Presence Of MnCl2 Research Articles

O6-methylguanine-induced replication blocks.

The ability of Klenow polymerase I, phage T7 polymerase (Sequenase), human polymerase alpha, and human polymerase beta to synthesize past (bypass) O6-methylguanine (O6-meG) lesions was studied in the presence of MgCl2 and MnCl2. An end-labeled 16-mer primer was annealed to the 3' end of gel-purified oligodeoxyribonucleotide templates (45-mers), each containing a single O6-meG in place of one G in the sequence -G1G2CG3G4T-. Extension products were analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. A fraction of the products extended by Klenow fragment terminated either opposite or one base before O6-meG located at sites 1 and 3. Termination occurred primarily one base before O6-meG located at sites 2 and 4. The remaining fractions that bypassed the lesions represented full-length product. In control reactions, the O6-meG-containing templates were annealed with complementary 45-mers, repaired with O6-alkylguanine DNA-alkyltransferase, annealed with an excess of labeled primer, and extended by Klenow fragment. Full-length extension of > 90% was observed with each template. Primer extension past O6-meG by DNA polymerase alpha and Sequenase was partially blocked in a manner which varied with the site of O6-meG in the template while primer extension by DNA polymerase beta was completely blocked (< 2% full length extension) with O6-meG at sites 1-4. Substitution of MnCl2 for MgCl2 in the reaction mixture greatly increased the bypass of O6-meG by Klenow fragment and DNA polymerase alpha but not Sequenase or DNA polymerase beta. The increased ability of Klenow fragment to bypass O6-meG in the presence of MnCl2 was found to result from an increased incorporation of G (O6-meG at sites 1 and 2) and A (O6-meG at sites 1, 2, and 3) opposite the lesion. The results indicate that O6-meG can block in vitro polymerization by several DNA polymerases and are consistent with the observed cytotoxic effects of methylating agents on mammalian cells.

Identification of an Ectokinase Activity in Cerebellar Granule Primary Neuronal Cultures

Primary neuronal cultures from 8-day-old rat cerebellum were incubated in the presence of exogenously added 16 nM [gamma-32P]ATP. Phosphorylation of a 45-kDa endogenous protein was detected within 1 min and increased linearly for approximately 20 min. Unlike what was seen with [gamma-32P]ATP, in the presence of [32P]orthophosphate no visible phosphorylation of protein was detected after 10 min, but a different pattern of phosphorylation was obtained in 30 min. The phosphorylation of the 45-kDa protein was reduced by 80-90% in the presence of 1 microM unlabeled ATP, 5 U/ml of apyrase, or 0.01% trypsin but not 1 mM PO4(3-). Phosphorylation was inversely proportional to cell density and was unaffected by addition to the cells of 56 mM KCl or 100 microM glutamate for 3 min. The presence of exogenously added cellular protein extracts or pretreatment of the cells for up to 20 min in phosphorylation buffer also did not affect the observed phosphorylation of the 45-kDa protein. The phosphorylation was found to be insensitive to MgCl2 but inhibited in the presence of MnCl2 or NaF and in the absence of CaCl2. Analogues of ATP suppressed phosphorylation of the 45-kDa protein by 80-90%. A similar inhibition was obtained in the presence of ADP or AMP. In this study, we establish via several different means that the phosphorylation of the 45-kDa protein in primary neuronal granule cultures occurs extracellularly through an ectokinase activity, which is furthermore distinguishable from a series of other presently characterized ecto-protein enzymes and intracellular kinases.

