Purification and properties of two endonucleases specific for apurinic/apyrimidinic sites in DNA from Saccharomyces cerevisiae

Two distinct endonucleases from Saccharomyces cerevisiae, specific for apurinic/apyrimidinic sites (AP-endonucleases A and B), have been extensively purified and characterized. Both are free from unspecific and ultraviolet-specific endonucleases and exonucleases. The two enzymes are monomeric proteins of around 24 000 daltons. Both are sensitive to ionic strength and most active in the presence of 150 and 100 mM NaCl for AP-endonucleases A and B, respectively. They are not absolutely dependent on divalent cations, since they are insensitive to EDTA, although AP-endonuclease A is activated by Ca²⁺ or Mg²⁺ and AP-endonuclease B by Mg²⁺ only. ATP inhibits the enzymes. AP-endonuclease A reacts optimally between pH 6 and 8, and AP-endonuclease B at pH 8. AP-endonuclease A is more stable at 60°C (half-life of 17 min) than B (half-life of 4 min). AP-endonucleuase A is insensitive to N-ethylmaleimide or ρ-chloromercuribenzoate. AP-endonuclease B is also insensitive to N-ethylmaleimide, but ρ-chloromercuribenzoate inhibits its activity.     

An analysis of the repair processes in ultraviolet-irradiated Micrococcus luteus using purified ultraviolet-endonuclease

The measurement of the frequency of endonucleolytic incisions in ultraviolet-irradiated DNA serves as the test for the presence of pyrimidine dimers. In accordance with this approach, the lysates of three Micrococcus luteus strains containing radioactively labeled chromosomes were treated with purified M. luteus ultraviolet-endonuclease to trace segregation of dimers amongst parental and newly synthesized DNA and their removal during postreplication and excision DNA repair. A considerable proportion of the dimers in all strains tested proved to be insensitive to the action of exogenous incising enzyme. The use of chloramphenicol as an inhibitor of postirradiation protein synthesis in combination with ultraviolet-endonuclease treatment of DNA allowed to reveal at least two alternative pathways of postreplication repair: constitutively active recombinational pathway and inducible nonrecombinational one.     

Large-scale preparation of plasma membrane vesicles from PC-12 Pheochromocytoma cells and their use in noradrenaline transport studies

Plasma membranes were isolated from rat pheochromocytoma cells (PC-12) grown in spinner culture. The rapid and simple isolation procedure consisted of a differential and isopycnic centrifugation (in a linear sucrose gradient) with the aid of a high capacity fixed angle rotor equipped with siliconized centrifuge tubes. The isolated membranes were closed and osmotically active vesicles (about 0.3 μm in diameter) with a mean intravesicular water space of 1.84 μl / mg protein. In the presence of an inward gradient of sodium chloride and an outward gradient of potassium, [³H]noradrenaline (50 nM) was taken up and accumulated 550-fold (at 31°C). The uptake and accumulation of [³H]noradrenaline was temperature-sensitive and inhibited by the tricyclic antidepressant desipramine. Membrane vesicles isolated from PC-12 cells represent a useful model for the investigation of the molecular mechanism of the neuronal noradrenaline transport system.     

Purification and characterization of the membrane (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B

The membrane (Na⁺ + Mg²⁺)-ATPase of Acholeplasma laidlawii B has been solubilized with a Brij-58/sodium deoxycholate mixture and purified by a combination of gel filtration and ion-exchange chromatography. The purified, partially delipidated ATPase has a specific activity of 195 μmol P_i/mg protein per h, which could be enhanced by 25% upon the addition of exogenous phospholipids. The kinetic properties of the purified enzyme are similar to those of the native membrane-bound enzyme, suggesting that it has not been substantially altered during the purification procedure. The enzyme is an assembly of five polypeptide species and its kinetic properties suggest that it is dissimilar to other known ATPases.     

Demonstration in vitro of two intracellular inactivators of nitrate reductase from Neurospora

Two different inactivators of nitrate reductase have been found in cell free preparations of Neurospora. T he first (Inactivator I) is very active at pH9, is inhibited by disodium ethylene diamine tetraacetate (EDTA) and is present in all mycelia incubated under all conditions tested; the second (inactivator II) is very active at pH 5, is repressed by ammonia or by a metabilic product of ammonia and derepssed by nitrogen starvation, cannot be derepressed by nitrogen starvation in strain nit-2, in which a number of “ammonia-repressible” enzymes are permanently repressed, and is sensitive to phenyl methyl sulfonyl fluoride.Crude extracts of mycelia contain inhibitors(s) of both inactivators.     

