Base sequence studies of 300 nucleotide renatured repeated human DNA clones

A band of 300 nucleotide long duplex DNA is released by treating renatured repeated human DNA with the single strand-specific endonuclease S₁. Since many of the interspersed repeated sequences in human DNA are 300 nucleotides long, this band should be enriched in such repeats. We have determined the nucleotide sequences of 15 clones constructed from these 300 nucleotide S₁-resistant repeats. Ten of these cloned sequences are members of the Alu family of interspersed repeats. These ten sequences share a recognizable consensus sequence from which individual clones have an average divergence of 12.8%. The 300 nucleotide Alu family consensus sequence has a dimeric structure and was evidently formed from a head to tail duplication of an ancestral monomeric sequence. Three of the remaining clones are variations on a simple pentanucleotide sequence previously reported for human satellite III DNA. Two of the 15 clones have distinct and complex sequences and may represent other families of interspersed repeated sequences.     

Antibodies directed toward human erythrocyte Ca2+-ATPase: effect on enzyme function and immunoreactivity of Ca2+-ATPases from other sources

Antibodies directed against purified human erythrocyte Ca²⁺-ATPase (purified according to a procedure modified from V. Niggli, J. T. Penniston, and E. Carafoli, 1979, J. Biol. Chem., 254, 9955–9958) were raised in rabbits. In competitive radioimmunoassay tests of immunological cross-reactivity, human erythrocyte Ca²⁺-ATPase shows a consistent pattern of immunological similarity to the Ca²⁺-ATPases derived from cell surface fractions of other species, such as rat and dog erythrocyte ghosts, rat corpus luteum plasma membranes, and rat brain synaptic plasma membranes. On the other hand, a purified Ca²⁺-ATPase preparation from rabbit skeletal muscle sarcoplasmic reticulum failed to show any immunological similarity to the human enzyme. The amount of Ca²⁺-ATPase protein in the erythrocyte ghosts was estimated to be about 0.6 μg/mg ghost protein, which was not too different from the calculated value of 1.2 ± 0.2 μg/mg ghost protein (mean ± SD, n = 6) based on the calmodulin binding studies of the erythrocyte ghosts. Anti-Ca²⁺-ATPase immunoglobulin G inhibited enzyme activity and calcium transport, showing that at least one subpopulation of antibodies can block the active site of the enzyme. The antibodies had no effect on the binding of calmodulin to erythrocyte membranes.     

Interaction of epoxide hydrolase with itself and other microsomal proteins

The interactions of rat liver epoxide hydrolase (EC 3.3.2.3) with itself and with cytochromes P-450 and NADPH-cytochrome P-450 reductase were investigated in microsomal preparations and in reconstituted systems in which all of the enzymes are functionally active. Hydrodynamic measurements indicated that purified epoxide hydrolase behaves as a single aggregate of approximately 16 monomeric units and that further aggregation of the protein only occurs in the presence of high concentrations of phospholipid. Neither guanidine-HCl nor the nonionic detergent Lubrol PX was able to completely dissociate the aggregate into monomers. The interactions of epoxide hydrolase with NADPH-cytochrome P-450 reductase and the major forms of cytochrome P-450 isolated from phenobarbital- and 5,6-benzoflavone-treated rats were studied by Soret difference spectroscopy, by perturbation of the fluorescence of NADPH-cytochrome P-450 reductase and fluorescein-labeled epoxide hydrolase, and by CD spectroscopy. The spectra provided evidence that binding of the proteins to each other occurs and some of the results suggest that affinity constants are on the order of 10⁷, m^?1. The spectral perturbations were not observed with other intrinsic membrane proteins. When microsomes were treated with the crosslinking reagent dimethylsuberimidate and solubilized with detergents, epoxide hydrolase could be precipitated with antibodies raised to cytochromes P-450 or NADPH-cytochrome P-450 reductase. Transient times were determined for the conversion of 1-octene to octene-1,2-dihydrodiol in a reconstituted enzyme system and for the conversion of naphthalene to naphthalene-1,2-dihydrodiol in rat liver microscomes and compared to the transient times predicted from the enzymatic rates of hydrolysis of the intermediate epoxides. In all cases the observed transient times were shorter than expected, in support of the view that coupling of epoxide hydrolase with cytochromes P-450 occurs. These results support the view that epoxide hydrolase couples with cytochrome P-450-containing mixed-function oxidase systems and may have relevance to the metabolism of potentially harmful xenobiotics by these enzymes.     