Crystallization and structural analysis of bullfrog red cell L‐subunit ferritins

Ferritin is a 24 subunit protein that controls biomineralization of iron in animals, bacteria, and plants. Rates of mineralization vary among members of the ferritin family, particularly between L and H type subunits of animal ferritins which are differentially expressed in various cell types. To examine ferritin from a highly differentiated cell type and to clarify the relationship between ferritin structure and function, bullfrog red cell L ferritin has been cloned, overexpressed in E. coli, and crystallized under two conditions. Crystals were obtained at high ionic strength in the presence of MnCl2 at a concentration comparable to that of the protein and in the presence of MgCl2 at a concentration much higher than that of the protein. Under both crystallization conditions, the crystals are tetragonal bipyramids in the space group F432 with unit cell dimensions a = b = c = 182 +/- 0.5 A. Crystals obtained in the presence of manganese and ammonium sulfate diffract to 1.9 A, while those obtained in the presence of magnesium and sodium tartrate diffract to 1.6 A. Isomorphous crystals have been obtained under similar conditions for a site-directed mutant with a reduced mineralization rate in which Glu-57, -58, -59, and -61 are all replaced by Ala. The structure of wild type L-subunit with magnesium has been solved by molecular replacement using the calcium salt of human liver H subunit (Lawson et al., Nature (London) 349:541-544, 1991) as the model. The crystallographic R factor for the 6-2.2 A shell is 0.21. The overall fold of human H and bullfrog L ferritins is similar with an rms difference in backbone atomic positions of 0.97 A. The largest structural differences occur in the D helix and the loop connecting the D and E helices of the four helix bundle. Because red cell L ferritin and liver H ferritin show differences in both rates of mineralization and three-dimensional structure, more detailed comparisons of these structures are likely to shed new light on the relationship between conformation and function.

Purification and characterization of adenine phosphoribosyltransferase from Arabidopsis thaliana

Adenine phosphoribosyltransferase (APRT; EC 2. 4,2. 7) from Arabidopsis thaliana was purified approximately 3800‐fold, to apparent homogeneity. The purification procedure involved subjecting a leaf extract to heat denaturation, (NH4)2SO4 precipitation, Sephadex G‐25 salt separation, ultracentrifugation and liquid chromatography on Diethylaminoethyl Sephacel, Phenyl Sepharose CL‐4B, Blue Sepharose CL‐6B and adenosine 5′‐monophosphate‐Agarose. The purified APRT was a homodimer of approximately 54 kDa and it had a specific activity of approximately 300 μmol (mg total protein)‐1 min‐1. Under standard assay conditions, the temperature optimum for APRT activity was 65°C and the pH optimum was temperature dependent. High enzyme activity was dependent upon the presence of divalent cations (Mn2+ or Mg2+). In the presence of MnCl2+ other divalent cations (Mg2+, Ca2+, Ba2+, Hg2+ and Cd2+) inhibited the APRT reaction. Kinetic studies indicated that 5‐phosphoribose‐1‐pyrophosphate (PRPP) caused substrate inhibition whereas adenine did not. The Km for adenine was 4.5±1.5 μM, the Km for PRPP was 0.29±0.06 mM and the Ki for PRPP was 1.96±0.45 mM. Assays using radiolabelled cytokinins showed that purified APRT can also catalyze the phosphoribosylation of isopentenyladenine and benzyladenine. The Km for benzyladenine was approximately 0.73±0.06 mM

Evidence for a regulatory role of a phosphorylation-dephosphorylation cycle in hypothalamic and lung histidine decarboxylase (HisDC) activity

To investigate the modulation of histidine decarboxylase (HisDC) activity by the degree of phosphorylation of a modulatory (or enzyme) protein, HisDC was partially purified from the cytosol of the hypothalamus, the lungs and the glandular stomach of rats by ammonium sulphate precipitation (25–45% saturation) and incubated with highly purified phosphoprotein phosphatase (Type 2-A) under control and phosphorylating conditions by adding ATP, MgCl2, cAMP and purified protein kinase A to reaction mixtures. The presence of phosphoprotein, phosphatase 2A in reaction mixtures resulted in a partial reversal of HisDC inhibition induced by phosphorylating conditions, and a markedly enhanced basal enzyme activity in control conditions. In contrast, the glandular stomach HisDC failed to response strikingly either to phosphorylating or to dephosphorylating conditions. In the cytosol, the time-course of preincubations showed an increased HisDC activity during the first 1–20, min incubation period and more enhanced enzyme activity in the presence of MnCl2, a stimulator of phosphoprotein phosphatase 2A. The addition of NaF (a common inhibitor of phosphoprotein phosphatases) to the preincubation mixtures resulted in instant, marked and long-lasting decreases in HisDC activity.

Biochemical characterization of Epstein-Barr virus nuclear antigen 2A and an associated ATPase activity.