Steady-state fluorescence anisotropy changes of 1,6-diphenyl-1,3,5,-hexatriene in membranes from Bacillus megaterium spores

The steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene incorporated into isolated Bacillus megaterium spore membranes was measured. Compounds capable of triggering spore germination in vivo caused an increase in the anisotropy of diphenylhexatriene. These increases in anisotropy of diphenylhexatriene in spore membranes are likely to represent at least a portion of the trigger mechanism for spore germination based on the following observations. First, there was an exceptional positive correlation between compounds that both triggered germination in vivo and caused changes in anisotropy in vitro. Second. the capacity of membranes to respond to germinants by increases in anisotropy was unique to membranes from spores but disappeared after germination. Third, alteration of spores chemically or genetically to block the in vivo triggering of germination by l-proline also blocked the in vitro anisotropy change with l-proline but not d-glucose. Finally, there was no correlation between the transport activities of specific compounds and the ability of these compounds to either trigger germination or alter the anisotropy of diphenylhexatriene in the membranes. Although we do not known the nature of the molecular interactions giving rise to the anisotropy changes, we hypothesize that they are due to changes in protein conformation that alter protein-protein and/or protein-lipid interactions. Such modifications of membrane structures could account for the rapid release of small molecular weight compounds such as K⁺ and Ca²⁺ early in germination.     

Characterization of a site-specific restriction endonuclease SphI from Streptomyces phaeochromogenes

A new type-II restriction endonuclease SphI, has been partially purified from Streptomyces phaeochromogenes. SphI recognizes the hexanucleotide sequence 5′-GCATG↓C and cleaves it at the position marked by the arrow. This nucleotide sequence is present twice in SV40 DNA, four times in λ DNA and only once in the cloning vehicles pBR322, pBR325, pBR327 and pBR328.     

Lectin receptors in Trypanosoma cruzi an N-acetyl-d-glucosamine-containing surface glycoprotein specific for the trypomastigote stage

We have investigated the interaction of three lectins, differing in their sugar specificities, with the surface of the three differentiation stages of Trypanosoma cruzi. The Scatchard constants for each lectin and parasite stage imply that differentiation of T. cruzi is accompanied by changes in the cell surface saccharides. Trypomastigotes obtained from two different sources do not differ appreciably as to the number and affinity of binding sites for the three lectins employed, suggesting a similar cell-surface saccharide composition. These conclusions are reinforced by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the ¹³¹I-labeled surface glycoproteins, following isolation by affinity chromatography. The surface membrane of trypomastigotes, the infective stage to T. cruzi for mammalian cells, possesses a specific glycoprotein of apparent M_r 85 000 (Tc-85) which is absent from the other two stages and can be isolated by affinity chromatography on wheat germ agglutinin-Sepharose columns. This glycoprotein also binds to concanavalin A, but not to Lens culinaris lectin. The binding of Tc-85 to wheat germ agglutinin is unnafected by treatment of either the isolated glycoprotein or intact living trypomastigotes with neuraminidase. Since $\text{N-}\text{acetyl}\text{-}\text{d}\text{-}\text{glucosamine}$ inhibits internalization of trypomastigotes by cultured mammalian cells, it is suggested that Tc-85 might be involved in adhesion and/or interiorization of T. cruzi into mammalian cells, possibly via recognition of an ubiquitous host-cell surface $\text{N-}\text{acetyl}\text{-}\text{d}\text{-}\text{glucosamine}\text{-}\text{specific}$ receptor activity.     

D-Ribulose-1,5-bisphosphate carboxylase/oxygenase from chemolithotrophically grown Rhizobium japonicum

Ribulose-1,5-bisphosphate carboxylase/oxygenase has been purified from chemolithotrophically grown Rhizobium japonicum SR and ribulose-5-phosphate kinase activity has also been detected in extracts of such cells. Electrophoretically homogeneous ribulosebisphosphate carboxylase/oxygenase purified in the presence of PMSF showed two types of large subunits of 55 000 and 53 000 daltons and small subunits of 14 200 daltons. The heterogeneity of large subunits was not observed when the enzyme was prepared in the presence of PMSF and DIFP. Ribulose-1,5-bisphosphate carboxylase from R. japonicum was inhibited by antibodies to this enzyme and a single precipitin band from the antibody-enzyme interaction was observed on double diffusion plates. Antibodies to R. japonicum enzyme did not cross-react on immunodiffusion plates with the ribulosebisphosphate carboxylase/oxygenases from wheat, spinach, soybean and tobacco.     

