-binding region of the receptor.     

A missense mutation (Asp250----Asn) in exon 6 of the human lipoprotein lipase gene causes chylomicronemia in patients of different ancestries.

We have previously reported two common lipoprotein lipase (LPL) gene mutations underlying LPL deficiency in the majority of 37 French Canadians (Monsalve et al., 1990. J. Clin. Invest. 86: 728-734; Ma et al., 1991. N. Engl. J. Med. 324: 1761-1766). By examining the 10 coding exons of the LPL gene in another French Canadian patient, we have identified a third missense mutation that is found in two of the three remaining patients for whom mutations are undefined. This is a G to A transition in exon 6 that results in a substitution of asparagine for aspartic acid at residue 250. Using in vitro site-directed mutagenesis, we have confirmed that this mutation causes a catalytically defective LPL protein. In addition, the Asp250----Asn mutation was also found on the same haplotype in an LPL-deficient patient of Dutch ancestry, suggesting a common origin. This mutation alters a TaqI restriction site in exon 6 and will allow for rapid screening in patients with LPL deficiency.     

A bovine papillomavirus E1-related protein binds specifically to bovine papillomavirus DNA.

The E1 open reading frame of bovine papillomavirus (BPV) was expressed as a RecA-E1 fusion protein in Escherichia coli. The bacterially expressed RecA-E1 protein exhibited sequence-specific DNA binding activity; strong binding to the region from nucleotides 7819 to 93 on the BPV genome (designated region A) and weak binding to the adjacent region from nucleotides 7457 to 7818 (region B) were observed. The interaction between the BPV-derived RecA-E1 protein and region A appeared to be highly specific for BPV DNA, as no comparable binding was detected with heterologous papillomavirus DNAs. Binding to region A was eliminated by digestion of region A at the unique HpaI site, which suggests that the RecA-E1 binding site(s) was at or near the HpaI recognition sequence. Binding to region B but not region A was observed when nuclear extracts from ID13 cells were used as a source of E1 proteins. The absence of region A binding by ID13 extracts may reflect a negative regulation of E1 DNA binding activity.     

Multiple zeins from maize endosperms characterized by reversed-phase high performance liquid chromatography

The major storage proteins of maize (Zea mays L.) endosperm are located in protein bodies, and may be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) into two major classes and four minor classes of polypeptides. The two major classes (commonly known as zeins) have been separated previously into a large number of components by isoelectric focusing (IEF). Reversed-phase high performance liquid chromatography (HPLC) further separated the major classes into additional components, and gave distinctive peaks for each minor zein class. Some IEF bands produced two or more HPLC fractions, while some HPLC fractions produced two or more IEF bands. Apparently identical IEF bands from different inbreds may appear in different fractions after HPLC. Thus the total number of zeins revealed by separations based on apparent size (SDS-PAGE), net charge (IEF), and hydrophobicity (HPLC) is very large. Different laboratories have developed diverse nomenclatures which cause much confusion. A key is presented to provide a flexible and expandable nomenclature for this complex group of proteins.     

Preliminary crystallographic data, primary sequence, and binding data for an anti-peptide Fab and its complex with a synthetic peptide from influenza virus hemagglutinin

X-ray quality crystals which diffract to high resolution (less than or equal to 1.9-2.1 A) have been grown of an anti-peptide Fab and its complex with a 9-residue peptide antigen. Both crystals are monoclinic P2(1), with unit cell dimensions a = 90.3 A, b = 82.9 A, c = 73.4 A, beta = 122.5 degrees for the native Fab and a = 63.9 A, b = 73.0 A, c = 49.1 A, beta = 120.6 degrees for the complex. The peptide sequence corresponds to residues 100-108 of all influenza virus hemagglutinins (HA1) of the H3 subtype (1968-1987). The peptide antigen has been well characterized immunologically (Wilson, I.A., Niman, H.L., Houghton, R.A., Cherenson, A.R., Connolly, M.L., and Lerner, R.A. (1984) Cell 37, 767-778; Wilson, I.A., Bergmann, K.F., and Stura, E.A. (1986) in Vaccines '86 (Channock, R.M., Lerner, R.A., and Brown, F., eds) pp. 33-37, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), structurally, as a free peptide by NMR (Dyson, J.H., Cross, K.J., Houghton, R.A., Wilson, I.A., Wright, P.E., and Lerner, R.A. (1985) Nature 318, 480-483; Dyson, J.H., Lerner, R.A., and Wright, P.E., (1988) Annu. Rev. Biophys. Chem. 17, 305-324), as part of the intact antigen by x-ray crystallography (Wilson, I.A., Skehel, J.J., and Wiley, D. C. (1981) Nature 289, 366-373) and by binding studies to the HA molecule (White, J.M., and Wilson, I.A. (1987) J. Cell Biol. 105, 2887-2896). Knowledge of the three-dimensional structure of the complex will elucidate the details of how anti-peptide antibodies recognize a small peptide antigen and provide insights into the recognition of the same sequence in the intact protein antigen. As both native Fab and the peptide-Fab complex have been crystallized, we can also determine in addition whether changes in the structure of the antibody accompany antigen binding. The nucleotide sequence of the mRNA coding region of the anti-peptide Fab has been determined to provide the amino acid sequence ultimately required for the high resolution three-dimensional structure determination.     