A partial purification of the Epstein-Barr-virus nuclear antigen 2A (EBNA 2A) protein from the Epstein-Barr-virus-infected lymphoblastoid cell line, Cherry, has been designed. The main purification step was immunoaffinity chromatography, based on the mAb, 115E, directed towards the carboxy terminus of EBNA 2A. This was followed by chromatography over a Blue Sepharose column. According to silver-stained SDS/PAGE, EBNA 2A was estimated to be 20% pure. The purified fractions contained an ATPase activity that was inhibited by the mAb 115E. Immunopurification of six EBNA-2A-positive cell lines and their negative counterpart showed that only fractions from EBNA-2A-positive lines contained ATPase activity. In gel-filtration experiments EBNA 2A eluted as a 75-kDa protein in conjunction with an ATPase activity. The EBNA 2A protein was covalently labeled by the ATP analog [14C]5'-[p-(fluorosulfonyl)benzoyl]adenosine. The ATPase activity was found to be optimal in the presence of 0.25 mM MgCl2 or CaCl2, whereas, in the presence of MnCl2 and ZnCl2, the activity was only about 50% of the control. High concentrations of Na2VO3 and heparin do not interfere with the activity, while 2.5 mM NaF or 0.5 M NaCl give a 50% reduction of the activity. The Km for ATP and for GTP was 13 microM and 11 microM, respectively, and the Vmax for ATP was about six-times higher than with GTP as substrate. Other low-molecular-mass non-protein phosphate esters, such as phosphoserine or phosphothreonine inhibited the ATPase activity with a Ki of 18 and 32 microM, respectively. Phosphotyrosine had a Ki of 480 microM. Serine, threonine and tyrosine had no inhibitory effect on the ATPase activity.

Synthese und Kristallstruktur von (PPh4)2Te4 / Synthesis and Crystal Structure of (PPh4)2Te4

(PPh4)2Te4 has been prepared by the reaction of Na2Te3 with PPh4Cl in DMF solution in the presence of MnCl2 or FeCl2. The compound forms dark red crystals, which were characterized by IR spectroscopy as well as by an X-ray crystal structure determination (6109 observed independent reflections, R = 0.030). Crystal data: triclinic, space group PĪ, Z = 2, a = 1046.7(2), b = 1130.1(2), c = 1967.8(4) pm, α = 93.63(3), β = 90.43(3), γ = 108.89(3)° at —60 °C. The cations are grouped in pairs (PPh4 +)2 around inversion centres, each pair being surrounded by eight Te4 2- ions, arranged as in the CsCl type lattice. The chain-like Te4 2- ions have no close contacts with one another.

Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase

A recombinant DNA polymerase derived from the thermophilic eubacterium Thermus thermophilus (Tth pol) was found to possess very efficient reverse transcriptase (RT) activity in the presence of MnCl2. Many of the problems typically associated with the high degree of secondary structure present in RNA are minimized by using a thermostable DNA polymerase for reverse transcription, and predominantly full-length products can be obtained. The cDNA can also be amplified in the polymerase chain reaction (PCR) with the same enzyme. The Tth pol was observed to be greater than 100-fold more efficient in a coupled RT/PCR than the analogous DNA polymerase from Thermus aquaticus (Taq pol). The sensitivity of the reactions performed by Tth pol allowed for the detection of ethidium bromide stained products starting with as little as 100 copies of synthetic cRNA. Similar results were also obtained with RNA from a Philadelphia-chromosome positive cell line. Detection of IL-1 alpha mRNA was possible starting with 80 pg of total cellular RNA. The ability of Tth pol to perform both reverse transcription and DNA amplification will undoubtedly prove useful in the detection, quantitation, and cloning of cellular and viral RNA.

Kinetic Characterization of Ribonuclease-Resistant 2′-Modified Hammerhead Ribozymes

The incorporation of 2'-fluoro- and 2'-aminonucleotides into a hammerhead ribozyme was accomplished by automated chemical synthesis. The presence of 2'-fluorouridines, 2'-fluorocytidines, or 2'-aminouridines did not appreciably decrease catalytic efficiency. Incorporation of 2'-aminocytidines decreased ribozyme activity approximately by a factor of 20. The replacement of all adenosines with 2'-fluoroadenosines abolished catalysis in the presence of MgCl2 within the limits of detection, but some activity was retained in the presence of MnCl2. This effect on catalysis was localized to a specific group of adenines within the conserved single-stranded region of the ribozyme. The decrease in catalytic efficiency was caused by a decrease in the rate constant; the Michaelis constant was unaltered. The 2'-fluoro and 2'-amino modifications conferred resistance toward ribonuclease degradation. Ribozymes containing 2'-fluoro- or 2'-aminonucleotides at all uridine and cytidine positions were stabilized against degradation in rabbit serum by a factor of at least 10(3) compared to unmodified ribozyme.