The complete amino acid sequence of bovine liver catalase and the partial sequence of bovine erythrocyte catalase

The data upon which the sequence of the 506 residues in the subunit of bovine liver catalase (BLC) is based are presented in detail. A partial sequence of bovine erythrocyte catalase (BEC) which accounts for 493 residues shows complete concordance with the BLC data. On the other hand, BEC has at least 517 residues, that is, an extension beyond the C terminus of the BLC data. Although normally BLC has only 506 residues, there is evidence that, at some point in its history, it also had the C-terminal extension. It is speculated that this extension is lost in BLC either through a different processing of the molecule in liver than in erythrocytes or by partial degradation in the first stages of catabolism.     

Isolation of Paracoccus denitrificans cytochrome cd1: comparative kinetics with other nitrite reductases

The dissimilatory nitrite reductase of the cytochrome cd₁ type was purified from Paracoccus denitrificans (ATCC 13543) by a novel procedure that avoided conventional ion-exchange techniques. The characterization of this enzyme was extended to include amino acid composition, extinction coefficients, and kinetic properties not previously reported. Cytochromes cd₁ from Alicaligenes faecalis and Pseudomonas aeruginosa were also isolated and assayed with electron donor proteins. The enzymes from all three sources were shown to obey the same integrated rate law. Cross-reactivities were measured in which a reduced donor protein from one strain was assayed with cytochrome cd₁ from another strain using nitrite as ultimate acceptor. Donors included c-type cytochromes and azurins. In general, the enzymes showed specificity for a donor from the same strain; interspecies cross-reactions were typically slower on the order of 10-fold than corresponding native rates. Notable exceptions were Paracoccus cytochrome cd₁, which alone reacted with eukaryotic horse cytochrome c at appreciable rates, and the Pseudomonas cd₁-Alcaligenesc554 reaction, which was 4-fold faster than the native Alcaligenes cd₁-Alcaligenesc554 reaction. For all three enzymes, competitive kinetics were measured in which the alternative substrates, nitrite and oxygen, competed for enzyme in the same assay. It was found that the competitive kinetics were dominated by nonenzymatic reactions involving an enzyme product, nitric oxide.     

Synthesis of a branched pentasaccharide sequence of "bisected" structure of N-glycoproteins

For the synthesis of the threefold-branched pentasaccharide, O-alpha-D-mannopyranosyl-(1----3)-O-[(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-(1----4)]-O-[alpha-D-mannopyranosyl-(1----6)]-O-beta-D- mannopyranosyl-(1----4)-2-acetamido-2-deoxy-D-glucopyranose (20), which is a part of the structure of the N-glycoproteins, the disaccharide 4-O-(4-O-acetyl-3,6-di-O-allyl-2-O-benzyl-beta-D-mannopyranosyl) -1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-beta-D-glucopyranose was synthesized as a key compound by use of the silver silicate-catalyst procedure. After elimination of the 4-O-acetyl group, a 2-acetamido-2-deoxy-beta-D-glucopyranosyl group was attached according to the phthalimido method. Further elimination of the allyl groups allowed the linkage of two alpha-D-mannopyranosyl groups in the presence of mercury salt. A deblocking sequence consisting of four steps gave 20.     