Structure and action of heteronemertine polypeptide toxins Binding of cerebratulus lacteus toxin B-IV to axon membrane vesicles

The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of ¹²⁵I-labeled toxin B-IV by native toxin B-IV have shown specific binding of ¹²⁵I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [³H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association ($\text{k}{}_{\mathrm{+1}}\text{=7.3·10}{}^5\text{M}{}^{\mathrm{?1}}\text{·s}{}^{\mathrm{?1}}$) and dissociation ($\text{k}{}_{\mathrm{?\; 1}}\text{=2·10}{}^{\mathrm{?3}}\text{s}{}^{\mathrm{?1}}$) shows a nearly identical $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding.     

Purification and properties of a transketolase responsible for formaldehyde fixation in a methanol-utilizing yeast, Candida boidinii (Kloeckera sp.) No. 2201

Dihydroxyacetone synthase, present in methanol-grown Candida boidinii (Kloeckera sp.) No. 2201, catalyzes the transfer of the glycolaldehyde group from xylulose 5-phosphate to formaldehyde to form glyceraldehyde 3-phosphate and dihydroxyacetone. This enzyme was purified to electrophoretic homogeneity and found to be a new type of transketolase. The molecular weight of the enzyme was estimated to be 190 000 by gel filtration. The enzyme appeared to be composed of four identical subunits (M_r, 55 000). Thiamin pyrophosphate and Mg²⁺ were required for the activity. The optimum pH was found to be 7.0. With xylulose 5-phosphate as the ketol-donor, aliphatic aldehydes (C₁?C₇), glycolaldehyde and glyceraldehyde were better acceptors than ribose 5-phosphate. The kinetic data were consistent with a ping-pong bi-bi mechanism. The K_m values obtained were as follows: xylulose 5-phosphate, 1.0 nM; formaldehyde, 0.43 mM; glyceraldehyde 3-phosphate, 0.42 mM; and dihydroxyacetone, 0.52 mM.     

A potent platelet aggregation inducer from Trimeresurus gramineus snake venom

A potent platelet aggregation inducer (platelet aggregoserpentin) was purified from Trimeresurus gramineus snake venom by DEAE-Sephadex A-50 and Sepharyl S-300 column chromatography. It was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It elicited dose-dependently platelet aggregation and serotonin release action in rabbit platelet suspension. Exogenous calcium was required for its activity. Creatine phosphate/creatine phosphokinase and apyrase showed no significant inhibitory effect on aggregoserpentin-induced platelet aggregation in platelet suspension. Aggregoserpentin induced aggregation in ADP-refractory platelet-rich plasma. It caused no detectable molonic dialdehyde formation in the process of platelet aggregation. Indomethacin did not inhibit aggregoserpentin-induced platelet aggregation. Mepacrine abolished preferentially its aggregating activity, while prostaglandin E₁ completely blocked both aggregoserpentine-induced aggregation and release reaction. Furthermore, platelet aggregoserpentine lowered basal and prostaglandin E₁-stimulated cAMP levels in platelet suspension. Nitroprusside inhibited both its aggregating and releasing activity, while verapamil preferentially blocked its aggregating activity. It is concluded that aggregoserpentin activated platelets through lowering cAMP levels or the activation of endogenous phospholipase A₂, resulting in the formation of platelet activating factor, but not of prostaglandins.     

Immunoreactivity of subunits of the (Na+ + K+)-ATPase Cross-reactivity of the α,α+ and β forms in different organs and species

The immunologic cross-reactivity of the α and α+ forms of the large subunit and the β subunit of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from brain and kidney preparations was examined using rabbit antiserum prepared against the purified holo lamb kidney enzyme. As previously reported by Sweadner ((1979) J. Biol. Chem. 254, 6060–6067) phosphorylation of the large subunit of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in the presence of Na⁺, Mg²⁺, and [$\text{γ-}{}^{32}\text{P}\text{]}\text{ATP}$ revealed that dog and, very likely, rat brain contain two forms of the large subunit (designated α and α+) while dog, rat, and lamb kidney contain only one form (α). The cross-reactivity of the α and α+ forms in these preparations was investigated by resolving the subunits by SDS-polyacrylamide gel electrophoresis. The separated polypeptides were transferred to unmodified nitrocellulose paper, and reacted with rabbit anti-lamb kidney serum, followed by detection of the antigen-antibody complex with ¹²⁵I-labeled protein A and autoradiography. By this method, the α and α+ forms of rat and dog brain, as well as the α form found in kidney, were shown to cross-react. In addition, membranes from human cerebral cortex were shown to contain two immunoreactive bands corresponding to the α and α+ forms of dog brain. In contrast, the brain of the insect Manduca sexta contains only one immunoreactive polypeptide with a molecular weight intermediate to the α and α+ forms of dog brain. The β subunit from lamb, dog and rat kidney and from dog and rat brain cross-reacts with anti-lamb kidney ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ serum. The mobility of the β subunit from dog and rat brain on SDS-polyacrylamide electrophoresis gels is greater than the mobility of the β subunit from lamb, rat or dog kidney.     

