Amino acid sequence and relative biological activity of eel atrial natriuretic peptide

A peptide exhibiting vasodepressor and natriuretic activities in rats was isolated from eel atria, and its primary structure was determined as H-Ser-Lys-Ser-Ser-Ser-Pro-Cys-Phe-Gly-Gly-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Tyr-Ser- Gly-Leu-Gly-Cys-Asn-Ser-Arg-Lys-OH. This peptide, termed eel atrial natriuretic peptide (ANP), has sequence homology of 59% to mammalian (human or rat) ANP, 52% to fowl ANP, and 46% to frog ANP. When the biological activity of synthetic eel ANP was compared with that of human ANP, the eel peptide was 110 times more potent for the vasodepressor activity in eels, nearly equipotent for the vasodepressor activity in quails, and 20 times less potent for the vasodepressor and natriuretic activity in rats.     

Isolation and characterization of thymosin beta 9 Met from pork spleen

We have identified a new thymosin beta 4-like peptide in pork spleen. The new peptide (12 mg) and thymosin beta 4 (33 mg) were isolated from 230 g of spleen by solid phase extraction, preparative isoelectric focusing, and HPLC. The new peptide was termed thymosin beta 9 Met to indicate its close relationship to thymosin beta 9 from calf. The only difference from thymosin beta 9 is the substitution of leucine by methionine at position 6. This peptide replaces thymosin beta 10 which is the minor thymosin beta 4-like peptide in most mammals, e.g., in man, rat, mouse, cat, and rabbit. The structure was determined by amino acid analysis, tryptic digestion, and carboxypeptidase digestion. Pork spleen contains 192 micrograms of thymosin beta 4 and 117 micrograms of thymosin beta 9 Met per gram of tissue.     

Cleavage of a 100 kDa membrane protein (aggregin) during thrombin-induced platelet aggregation is mediated by the high affinity thrombin receptors

Thrombin-induced platelet aggregation is accompanied by cleavage of aggregin, a surface membrane protein (Mr = 100 kDa), and is mediated by the intracellular activation of calpain. We now find that agents that increase intracellular levels of platelet cAMP by stimulating adenylate cyclase, also inhibit thrombin binding and platelet activation by destabilizing thrombin receptors on the platelet surface. Iloprost (a stable analog of PGI2) and forskolin each completely inhibited platelet aggregation by 2 nM thrombin and markedly decreased cleavage of aggregin. Thrombin inactivated by D-phenylalanine-L-prolyl-L-arginine chloromethyl ketone (PPACK-thrombin) binds to the highest affinity site for thrombin on the platelet surface, but thrombin modified by N alpha-tosyl-L-lysine chloromethylketone (TLCK-thrombin) does not. We now demonstrate that preincubation of platelets with PPACK-thrombin blocked platelet aggregation and cleavage of aggregin induced by 2 nM thrombin. In contrast, TLCK-thrombin neither blocked platelet aggregation nor the cleavage of aggregin. These results show that a) platelet aggregation and cleavage of aggregin by thrombin (2nm) involves the occupancy of high affinity alpha-thrombin receptors on the platelet surface, and b) stimulators of adenylate cyclase which increase cAMP, inhibit thrombin-induced platelet aggregation and cleavage of aggregin by mechanisms which include inhibiting the binding of thrombin to its receptors.     

Chromium (V) and hydroxyl radical formation during the glutathione reductase-catalyzed reduction of chromium (VI)

Electron spin resonance measurements provide evidence for the formation of long-lived Cr(V) intermediates in the reduction of Cr(VI) by glutathione reductase in the presence of NADPH and for the hydroxyl radical formation during the glutathione reductase catalyzed reduction of Cr(VI). Hydrogen peroxide suppresses Cr(V) and enhances the formation of hydroxyl radicals. Thus Cr(V) intermediates catalyze generation of hydroxyl radicals from hydrogen peroxide through a Fenton-like reaction. Thus the mechanism of Cr(VI) toxicity might involve the interaction between macromolecules and the hydroxyl radicals.     

Solution structure of the chromomycin-DNA complex

The structure of the chromomycin-DNA complex at the deoxyoctanucleotide duplex level has been determined from one- and two-dimensional proton NMR studies in Mg-containing aqueous solution. The NMR results demonstrate that the antitumor agent binds as a symmetrical dimer to the self-complementary d[T-T-G-G-C-C-A-A] duplex with retention of the 2-fold symmetry in the complex. A set of intermolecular nuclear Overhauser enhancements (NOEs) establishes that two chromomycin molecules in the dimer share the minor groove at the G-G-C-C.G-G-C-C segment in such a way that each hydrophilic edge of the chromophore is located next to the G-G.C-C half-site and each C-D-E trisaccharide chain extends toward the 3'-direction of the octanucleotide duplex. In addition, the A-B disaccharide segment and the hydrophilic side chain of the antitumor agent are directed toward the phosphate backbone. The observed changes in nucleic acid NOEs and coupling patterns on complex formation establish a transition to a wider and shallower minor groove at the central G-G-C-C.G-G-C-C segment required for accommodating the chromomycin dimer. The present demonstration that chromomycin binds as a dimer and switches the conformation of the DNA at its G.C-rich minor groove binding site provides new insights into antitumor agent design and the sequence specificity of antitumor agent-DNA recognition.     

