Showing papers in "Journal of Biological Chemistry in 1982"

Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters

Abstract: Tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) directly activate in vitro Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), which normally requires unsaturated diacylglycerol. Kinetic analysis indicates that TPA can substitute for diacylglycerol and greatly increases the affinity of the enzyme for Ca2+ as well as for phospholipid. Under physiological conditions, the activation of this enzyme appears to be linked to the receptor-mediated phosphatidylinositol breakdown which may be provoked by a wide variety of extracellular messengers, eventually leading to the activation of specific cellular functions or proliferation. Using human platelets as a model system, TPA is shown to enhance the protein kinase C-specific phosphorylation associated with the release reaction in the total absence of phosphatidylinositol breakdown. Various phorbol derivatives which have been shown to be active in tumor promotion are also capable of activating this protein kinase in in vitro systems.

Codon selection in yeast.

Abstract: Extreme codon bias is seen for the Saccharomyces cerevisiae genes for the fermentative alcohol dehydrogenase isozyme I (ADH-I) and glyceraldehyde-3-phosphate dehydrogenase. Over 98% of the 1004 amino acid residues analyzed by DNA sequencing are coded for by a select 25 of the 61 possible coding triplets. These preferred codons tend to be highly homologous to the anticodons of the major yeast isoacceptor tRNA species. Codons which necessitate site by side GC base pairs between the codons and the tRNA anticodons are always avoided whenever possible. Codons containing 100% G, C, A, U, GC, or AU are also avoided. This provides for approximately equivalent codon-anticodon binding energies for all preferred triplets. All sequenced yeast genes show a distinct preference for these same 25 codons. The degree of preference varies from greater than 90% for glyceraldehyde-3-phosphate dehydrogenase and ADH-I to less than 20% for iso-2 cytochrome c. The degree of bias for these 25 preferred triplets in each gene is correlated with the level of its mRNA in the cytoplasm. Genes which are strongly expressed are more biased than genes with a lower level of expression. A similar phenomenon is observed in the codon preferences of highly expressed genes in Escherichia coli. High levels of gene expression are well correlated with high levels of codon bias toward 22 of the 61 coding triplets. As in yeast, these preferred codons are highly complementary to the major cellular isoacceptor tRNA species. In at least four cases (Ala, Arg, Leu, and Val), these preferred E. coli codons are incompatible with the preferred yeast codons.

Superoxide radical inhibits catalase.

Abstract: Catalase was inhibited by a flux of O2- generated in situ by the aerobic xanthine oxidase reaction. Two distinct types of inhibition could be distinguished. One of these was rapidly established and could be as rapidly reversed by the addition of superoxide dismutase. The second developed slowly and was reversed by ethanol, but not by superoxide dismutase. The rapid inhibition was probably due to conversion of catalase to the ferrooxy state (compound III), while the slow inhibition was due to conversion to the ferryl state (compound II). Since neither compound III nor compound II occurs in the catalatic reaction pathway, they are inactive. This inhibition of catalase by O2- provides the basis for a synergism between superoxide dismutase and catalase. Such synergisms have been observed in vitro and may be significant in vivo.

Cytoplasmic dot hybridization. Simple analysis of relative mRNA levels in multiple small cell or tissue samples.

Abstract: A simple technique for the simultaneous measurement of relative levels of a specific mRNA in numerous small samples of animal cells or tissue is described The technique involves denaturation of cytoplasmic preparations, followed by dotting of up to 96 samples onto a single sheet of nitrocellulose, hybridization with a 32P-labeled cDNA plasmid, autoradiography, and scanning By analyzing cytoplasmic preparations instead of purified RNA, manipulations of multiple samples prior to analysis is minimized Experiments with a clonal line of rat pituitary tumor (GH3) cells showed that this technique can be employed to follow the induction by Ca2+ of prolactin mRNA sequences, employing cytoplasm prepared from as little as 25 x 10(4) cells The specificity of the technique for prolactin mRNA was shown by employing GC cells, a GH3 cell variant lacking detectable prolactin mRNA sequences Experiments with cultured rat hemipituitaries showed that the prolactin mRNA present in cytoplasm corresponding to as little as 1/100 of a pituitary could be readily detected This technique is quite simple, requires very small amounts of cells or tissue, and permits the simultaneous analysis of multiple samples Hence, it should be quite useful for studies with various experimental systems of the regulation of specific mRNA levels

Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin.