A new site-specific endonuclease from Streptomyces lavendulae (SlaI)

A new restriction-like endonuclease, SlaI, was found and partially purified from Streptomyces lavendulae ATCC8664. This endonuclease cleaved bacteriophage lambda DNA at only one site, and cytosine-substituted bacteriophage T4 DNA at 16 sites. The recognition sequence was determined by using SlaI fragments of cytosine-substituted bacteriophage T4 DNA. The hexanucleotide recognized by SlaI endonuclease was

$\text{5′-}\text{C}\text{?}\text{T}\text{-}\text{C}\text{-}\text{G}\text{-}\text{A}\text{-}\text{G}\text{-3′}$

$\text{3′-}\text{G}\text{-}\text{A}\text{-}\text{G}\text{-}\text{C}\text{-}\text{A}\text{-↑}\text{C}\text{-5′}$

with the sites of cleavage as indicated by the arrows. Therefore, SlaI endonuclease was an isochizomer of XhoI endonuclease.     

Sequence of the cryptic satellite DNA from the plant Sinapis alba

A satellite DNA with a buoyant density equal to that of main band DNA in neutral cesium chloride (‘cryptic satellite’) can be isolated from the DNA of mustard (Sinapis alba) nuclei by Ag⁺/Cs₂SO₄ density gradient centrifugation. This satellite is cleaved into 172 bp repeat units by HinfI, AluI or HaeIII. The HinfI fragments have been further cleaved by AluI, and seven AluI subfragments have been sequenced. As a result two versions of a basic 172 HinfI repeat have been found, one (A + B) with an additional HinfI site. These two sequences (A + B and C) are the most frequent versions of the basic repeat of mustard satellite DNA. The basic 172 bp unit does not contain subrepeats or palindromic sequences. It is not similar (at a criterion of 15 common bases) with any known satellite sequence. It is not unusually highly methylated in the native state.     

Analysis of membrane fractions from Mycoplasma gallisepticum

Membrane fractions have been isolated from Mycoplasma gallisepticum following a procedure derived from that described by Maniloff, J. and Quinlan, D.C. (J. Bacteriol. (1974) 120, 495–501). A light fraction F₁ was obtained which contained structures resembling the bleb-infrableb apparatus characteristic of M. gallisepticum. It was enriched in DNA and had an electrophoretic profile different from that of unfractionated membranes. Cholesterol-to-phospholipid ratios higher than two and elevated values of the ratio of saturated to unsaturated fatty acids were other characteristics of this fraction. The two other fractions isolated (F_II and F_IV) also differed from intact membranes by their cholesterol and phospholipid content as well as by their saturation ratios. The membrane fluidity of F_II and F_IV, estimated by fluorescence polarization, was similar to that of unfractionated membranes while a slight but significant difference was recorded for the light fraction. Possible relationships between the lateral heterogeneity of the M. gallisepticum membrane and the obtainment of fractions are discussed.     

A versatile endonuclease IV from Thermus thermophilus has uracil-excising and 3'-5' exonuclease activity

Apurinic/apyrimidinic (AP) sites arise in DNA through the spontaneous loss of bases or through the release of damaged bases from DNA by DNA glycosylases. AP sites in DNA can be catalyzed by AP endonucleases such as exonuclease III and endonuclease IV, generating a 3'-hydroxyl group and a 5'-terminal sugar phosphate. Here, we have identified and characterized a novel endonuclease IV from a hyperthermophilic bacterium Thermus thermophilus designated as TthNfo. TthNfo efficiently removed AP site from double-stranded oligonucleotide substrate. No significant difference was observed in the rate of reaction of four bases opposite AP site with TthNfo. In addition, TthNfo possesses a 3'-5' exonuclease activity similar to that of Escherichia coli exonuclease III. Surprisingly, we found that TthNfo also catalyzes the excision of uracil from DNA. In comparison with other endonuclease IV proteins, the removal of uracil residue was unique to TthNfo. Based on these observations and the absence of exonuclease III in T. thermophilus, we suggest that versatile enzyme activities of TthNfo play an important role in counteracting DNA base damage in vivo.