Purification and characterization of the polycation-stimulated protein phosphatase catalytic subunit from porcine renal cortex

The predominant form of phosphorylase phosphatase activity in porcine renal cortical extracts was a polycation-stimulated protein phosphatase. This activity was present in extracts in a high-molecular-weight form which could be converted to a free catalytic subunit by treatment with ethanol, urea, or freezing and thawing in the presence of beta-mercaptoethanol. The catalytic subunit of the polycation-stimulated phosphatase was purified by chromatography on DEAE-Sephacel, heparin-Sepharose, and Sephadex G-75. The phosphatase appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis. The enzyme had an apparent Mr of 35 000 on gel filtration and SDS-polyacrylamide gel electrophoresis. The purified phosphatase could be stimulated by histone H1, protamine, poly(D-lysine), poly(L-lysine) or polybrene utilizing phosphorylase a as the substrate. It preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. The phosphatase was highly sensitive to inhibition by ATP. These results suggest that the renal polycation-stimulated phosphatase catalytic subunit is very similar to or identical with the skeletal muscle phosphatase form which has been previously designated phosphatase-2Ac.     

Anion effects on the reaction of lecithin-cholesterol acyltransferase with discoidal complexes of phosphatidylcholines . apolipoprotein A-I . cholesterol

Discoidal complexes of phosphatidylcholine (PC) . apolipoprotein A-I . cholesterol were prepared with egg PC, palmitoyloleoylPC, dipalmitoylPC, or dimyristoylPC, and were used as substrates of purified lecithin-cholesterol acyltransferase to investigate the effects of neutral salts on the enzymatic reaction. Sodium fluoride, chloride and bromide concentrations up to 1 M, did not affect the properties of the substrate particles, but caused marked and distinct changes in the activity of the enzyme with the various PC particles. The effects of salts were largely due to the anions, which followed the order of the lyotropic series in their inactivating capacity: F- less than Cl- less than Br- less than NO3- less than I- less than SCN-. Sodium salts (F-, Cl-, and Br-) produced a very large increase in the pH optimum of the enzymatic reaction (7.4 to at least 8.5) essentially obliterating the ionization of a functional group with pK of 8.1. The kinetics of the enzymatic reaction revealed major differences among the PC particles, and different responses of their kinetic parameters with increasing salt concentrations. The conclusions reached in this work are the following: (1) The relative reactivity of PC substrates, in discoidal particles, with lecithin-cholesterol acyltransferase depends strongly on the concentration and type of salts in the medium. (2) Anions (in lyotropic series) rather than cations affect the enzymatic reaction. (3) There are functional groups with pK of 8.1 which are affected markedly in their ionization behavior by anion binding. (4) The active site of lecithin-cholesterol acyltransferase and its interaction with anions are affected by the exact nature of the PC-apolipoprotein interface.     

Chemical and physical characterization of rabbit sperm acrosome stabilizing factor

Rabbit Acrosome Stabilizing Factor (ASF) is an epididymal product that reversibly inhibits the process of sperm capacitation. The native molecular weights of the monomer and dimer ASF were determined from sedimentation and diffusion data at 129,000 and 259,000 Mr. The monomer is composed of 92,000 and 38,000 Mr subunits according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with size heterogeneity demonstrated for the latter. The stoichiometry of the subunits appears to be one-to-one by gel scanning. Amino acid and carbohydrate compositions are characteristic of a globular glycoprotein, which is high in cysteine content and is 8.3% carbohydrate by weight. The sugar composition suggests the presence of both high mannose and complex N-linked oligosaccharides with the unusual feature of appreciable amounts of glucose. The isoelectric character of ASF spans a range from 5.3 to 7.0.     

An immunopathologic study of a 330-kD protein defined by monoclonal antibodies and reactive with anti-RTE alpha 5 antibodies and kidney eluates from active Heymann nephritis

There is evidence indicating that the glomerular Ig deposits of Heymann's nephritis (HN)--a model of epimembranous glomerulonephritis--may be formed at least in part in situ by binding of free circulating antibody with brush border (BB) antigen expressed by glomerular epithelial cells. In this work, we provide evidence that a 330-kD protein defined by seven monoclonal antibodies is responsible for HN. 1) Ig eluted from glomeruli of rats with HN induced classically with crude BB preparation bind specifically the 330-kD antigen; 2) passive immunization with monoclonal antibodies induces epimembranous glomerular Ig deposits; 3) active immunization with the 330-kD antigen induces proteinuric glomerulonephritis; 4) the 330-kD antigen was present in the nephritogenic preparation purified by Edgington, Glassock, and Dixon, because it was identified by the corresponding heterologous antisera. These results, obtained by a completely different approach, confirm and extend those of Kerjaschki and Farquhar and provide a link with the classical studies on HN.