Abstract: The kinetics of the activation of Glu-plasminogen and Lys-plasminogen (P) by a two-chain form of human tissue plasminogen activator (A) were studied in purified systems, and in the presence of fibrinogen (f) and of fibrin films (F) of increasing size and surface density. The activation in the purified systems followed Michaelis-Menten kinetics with a Michaelis constant of 65 microM and a catalytic rate constant of 0.06 s-1 for Glu-plasminogen as compared to 19 microM 0.2 s-1 for Lys-plasminogen. In the presence of fibrinogen plots of 1/v versus 1/[P] or 1/v versus 1/[f] yielded straight lines with an apparent Michaelis constant at infinite [f] of 28 microM and a catalytic rate constant of 0.3 s-1 for Glu-plasminogen as compared to 1.8 microM and 0.3 s-1 for Lys-plasminogen. In the systems with fibrin, plasmin was estimated from the rate of release of 125I from 125I-labeled fibrin films. The initial rate of activation (v) was calculated and Lineweaver-Burk plots of 1/v versus 1/[P] or 1/v versus 1/[F] yielded straight lines. Activation occurred with an intrinsic Michaelis constant of 0.16 microM and a catalytic rate constant of 0.1 s-1 for Glu-plasminogen as compared to 0.02 microM and 0.2 s-1 for Lys-plasminogen. The kinetic analysis suggested that the activation in the presence of fibrin occurs through binding of an activator molecule to the clot surface and subsequent addition of plasminogen (sequential ordered mechanism) to form a cyclic ternary complex. The Low Michaelis constant in the presence of fibrin allows efficient plasminogen activation on a fibrin clot, while its high value in the absence of fibrin prevents efficient activation in plasma.

Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria.

Abstract: Yeast mitochondria were fractionated into inner membrane, outer membrane, matrix, and intermembrane space. Identity and purity of each fraction were monitored by enzyme assays, dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological detection of characteristic mitochondrial polypeptides. Cytochrome b2 and cytochrome c peroxidase were found to be components of the intermembrane space. The most reliable marker of the outer membrane was a major 29,000-dalton polypeptide component. The availability of submitochondrial fractions provides a basis for studying import of precursor polypeptides into isolated yeast mitochondria.

Calcium-activated, phospholipid-dependent protein kinase from rat brain. Subcellular distribution, purification, and properties.

Abstract: The subcellular distribution of Ca2+-activated, phospholipid-dependent protein kinase (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and Nishia k a , Y. (1979) J. Biol. Chem 254, 3692-3695) in rat brain was investigated. Approximately one-third of the protein kinase was recovered in the soluble cytosol fraction, another one-third was in the crude mitochondrial fraction, and the rest was in nuclear and microsomal fractions. Upon further analysis of the crude mitochondrial fraction, most of the enzyme was found to be associated with synaptosomal membranes. The cytosol protein kinase was purified approximately 800fold to apparent homogeneity by DEAE-cellulose and Sephadex G-150 column chromatographies, followed by isoelectrofocusing electrophoresis, blue-Sepharose CL-GB, and phenyl-Sepharose CG4B column chromatographies. The molecular weight of the protein kinase was about 77,000 as estimated by sucrose density gradient ultracentrifugation. The enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M, 82,000, indicating that the enzyme is composed of one polypeptide chain. The enzyme was free of calmodulin. The protein kinase associated with membrane was solubilized with Triton X100, and partially purified by DEAE-cellulose and Sephadex G-150 column chromatographies. The membraneassociated protein kinase thus obtained was indistinguishable from the cytosol protein kinase in physical, kinetic, and catalytic properties. Both enzymes were fully activated by diacylglycerol in the presence of phospholipid and less than micromolar concentrations of Ca2+.

A monoclonal antibody-defined antigen associated with gastrointestinal cancer is a ganglioside containing sialylated lacto-N-fucopentaose II.