Possible regulation of hypothalamus and lung histidine decarboxylase activity by cAMP‐dependent protein kinase

Activity of crude histidine decarboxylases (HisDC) from the hypothalamus and the lungs, was markedly reduced by incubating with ATP.Mg, cAMP and cAMP-dependent protein kinase A, whereas activity of the crude glandular stomach enzyme changed only slightly under equal condition. The omission of one of these components failed to reduce HisDC activity by as much as the complete system. Addition of bovine heart (type II) or rat cerebellum protein kinase A (types I and II) inhibitor to the assay prevented enzyme inactivation; moreover, protein kinase A inhibitors permitted moderate activation under phosphorylating and control conditions. Cytosolic hypothalamus HisDC activity was elevated 2-2.2-fold by incubating the cytosol for 15 min in the presence of MnCl2, a known stimulator of phosphoprotein phosphatase; this was prevented when 20 mM NaF, a common inhibitor of phosphoprotein phosphatase, was added to the cytosol. The apparent Km of ATP.Mg-treated hypothalamus HisDC for histidine was elevated 5-10-fold compared to controls, whereas the Vmax was approximately the same. Under this condition, the Km was calculated as high as 0.5-2.2 mM (depending on phosphorylating conditions), while controls had a Km of 0.1-0.3 mM (depending on the initial phosphorylating states). Addition of rabbit muscle (type I), bovine heart (type II) or rat cerebellum (types I and II) inhibitor of protein kinase A, to the phosphorylating mixture, abolished the difference in Km between control and ATP.Mg-treated HisDC. Moreover, rat cerebellum protein kinase A inhibitors increased Vmax to above the control level; while 20 mM NaF (inhibitor of phosphoprotein phosphatase) decreased Vmax to approximately one half of that of the controls. These data indicate that HisDC activity in the hypothalamus and the lungs, but not in the stomach, is affected in oppositely by protein kinase A and phosphoprotein phosphatases.

A metal‐mediated hydride shift mechanism for xylose isomerase based on the 1.6 Å Streptomycs rubiginosus structure with xylitol and D‐xylose

The crystal structure of recombinant Streptomyces rubiginosus D-xylose isomerase (D-xylose keto-isomerase, EC 5.3.1.5) solved by the multiple isomorphous replacement technique has been refined to R = 0.16 at 1.64 A resolution. As observed in an earlier study at 4.0 A (Carrell et al., J. Biol. Chem. 259: 3230-3236, 1984), xylose isomerase is a tetramer composed of four identical subunits. The monomer consists of an eight-stranded parallel beta-barrel surrounded by eight helices with an extended C-terminal tail that provides extensive contacts with a neighboring monomer. The active site pocket is defined by an opening in the barrel whose entrance is lined with hydrophobic residues while the bottom of the pocket consists mainly of glutamate, aspartate, and histidine residues coordinated to two manganese ions. The structures of the enzyme in the presence of MnCl2, the inhibitor xylitol, and the substrate D-xylose in the presence and absence of MnCl2 have also been refined to R = 0.14 at 1.60 A, R = 0.15 at 1.71 A, R = 0.15 at 1.60 A, and R = 0.14 at 1.60 A, respectively. Both the ring oxygen of the cyclic alpha-D-xylose and its C1 hydroxyl are within hydrogen bonding distance of NE2 of His-54 in the structure crystallized in the presence of D-xylose. Both the inhibitor, xylitol, and the extended form of the substrate, D-xylose, bind such that the C2 and C4 OH groups interact with one of the two divalent cations found in the active site and the C1 OH with the other cation. The remainder of the OH groups hydrogen bond with neighboring amino acid side chains. A detailed mechanism for D-xylose isomerase is proposed. Upon binding of cyclic alpha-D-xylose to xylose isomerase, His-54 acts as the catalytic base in a ring opening reaction. The ring opening step is followed by binding of D-xylose, involving two divalent cations, in an extended conformation. The isomerization of D-xylose to D-xylulose involves a metal-mediated 1,2-hydride shift. The final step in the mechanism is a ring closure to produce alpha-D-xylulose. The ring closing is the reverse of the ring opening step. This mechanism accounts for the majority of xylose isomerase's biochemical properties, including (1) the lack of solvent exchange between the 2-position of D-xylose and the 1-pro-R position of D-xylulose, (2) the chemical modification of histidine and lysine, (3) the pH vs. activity profile, and (4) the requirement for two divalent cations in the mechanism.