The displacement of copper by iron at the specific binding sites of ovotransferrin

We have examined the kinetics and mechanism by which iron can displace copper at the specific metal-binding sites of ovotransferrin. Fe2+ was added to Cu2+-ovotransferrin-CO3(2-) in the presence of NaHCO3 and ambient O2. The reaction has been followed by standard and stopped-flow spectrophotometry, EPR spectroscopy and analysis of chromogen-reactive Fe2+. The reaction is best described as triphasic. An initial jump in absorbance takes place in the first 2 s. In the next minute there is a further increase in absorbance and shift in the spectral maximum from 440 to 446 nm. The third phase is complex. The bulk of the spectrophotometric change, a decrease in absorbance with a shift to a maximum of 453 nm, lasts approx. 3 min. Minor spectral and EPR changes, however, take place over the next several hours. Chromogenic analysis of Fe2+ indicates that approx. 1 min is required to oxidize the Fe2+. EPR spectra reveal the formation of an Fe3+-ovotransferrin complex within the first 20 s; however, this lacks the characteristic doublet of specific Fe3+-ovotransferrin-CO3(2-). The simultaneous presence of specific Cu2+-ovotransferrin-CO3(2-) and Fe3+-ovotransferrin-CO3(2-) signals suggests a period in which the protein specifically binds both metal ions perhaps resulting from a differential reactivity of the two metal-binding sites. The addition of Cu(NO3)2 to Fe3+-ovotransferrin-CO3(2-) resulted in a complex with specific Fe3+ and non-specific Cu2+. The EPR spectrum of this complex and the final product of our displacement reaction were virtually identical. Distinct parallels in reaction of Cu2+-ovotransferrin-CO3(2-) with Fe(NH4)2(SO4)2, Fe(NO3)3 and Fe3+-nitrilotriacetic acid were observed. A reaction sequence involving the binding and oxidation of non-specific Fe2+ followed by Cu2+ displacement by Fe3+ at the specific sites and binding of non-specific Cu2+ is suggested.     

Fetal lamb 3 beta, 20 alpha-hydroxysteroid oxidoreductase: dual activity at the same active site examined by affinity labeling with 16 alpha-(bromo[2'-14C]acetoxy)progesterone

3 beta,20 alpha-Hydroxysteroid oxidoreductase was purified to homogeneity from fetal lamb erythrocytes. The Mr 35,000 enzyme utilizes NADPH and reduces progesterone to 4-pregnen-20 alpha-ol-3-one [Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1] and 5 alpha-dihydrotestosterone to 5 alpha-androstane-3 beta, 17 beta-diol [Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1]. 5 alpha-Dihydrotestosterone competitively inhibits (Ki = 102 microM) 20 alpha-reductase activity, suggesting that both substrates may be reduced at the same active site. 16 alpha-(Bromoacetoxy)progesterone competitively inhibits 3 beta- and 20 alpha-reductase activities and also causes time-dependent and irreversible losses of both 3 beta-reductase and 20 alpha-reductase activities with the same pseudo-first order kinetic t1/2 value of 75 min. Progesterone and 5 alpha-dihydrotestosterone protect the enzyme against loss of the two reductase activities presumably by competing with the affinity alkylating steroid for the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase. 16 alpha-(Bromo[2'-14C]acetoxy) progesterone radiolabels the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase wherein 1 mol of steroid completely inactivates 1 mol of enzyme with complete loss of both reductase activities. Hydrolysis of the 14C-labeled enzyme with 6 N HCl at 110 degrees C and analysis of the amino acid hydrolysate identified predominantly N pi-(carboxy[2'-14C]methyl)histidine [His(pi-CM)].(ABSTRACT TRUNCATED AT 250 WORDS)     

The primary structure of human apolipoprotein A-IV

Human apolipoprotein (apo) A-IV was purified from chylous ascites fluid. Proteolytic peptides produced by trypsin and Staphylococcus aureus V8 proteinase digestions were purified by high-performance liquid chromatography and sequenced. Human apoA-IV contains 376 amino acid residues. The peptide-derived sequence generally matches two previously reported DNA-derived amino acid sequences except for discrepancies in five positions. In order to examine these discrepancies further, one complete apoA-IV cDNA clone and another partial clone were sequenced. Comparison of all the available information indicates that the peptide-derived sequence reported here is accurate. Sequencing errors probably account for some of the discrepancies between the two primary sequences predicted by earlier nucleotide analyses. In certain positions, however, bona fide sequence heterogeneity or cloning artifact cannot be excluded.     

A long range restriction map of the distal human X chromosome short arm around the steroid sulfatase locus.

The distal short arm of the human X chromosome is of interest because it contains genes which escape X chromosome inactivation and because it is subject to frequent deletions in human patients. The steroid sulfatase gene has been particularly well studied as an example of a gene which escapes X inactivation and which is included in a number of these deletion events. For these reasons a physical map of the region around the STS gene would be of interest. We have constructed a rare cutting enzyme map of this area and have determined the position of several nearby markers with respect to STS. We have also oriented the 5' and 3' ends of the STS gene on this map and have determined the centromeric and telomeric portions of the region. Finally, we have shown that this map can be used to locate deletion breakpoints in STS deficient patients.