Abstract: Two monoclonal antibodies produced by hybridomas obtained from a mouse immunized with a colorectal carcinoma cell line bind specifically to human gastrointestinal cancer cells. The antigen of this antibody in the carcinoma cell line, a monosialoganglioside, has been isolated. Its carbohydrate structure is probably (formula: see text). This oligosaccharide is a sialyl derivative of lacto-N-fucopentaose II, a hapten of the human Lea blood group antigen. About 30 micrograms of ganglioside is obtained from 1 g of cells, wet weight. The ganglioside was detected by autoradiography in lipid extracts of some carcinomas and of meconium, but not in lipid extracts of normal adult tissues. Antigen was detected by solid phase radioimmunoassay in lipid extracts from 12 out of 21 adenocarcinomas of the colon, from 4 out of 5 gastric adenocarcinomas, and from 4 out of 7 pancreatic carcinomas. Antigen was not detected in lipid extracts from 5 esophageal carcinomas or from normal colon and gastric mucosa, pancreas, kidney, liver, and bone marrow.

The metabolism of menadione (2-methyl-1,4-naphthoquinone) by isolated hepatocytes. A study of the implications of oxidative stress in intact cells.

Abstract: The cytotoxic effects of many quinones are thought to be mediated through their one-electron reduction to semiquinone radicals, which subsequently enter redox cycles with molecular oxygen to produce active oxygen species and oxidative stress. The two-electron reduction of quinones to diols, mediated by DT-diaphorase (NAD(P)H: (quinone-acceptor) oxidoreductase), may therefore represent a detoxifying pathway which protects the cell from the formation of these reactive intermediates. By using menadione (2-methyl-1,4-naphthoquinone) and isolated hepatocytes, the relative contribution of the two pathways to quinone metabolism has been studied and a protective role for DT-diaphorase demonstrated. Moreover, in the presence of cytotoxic concentrations of menadione rapid changes in intracellular thiol and Ca2+ homeostasis were observed. These were associated with alterations in the surface structure of the hepatocytes which may be an early indication of cytotoxicity.

The horseradish peroxidase-catalyzed oxidation of 3,5,3',5'-tetramethylbenzidine. Free radical and charge-transfer complex intermediates.

Abstract: Benzidine and related compounds are well known substrates for horseradish peroxidase/H2O2 oxidation. Typically, two different colored products are formed. In this paper, we study the oxidation of 3,5,3',5'-tetramethylbenzidine. The first colored product is a blue charge-transfer complex of the parent diamine and the diimine oxidation product. This species exists in rapid equilibrium with the radical cation. The radical was observed by ESR spectroscopy, and hyperfine splitting constants were determined. Addition of equimolar hydrogen peroxide yields the yellow diimine, which is stable at acid pH. At less than equimolar peroxide, all four species (diamine, radical cation, charge-transfer complex, and diimine) exist in equilibrium. A theoretical analysis of this redox system is presented, including a determination of the extinction coefficients and equilibrium constant for the nonradical species.

A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles.

Abstract: A method is presented for the preparation of a "native" epidermal growth factor (EGF) receptor-kinase complex of molecular weight 170,000 from A-431 cells. Although this receptor complex is capable of binding EGF, noncovalently, in quantities similar to the previously isolated 150,000 complex (Cohen, S., Carpenter, G., and King, L., Jr. (1980) J. Biol. Chem. 255, 4834-4842), the 170,000 preparation has 5 to 10 times the intrinsic kinase activity (autophosphorylation). However, the 170,000 kinase activity toward other proteins is lower than that of the 150,000 preparation. Both the 170,000 and 150,000 kinase activities are enhanced by EGF. The 170,000 and 150,000 proteins are also capable of forming covalent linkages to 125I-EGF, and each is precipitated by antisera directed against the 170,000 protein. We suggest the 150,000 protein is a proteolytic degradation product of the 170,000 protein. The EGF-enhanced kinase activity of the 170,000 preparation remains associated with the 125I-EGF-binding activity following EGF affinity chromatography, electrophoresis in nondenaturing gels, or immunoprecipitation with antisera directed against the sodium dodecyl sulfate (SDS) gel-purified 170,000 protein. These results indicate that the receptor, kinase, and substrate domains are linked, possibly covalently.

A one-step purification of membrane proteins using a high efficiency immunomatrix.