Reaction between iron(III) and pyrazoic acid

In the reaction of [Fe(H₂O)₆]³⁺ with pyrazoic acid, reduction of iron(III) to iron(II) is observed. When an excess of iron is present, the reaction involves a transfer of four electrons per mole of acid. At room temperature the redox reaction, which is dependent on hydrogen ion, iron(III) and pyrazoic acid concentrations, is rather slow and is the rate-determining step. The kinetic study was carried out at 50.0 ± 0.1 °C. The redox reaction is followed by a fast reaction of the iron(II) with an excess of ligand, resulting in the production of well-known complexes, where the acid acts as a chelating ligand through the nitrogen and oxygen atoms.     

A dynamical study on the interactions between the cytoskeleton components in the human erythrocyte as detected by saturation transfer electron paramagnetic resonance of spin-labeled spectrin, ankyrin, and protein 4.1

Isolated human erythrocyte spectrin, ankyrin, and protein 4.1 have been labeled with the maleimide spin label, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl, and studied by saturation transfer electron paramagnetic resonance spectroscopy. The presence of the labels does not affect the reassociation of these proteins with erythrocyte membranes selectively depleted of either spectrin-actin or of all the extrinsic proteins. When maleimide spin-labeled spectrin is reassociated with the erythrocyte membrane in presence of all the cytoskeleton components, including endogeneous or purified muscle actin, spectrin still preserves its flexible character. The rotational mobilities of maleimide spin-labeled ankyrin and maleimide spin-labeled protein 4.1 are of the same order of magnitude (tau c (L"/L) approximately 5 X 10(-5) and 8 X 10(-5) s, respectively, at 2 degrees C), while protein 4.1 is almost three times smaller in size than ankyrin. This result indicates that the movements of membrane-bound maleimide spin-labeled protein 4.1 are more restricted than those of ankyrin. This suggests that their respective binding sites have different structural properties. The rotational movements of both proteins are slowed down on the addition of spectrin indicating that protein 4.1 as well as ankyrin also represents one of the links of the cytoskeleton to the membrane.     

Monoclonal antibodies can discriminate between some active and inactive forms of leukocyte interferon

Antiviral activity of recombinant human leukocyte A interferon was inactivated by heating at 65 degrees C or by reduction of disulfide bonds. The specific immunoreactivity, as measured by radioimmunoassays measuring binding to monoclonal antibodies, decreased concomitantly with the antiviral activity. Although the monoclonal antibodies did bind to inactivated interferon, their binding affinity to inactivated interferon was in general very much lower than their binding affinity to active interferon. Therefore, this immunoassay could replace the antiviral assay for detection of biologically active interferon. In addition, most of these antibodies should be especially useful for purification of the interferons since they discriminate between the native active and inactive denatured species. Screening for such antibodies is convenient and simple. The general use of antibodies that preferentially interact with native molecules provides a powerful new principle for choosing monoclonal antibodies with extraordinary potential in assay and purification.     

Fatty acid synthetase, malic enzyme and other NADP+ binding dehydrogenases have similar antigenic determinant(s) at the NADPH binding domain

The sequence of tryptic and chymotryptic peptides from cytosolic and mitochondrial rabbit liver serine hydroxymethyltransferase are compared to the proposed sequence of a protein coded for by the glyA gene of Escherichia coli. The E. coli glyA gene is believed to code for serine hydroxymethyltransferase. Extensive sequence homology between these peptides were found for the proposed E. coli enzyme in the aminoterminal two-thirds of the molecule. All three proteins have identical sequences from residue 222-231. This sequence is known to contain the lysyl residue which forms a Schiff's base with pyridoxal-P in the two rabbit liver enzymes. These results support the interpretation that the proposed sequence of E. coli serine hydroxymethyltransferase is correct. The data also show that cytosolic and mitochondrial serine hydroxymethyltransferase are homologous proteins.     