Abstract: A method is described by which an immunoaffinity matrix was constructed by binding antibody directly or indirectly to protein A-Sepharose 4B followed by cross-linking of the complex with dimethyl pimelimidate. This allows optimal spatial orientation of antibodies and, thus, maximum antigen binding efficiency. The affinity matrices were stable to high and low pH buffers without any significant antibody loss. The optimal conditions of antibody saturation, cross-linker concentration, and elution system were established and affinity columns made with the monoclonal antibodies J5, W6/32, and OKT9 for one-step isolation of the common acute lymphoblastic leukemia-associated antigen, HLA-AB antigens, and transferrin receptor, respectively, from cell lysates. The same methodology was also applied to immobilize transferrin with polyvalent anti-transferrin antibodies. This was then used to isolate the transferrin receptor from cell lysates.

Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of protein C.

Abstract: The isolation from rabbit lung of a cofactor for thrombin-catalyzed Protein C activation is described. The lung is perfused to remove blood, homogenized, and extracted with Triton X-100. The cofactor is purified from the extract with two chromatographic columns of diisopropylphosphorothrombin-agarose. The purified cofactor is a protein with an apparent molecular weight on sodium dodecyl sulfate (SDS)-acrylamide gels of 68,000 k 5,000 before disulfide bond reduction and 74,000 k 6,000 after disulfide bond reduction. The cofactor activity is not inactivated permanently by SDS (l%), 8 M urea, or 6 M guanidinium C1, is stable to boiling for greater than 15 min, and is stable from pH 2.0-10. Stability is determined by assay immediately after dilutions of the cofactor from the perturbant. The cofactor activity is destroyed by pepsin or P-mercaptoethanol. The stability of the cofactor in SDS and SDS and 8 M urea solutions allows correlation of the protein species visualized by protein stain with the cofactor activity determined by assay of the extracted gel slices after electrophoresis with these denaturants. The cofactor activity also co-migrates with the major protein species on alkaline disc gel electrophoresis in the presence of Lubrol PX. Neither the cofactor activity nor the protein enters the gel in the absence of Lubrol. From 10 perfused rabbit lungs, 0.7-1.5 mg of cofactor is isolated with an overall yield of 10-20%. The purified cofactor requires thrombin to activate Protein C. The rate of activation of Protein C at constant thrombin is saturable with respect to cofactor. The isolated cofactor increases the rate of Protein C activation 1000-fold. Expression of cofactor activity requires Caz+ or other specific multivalent ions. The relationship between Ca2+ concentration and the initial rate of Protein C activation is a simple hyperbola with half-maximal stimulation at 0.2 k 0.1 m~ Ca2+. Other functional ions in order of effectiveness (in parentheses) are T b 3 + (71%), Mnz+ (52%), Sr2+ (36%), Cr2+ (23%), Co2+ (23%), Cd2+ (16%), and Gd3+ (12%). M 6 + , Ni”, Zn2+, Cu2+, and Ba2+ were less than 5% as active as Ca”.

Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 A resolution. I. General features and binding of methotrexate.

Abstract: X-ray data have been extended to 1.7 A for a binary complex of Escherichia coli dihydrofolate reductase with methotrexate and a ternary complex of Lactobacillus casei dihydrofolate reductase with methotrexate and NADPH. Models for both structures have been refined to R factors of 0.15 and include parameters for fixed and liquid solvent. The two species of dihydrofolate reductase resemble one another even more closely than was thought to be the case prior to refinement. Several new structural features have also been discovered. Among them are a cis peptide linking Gly-97 and Gly-98 (L. Casei numbering) in both species, an alpha helix involving residues 43 through 50 in the E. coli enzyme, and the existence of what may be a specific hydration site on exposed alpha helices. Refinement has led to a revised description of the details of methotrexate binding. We now see that a fixed water molecule mediates the interaction between methotrexate's 2-amino group and Thr-116 (L. casei numbering) and that the inhibitor's 4-amino group makes two hydrogen bonds with the enzyme (instead of one). Other revisions are also discussed. A hypothetical model for substrate binding is proposed in which the pteridine ring is turned upside down while all protein and solvent atoms remain fixed. Asp-26 in this model is hydrogen bonded to the substrate's 2-amino group and to N3.

Fibronectin adsorption on hydrophilic and hydrophobic surfaces detected by antibody binding and analyzed during cell adhesion in serum-containing medium.

Abstract: Studies have been carried out on the adsorption properties of plasma fibronectin (FN) on hydrophobic and hydrophilic surfaces. The hydrophobic and hydrophilic surfaces used were nonwettable bacteriological dishes and wettable tissue culture dishes, respectively. At low plasma FN concentrations, there was more plasma FN binding to hydrophobic surfaces than to hydrophilic surfaces. Under these conditions, the binding of antifibronectin to adsorbed fibronectin was much higher on the hydrophilic surfaces than the hydrophobic ones suggesting that adsorption of plasma FN on the two surfaces occurred in different conformations. In the presence of small amounts of serum albumin, however, antibody binding to plasma FN adsorbed on hydrophobic surfaces increased markedly indicating that a more favorable conformation had been attained. The adsorption of plasma FN onto both hydrophobic and hydrophilic surfaces from serum-containing solutions was found to depend on the serum concentration. At low serum concentrations in the incubations (up to 0.1%), there was increased adsorption of plasma FN with increasing serum concentrations. The highest concentration of adsorbed fibronectin was about 12 ng/cm2 and was sufficient to promote complete cell spreading. Above 1.0% serum there was a marked decrease in fibronectin adsorption and at 10% serum very little adsorption occurred. This indicated that at high serum concentrations other serum proteins were able to compete with fibronectin for surface adsorption sites. This was ccrfirmed by determining adsorption isotherms for fibro1:ectin in the presence of 0.5 and 10% fibronectin-depleted serum. In long term cell spreading experiments (1 h to 2 days), cell spreading eventually occurred on hydrophilic surfaces but not on hydrophobic ones in the presence of 10% serum. This could not be accounted for by exchange on the surfaces of fibronectin for adsorbed serum proteins in the absence of the cells. In the presence of the cells, however, there was an increase in fibronectin associated with the hydrophilic surfaces and a decrease in fibronectin associated with hydrophobic surfaces that occurred concomitantly with increased or decreased cell spreading. This result suggested the possibility that in long term cultures, cells deposit endogenous spreading factors on top of or in place of the adsorbed non-fibronectin serum proteins.

Inhibition of HeLa cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide associated with eucaryotic initiation factor 3 and a cap binding protein complex.

Abstract: Following poliovirus infection of HeLa cells, the synthesis of cellular proteins is inhibited but translation of poliovirus mRNA proceeds. The defect in the recognition of host cell mRNA may be due to a change in a cap recognition complex which, when added to an infected cell lysate, restores the ability to translate capped mRNAs. We employed immunoblotting techniques to examine initiation factors in crude lysates from uninfected and poliovirus-infected HeLa cells. Using an antiserum against eucaryotic initiation factor 3, we detected an antigen of approximate molecular weight 220,000 in uninfected cell lysates but not in infected cell lysates. Antigenically related polypeptides of 100,000 to 130,000 daltons, presumably degradation products, were detected in the infected cell lysate. The time course for degradation of the 220,000-dalton polypeptide correlates with that for inhibition of cellular protein synthesis in vivo. A portion of the population of 220,000-dalton polypeptides apparently associates with initiation factor eIF3 but is readily dissociated in buffers containing high salt. Affinity-purified antibodies against the polypeptide recognize a protein of the same size in a purified preparation of a cap binding protein complex obtained by cap-affinity chromatography. We postulate that the 220,000-dalton polypeptide is an essential component of the cap recognition complex and that its degradation in poliovirus-infected cells results in the inhibition of host cell translation. These results are in the first demonstration of a specific structural defect in an initiation factor resulting from poliovirus infection.

Quantification of the contribution of various steps to the control of mitochondrial respiration.

Abstract: Using principles developed by Kacser and Burns ((1973) in Rate Control of Biological Processes (Davies, D. D., ed) pp. 65-104, Cambridge University Press, London) and Heinrich and Rapoport ((1974) Eur. J. Biochem. 42, 97-105), inhibitor titration studies were carried out in order to quantify the amount of control (control strength) exerted by different steps in oxidative phosphorylation on the rate of mitochondrial oxygen uptake. In the resting state of respiration, nearly all control is exerted by the passive permeability of the mitochondrial inner membrane to protons. In the intermediate states and even in the active state of respiration, control is distributed among different steps including the adenine nucleotide translocator and cytochrome c oxidase. It is therefore not possible to speak of the rate-limiting step in oxidative phosphorylation. Since both the adenine nucleotide translocator and cytochrome c oxidase appear to be rate-controlling, hypotheses suggesting that respiration is fully controlled by either of these two steps alone need to be modified.