The interaction between vitamin B-12 and micelles in aqueous solution

The apparent pK for benzimidazole displacement of a number of cobalamins is markedly affected by the presence of sodium lauryl sulfate micelles. However, micelles of cetyltrimethylammonium bromide or Triton X have little or no effect on the pK. By measuring the apparent pK as a function of sodium lauryl sulfate concentration, the association constants between the micelles and both base on and base off methylcobalamin were calculated. This calculation indicates that the base off form is strongly associated with the micelle while the base on form is not.     

Effect of adenosylhomocysteine and other analog thioethers on a prokaryotic tRNA (guanine-7)-methyltransferase

S-Adenosylhomocysteine (SAH), a potent inhibitor of methyltransferases, and several thioethers structurally related to SAH, have been tested as potential inhibitors of tRNA (guanine-7)-methyltransferase from Salmonella typhimurium. The tested compounds are l-, d-, dl-S-adenosylhomocysteine, S-adenosylcysteine, methylthioadenosine, butylthioadenosine, thioethanoladenosine, isobutylthioadenosine, S-inosylhomocysteine, and methylthioinosine. Among them the highest inhibitory activity has been shown by SAH (K_i = 8 μM), whereas butylthioadenosine, isobutylthioadenosine, and thioethanoladenosine are almost inactive as inhibitors. The other compounds inhibit the enzyme with K_i values ranging between 400 and 800 μm. From these data it is possible to evaluate the importance of the -NH₂ and -COOH groups of the substrate in the binding to the enzyme molecule, as well as other features such as the chirality at the α-carbon atom and the length of the hydrocarbon chain connecting the -NH₂ and -COOH groups to the aromatic ring of adenosine. The aminic group of the adenosine is also critical, because S-inosylhornocysteine and methylthioinosine are poorer inhibitors in comparison with SAH and methylthioadenosine.     

The major sialoglycoprotein of human T-lympocytes

Human, peripheral-blood T-lymphocytes and human, T-lymphoblastoid cells of a MOLT 4B cell-line were surface-labeled by lactoperoxidase-catalyzed iodination, periodate and sodium borotritide, and galactose oxidase and sodium borotritide, and analyzed by dodecyl sodium sulfate-polyacrylamide gel-electrophoresis. Both types of cells were found to show a major, cell-surface sialoglycoprotein with an apparent mol. wt. of 95 000. After neuraminidase treatment, this glycoprotein showed a higher mol. wt. of 120 000. The major sialoglycoprotein of both types of cells bound to wheat-germ agglutinin and concanavalin A and, after neuraminidase treatment, to Arachis hypogaea agglutinin. The glycopeptides obtained from these glycoproteins by Pronase digestion gave similar elution-profiles on Sephadex G-50 gel filtration. These results suggest that the major sialoglycoprotein of normal T cells and that of MOLT 4B cells are very similar, if not identical.     

Inhibition of ornithine decarboxylase in human fibroblast cells by type I and type II interferons

Dilution of human fibroblast GM2767 cell cultures into fresh serum-containing growth medium induces ornithine decarboxylase activity 45-fold over a six-hour interval. When the fibroblast cultures are supplemented with human fibroblast α-, β-, or γ-interferon at the time of dilution into fresh growth medium, the induction of ornithine decarboxylase is inhibited 61%, 90%, and 65%, respectively. β-Interferon is the most effective type of interferon to inhibit induction of ornithine decarboxylase.     

Isolation and characterization of a DNA sequence complementary to rat liver glutathione S-transferase B mRNA

Total rat liver poly(A+)-RNA has been isolated from phenobarbital-treated rats and fractionated on sucrose gradients to enrich for glutathione S-transferase B mRNA. Poly(A+)-RNA fractions were assayed for glutathione S-transferase B mRNA activity by in vitro translation and those fractions enriched in glutathione S-transferase B mRNA were used as a template for cDNA synthesis. The cDNA was cloned into the PstI site of pBR322 by G-C tailing. Bacterial clones harboring inserts complementary to glutathione S-transferase mRNA were identified by colony hybridization using a [32P]cDNA probe reverse transcribed from poly(A+)-RNA enriched significantly in glutathione S-transferase B mRNA and by hybrid-select translation. Two recombinant clones, pGTB6 and pGTB15 hybrid-selected the mRNAs specific for the Ya and Yc subunits, indicating these two mRNAs share significant sequence homology. Radiolabeled pGTB6 was utilized in RNA gel-blot experiments to determine that the size of glutathione S-transferase B mRNA is 980 nucleotides and the degree of induction of the mRNA in response to 3-methylcholanthrene administration is threefold.     