ADP ribosylation of the specific membrane protein of C6 cells by islet-activating protein associated with modification of adenylate cyclase activity.

Abstract: Islet-activating protein (IAP), one of the pertussis toxins, exerted dual actions on crude membrane preparations from rat C6 glioma cells; an Mr = 41,000 membrane protein was ADP-ribosylated while GTP (and GTP-dependent isoproterenol) activation of membrane adenylate cyclase was enhanced when membranes were incubated with IaP. Both actions of IaP were dependent on the incubation time and the concentrations of NAD and IAP, and were inhibited by nicotinamide; the one action was strictly paralleled by the other in magnitude. Tryptic digestion of the Mr = 41,000 protein was markedly influenced by the presence of guanyl-5'-yl beta-gamma-imidodiphosphate or NaF, the specific ligands of the regulatory component of the adenylate cyclase system. No ADP ribosylation occurred in the membranes prepared from intact C6 cells that had been incubated with IAP, suggesting that the IAP substrate had already been ADP-ribosylated by the intracellular NAD during incubation of the intact cells. Cholera toxin catalyzed ADP ribosylation of other proteins with Mr = 45,000 and 48,000/49,000 (doublet). It is concluded that IAP, added to intact cells or isolated membranes, causes unique modification of the receptor-adenylate cyclase coupling mechanism as a result of ADP ribosylation of the Mr = 41,000 protein which is presumably one of the subunits, other than the cholera toxin substrates, of the guanine nucleotide regulatory component of the cyclase system.

Inhibition of DNA methyltransferase and induction of Friend erythroleukemia cell differentiation by 5-azacytidine and 5-aza-2'-deoxycytidine.

Abstract: Treatment of Friend erythroleukemia cells with the antileukemic drugs 5-azacytidine and 5-aza-2'-deoxycytidine leads to rapid, time-dependent, and dose-dependent decrease of DNA methyltransferase activity and synthesis of markedly undermethylated DNA. Since this DNA is at least partially methylated in vivo and serves as an excellent substrate for methylation in vitro, hypomethylation of DNA in analog-treated cells appears to result from the loss of DNA methyltransferase, rather than from an inherent inability of 5-azacytosine- substituted DNA to serve as a methyl acceptor. Inhibition of DNA synthesis blocks the loss of DNA methyltransferase activity while inhibitors of RNA synthesis do not, suggesting that the analogs must be incorporated into DNA to mediate their effect on the enzyme, and that minor substitution of 5-azacytosine for cytosine in DNA (approximately 0.3%) suffices to inactivate more than 95% of the enzyme in the cell. Several lines of evidence link changes in the pattern of DNA modification with differentiation. In this regard, it is significant that 5-azacytidine and 5-aza-2'-deoxycytidine act as weak inducers of erythroid differentiation of Friend erythroleukemia cells in the same concentration range where they affect DNA methyltransferase activity. For differentiation to proceed, the cells must be washed free of the drugs. Less than 24 h later, normal levels of DNA methyltransferase activity are restored and within 48 h, DNA isolated from the cells is not detectably undermethylated. This may in part explain why 5-azacytidine and 5-aza-2'-deoxycytidine induce differentiation in less than 15% of the population despite their initial profound effect on DNA methylation.

Reversible bending and helix geometry in a B-DNA dodecamer: CGCGAATTBrCGCG.

Abstract: A double-helical B-DNA dodecamer has been analyzed by single crystal x-ray diffraction methods and refined independently in four variants: sequence CGCGAATTCGCG at 20 degrees C and at 16 K, and CGCGAATTBrCGCG in 60% methylpentanediol at 20 and at 7 degrees C. The first three forms show a 14-19 degrees bend in overall helix axis, but the fourth is straight and unbent. Detailed comparisons of the various forms have led to a better understanding of helix geometry and bending. Structural principles can be understood best if organized under four headings: 1) intrinsic geometry of the sugar rings, 2) stacking and relative motion of base pairs, 3) geometry of the connecting phosphate backbone, and 4) mechanics of bending in B-DNA. The observed bending is neither completely localized nor smooth and continuous, but an intermediate compromise that can be termed "annealed kinking."

Halorhodopsin is a light-driven chloride pump.

Abstract: Light-dependent membrane potentials, ionic fluxes, and volume changes were measured in two kinds of Halobacterium halobium cell envelope vesicles: one containing bacteriorhodopsin and another halorhodopsin. Bacteriorhodopsin-containing vesicles extruded protons by a primary electrogenic mechanism and an energized volume decrease was observed. This was shown to be the consequences of sodium extrusion via proton/sodium antiport (which recirculated protons) and the accompanying passive chloride extrusion. Halorhodopsin-containing vesicles, in contrast, exhibited a volume increase during illumination, apparently caused by primary inward transport of chloride, and accompanied by passive cation (sodium or potassium, and proton) uptake. It was demonstrated that the chloride transport will occur against both electrical and concentration gradients across the vesicle membrane. Moreover, chloride was required on the vesicle exterior for the light-dependent generation of membrane potential, pH change, and swelling. These observations are inconsistent with an earlier proposal that halorhodopsin is an outward directed sodium pump, but suggest very strongly that it is an inward directed chloride pump. Quantitative arguments from the present work rule out a significant role of sodium in the functioning of halorhodopsin.

Characterization of the structural determinants required for the high affinity interaction of asparagine-linked oligosaccharides with immobilized Phaseolus vulgaris leukoagglutinating and erythroagglutinating lectins.

Abstract: The carbohydrate binding specificities of the leukoagglutinating phytohemagglutinin (L-PHA) and erythroagglutinating phytohemagglutinin (E-PHA) lectins of the red kidney bean, Phaseolus vulgaris, have been investigated by lectin-agarose affinity chromatography of Asn-linked oligosaccharides. High affinity binding to E-PHA-agarose occurs only with biantennary glycopeptides containing 2 outer galactose residues and a residue of N-acetylglucosamine linked beta 1,4 to the beta-linked mannose residue in the core. This species is not retarded on L-PHA-agarose. In contrast, tri- and tetraanternnary glycopeptides containing outer galactose residues and an alpha-linked mannose residue substituted at positions C-2 and C-6 are specifically retarded on L-PHA-agarose. Triantennary glycopeptides containing outer galactose residues and an alpha-linked mannose residue substituted at positions C-2 and C-4 are not retarded on L-PHA-agarose. Additionally, the presence of outer sialic acid residues or a core fucose residue does not influence the behavior of complex glycopeptides on either of these lectin-agarose conjugates. This ability of E-PHA and L-PHA to discriminate between Asn-linked oligosaccharides with various branching patterns can be utilized in the fractionation of these glycopeptides (see paper following).

Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography.

Abstract: We describe methods for the complete analysis of cellular nucleotides from as few as 10(6) 32Pi-labeled cells in a simple 2-day experiment. Nucleotides are extracted with acid, neutralized, and resolved by two-dimensional thin layer chromatography on polyethyleneimine cellulose. In the first dimension the nucleotides are separated based on the negative charge of their phosphate groups (i.e. cyclic, mono-, di, and triphosphates) and in the second dimension on their content of nucleobases (i.e. Ura, Cyt, Thy, Gua, and Ade). Because the separation is logical, one can predict the chromatographic migration of most nucleotides. By running standards we have determined the chromatographic location of over 90 biologically important nucleotides, nucleotide derivatives, and modified nucleotides from tRNA. We also developed a set of enzymatic and chemical methods to be used in conjunction with the chromatographic separations for verifying the identity of nucleotides and characterizing novel nucleotides. In this paper we use these methods to analyze and inventory the nucleotide content of Salmonella typhimurium in balanced log phase growth. Other potential uses of the method are also described.

Purification and characterization of a unique isozyme of cytochrome P-450 from liver microsomes of ethanol-treated rabbits.