Kinetics of electron transfer between Ce(IV) nitrate and iron(II) complexes

The rate constants for the oxidation of some 1,10- phenanthroline and 2,2’-bipyridine complexes of iron(II) by cerium(IV) in nitrate media are reported. In the acidity range investigated (0.05–2.0 M), the predominant reactive species is Ce⁴⁺, although CeOH³⁺ also contributes to the reaction progress.The results are shown to be consistent with the Marcus theory for outer-sphere electron transfer reactions, and the intrinsic parameter for Ce^4+/3+ couple was estimated.     

Sensitive substrates for human leukocyte and porcine pancreatic elastase: a study of the merits of various chromophoric and fluorogenic leaving groups in assays for serine proteases.

Five sensitive substrates of human leukocyte and porcine pancreatic elastase having the sequence MeO-Suc-Ala-Ala-Pro-Val-X where -X is -NA (4-nitroanilide), -SBzl (thiobenzyl ester), -OEt, -AMC (4-methyl-7-coumarylamide), or -NNapOMe (1-methoxy-3-naphthylamide), were synthesized. The kinetic constants for the enzymatic hydrolysis as well as the sensitivity of each substrate are reported. Hydrolysis of the peptide -AMC and -NNapOMe derivatives were followed by monitoring spectrofluorometrically the release of H-AMC and H-NNapOMe, respectively. Cleavage of the thiobenzyl ester yields benzyl mercaptan as the hydrolysis product. Its release was monitored at 412 nm by reaction with Ellman's reagent [5,5′-dithiobis(2-nitrobenzoic acid)] in the assay mixture to produce the 3-carboxy-4-nitrothiophenoxide anion or at 324 nm by reaction with 4,4′-dithiodipyridine to produce 4-thiopyridone. Hydrolysis of the ethyl ester was measured using a coupled assay with NAD⁺ and liver alcohol dehydrogenase. The NADH⁺ formed upon oxidation of the ethanol released from cleavage of the ester was followed at 340 nm. MeO-Suc-Ala-Ala-Pro-Val-SBzl, the best substrate of the series, was capable of detecting as little as 2.4 pm (0.072 ng/ml) of active-site titrated human leukocyte elastase and 5.8 pm (0.15 ng/ml) of active-site titrated porcine pancreatic elastase using 4,4′-dithiodipyridine. The corresponding values with Ellman's reagent were 5.0 pm (0.15 ng/ml) and 7.4 pm (0.19 ng/ml), respectively. Advantages of this substrate are its high $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ values and ease of synthesis. One disadvantage is the interference of high concentration of thiols with the assay. The peptidyl-AMC is almost as sensitive as the thiobenzyl ester and can detect 11 pm of HL elastase and 18 pm of PP elastase. An advantage of this substrate is the fact that cleavage involves a peptide bond. Disadvantages are relatively low $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ values and greater difficulty in synthesis. The peptidyl-NNapOMe has possible utility in histochemical studies due to the low intrinsic fluorescence of the substrate relative to the peptidyl-AMC. For a rate assay it has a lower sensitivity than either the thiobenzyl ester or peptidyl-AMC. The coupled liver alcohol dehydrogenase-ethyl ester assay offers no advantages except in cases where the ester substrate is commercially available. The 4-nitroanilide assay enjoys moderate sensitivity and is extremely convenient for routine use. Except for the peptidyl ethyl ester assay, all of the human leukocyte elastase assays reported in this paper are vastly more sensitive than any other existing assay for this protease.