Abstract: A new isozyme of cytochrome P-450 has been purified to electrophoretic homogeneity from hepatic microsomes of rabbits treated chronically with ethanol. Several criteria indicate that the ethanol-inducible cytochrome, which has a minimal molecular weight of 51,000 and is designated form 3a on the basis of its relative electrophoretic mobility, is distinct from the known isozymes of P-450. As judged spectrally, the new isozyme is high spin in the oxidized state, as is form 4, but differs in that the spin state is unperturbed by nonionic detergents. The absolute spectrum of the ferrous carbonyl complex of form 3a is red shifted as compared to that of forms 2, 3b, 3c, 4, and 6 and exhibits a maximum at 452 nm. The amino acid composition of form 3a is different from that of the other isozymes, and both the NH2- and COOH-terminal sequences are distinct; form 3a has an NH2-terminal alanine and a carboxyl-terminal leucine residue. Peptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following treatment with papain, chymotrypsin, or Staphylococcus aureus V8 protease and by high performance liquid chromatography following trypsinolysis indicates that form 3a is a unique gene product. This cytochrome displays the highest activity of all of the rabbit isozymes in the oxidation of ethanol to acetaldehyde and the p-hydroxylation of aniline when reconstituted with NADPH-cytochrome P-450 reductase and phospholipid in the presence of NADPH and oxygen.

The primary structure of the Saccharomyces cerevisiae gene for alcohol dehydrogenase.

Abstract: The DNA sequence of the gene for the fermentative yeast alcohol dehydrogenase has been determined. The structural gene contains no introns. The amino acid sequence of the protein as determined from the nucleotide sequence disagrees with the published alcohol dehydrogenase isozyme I (ADH-I) sequence for 5 of the 347 amino acid residues. At least one, and perhaps as many as four, of these differences is probably due to ADH-I protein heterogeneity in different yeast strains and not to sequencing errors. S1 nuclease was used to map the 5' and 3' ends of the ADH-I mRNA. There are two discrete, mature 5' ends of the mRNA, mapping 27 and 37 nucleotides upstream of the translation initiating ATG. These two equally prevalent termini are 101 and 91 nucleotides, respectively, downstream from a TATAAA sequence. Analysis of the 3' end of ADH-I mRNA disclosed two minor ends upstream of the major poly(A) addition site. These three ends map 24, 67, and 83 nucleotides, respectively, downstream from the translation-terminating TAA triplet. The sequence AA-TAAG is found 28 to 34 nucleotides upstream of each ADH-I mRNA poly(A) addition site. Sequence comparisons of these three 3' ends with those for four other yeast mRNAs yielded a 13-nucleotide consensus sequence to which TAAATAAGA is central. All of the known yeast poly(A) addition sites map at or near the A residue of a CTA site 25 to 40 nucleotides downstream from this consensus octamer.

The guanine nucleotide activating site of the regulatory component of adenylate cyclase. Identification by ligand binding.

Abstract: The regulatory component of adenylate cyclase (G/ F) contains the site(s) for activation of the enzyme by guanine nucleotides. We have developed methods for the assessment of binding of radiolabeled guanosine-5’(3-0-thio)triphosphate (GTPyS) and guanosine-5‘-(P,yimin0)triphosphate (Gpp(NH)p) to homogeneous preparations of G/F. These methods detect binding specifically to the activating site of G/F as judged by the following criteria. 1) The kinetics of activation and binding are identical. 2) Apparent Kd values for [%] GTPyS (0.7 PM) and I3HJGpp(NH)p (5 PM) agree with K, values for activation of G/F. 3) Apparent Kd values for nonactivating nucleotides, determined by competition for binding of GTPyS, agree with Ki values for inhibition of activation by GTPyS. 4) Both binding and activation depend on divalent metal ions with the same specificity. Binding of [36S]GTPyS or [3H]Gpp(NH)p reveals a single class of sites with an apparent stoichiometry of 1 mol of sites/mol of G/F (Mr = 80,000). Photoaffinity labeling of G/F with [y-32P]8-azido guanosine-5’-triphosphate specifically incorporates 32P into the 52,000- and 45,000-dalton subunits of G/F. Activation of G/F by GTPyS or Gpp(NH)p does not conform to simple Michaelis-Menten kinetics. While Hofstee and Scatchard analyses of activation and binding isotherms at apparent steady state appear to behave in a Michaelis-Menten manner, there is no reversal of activation or binding under activating conditions and the rate constant for activation is unchanged from 10 mi to 100 PM GTPyS. Reversal of binding and activation, which occurs only in the absence of divalent cation, is not a first order process. These anomalous kinetics are explained by a model in which rapid equilibrium binding of GTPyS precedes a dissociation of the 45,000- and 35,000-dalton subunits of G/F. This latter reaction is the rate-limiting step in the activation pathway. This model predicts an inhibitory function of the 35,000-dalton subunit, which we have confirmed with the resolved protein.