In Vitro Oxidation of Indoleacetic Acid by Soluble Auxin-Oxidases and Peroxidases from Maize Roots

Soluble auxin-oxidases were extracted from Zea mays L. cv LG11 apical root segments and partially separated from peroxidases (EC 1.11.1.7) by size-exclusion chromatography. Auxin-oxidases were resolved into one main peak corresponding to a molecular mass of 32.5 kilodaltons and a minor peak at 54.5 kilodaltons. Peroxidases were separated into at least four peaks, with molecular masses from 32.5 to 78 kilodaltons. In vitro activity of indoleacetic acid-oxidases was dependent on the presence of MnCl(2) and p-coumaric acid. Compound(s) present in the crude extract and several synthetic auxin transport inhibitors (including 2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid) inhibited auxin-oxidase activity, but had no effect on peroxidases. The products resulting from the in vitro enzymatic oxidation of [(3)H] indoleacetic acid were separated by HPLC and the major metabolite was found to cochromatograph with indol-3yl-methanol.

Oligomerizations of deoxyadenosine bis-phosphates and of their 3′-5′, 3′-3′, and 5′-5′ dimers: Effects of a pyrophosphate-linked, poly(t) analog

A pyrophosphate-linked polynucleotide analog based on thymidine 3',5' bis-phosphate (pTp) catalyzes the oligomerization of activated dimers of pdAp in the presence of MgCl2. Although no catalysis of the oligomerization of the activated monomer (ImpdAplm) was observed in the presence of MgCl2, there was a significant stimulation of oligomerization by the template in the presence of MnCl2.

Studies on restriction endonucleases of acetic acid bacteria and allied organisms. PartXI. Purification of restriction endonuclease from Acetobacter aceti IFO 3281 (AatII)and its properties.

The restriction endonuclease AatII was purified from cell-free extracts of Acetobacter aceti IFO 3281 by streptomycin treatment, ammonium sulfate fractionation, combined column chromatographies on DEAE-Toyopearl 650S, heparin-Sepharose CL-6B and DEAE-Sepharose CL-6B and FPLC on Mono Q and on Superose 12 (gel filtration). The purified enzyme was homogeneous on SDS-polyacrylamide gel disk electrophoresis. The relative molecular mass of the purified enzyme was 190,000 daltons by gel filtration. The SDS-polyacrylamide gel disk electrophoresis gave the relative molecular mass of 47,500 daltons. These data indicated that the purified, native enzyme is a tetramer (190,000 daltons) composed of four 47,500-dalton subunits. The isoelectric point of the enzyme was 6.0. The purified enzyme was intensely activated by manganese ion (50-fold increase or more when compared with magnesium ion). The enzyme worked best at 37 degrees C and pH 8.5 in a reaction mixture (50 microliters) containing 1.0 micrograms lambda DNA, 10 mM Tris-HCl, 7 mM 2-mercaptoethanol, 7 mM MnCl2 and 50 mM NaCl. The enzyme recognizes the same palindromic hexanucleotide sequence 5'-GACGTC-3', cuts between T and C and produces a 3'-tetranucleotide extension in the presence of MnCl2, as it does in the presence of MgCl2.

Phosphorylation of Highly Purified Growth Hormone Receptors by a Growth Hormone Receptor-associated Tyrosine Kinase

We have shown previously that growth hormone (GH) promotes the phosphorylation of its receptor on tyrosyl residues (Foster, C. M., Shafer, J. A., Rozsa, F. W., Wang, X., Lewis, S. D., Renken, D. A., Natale, J. E., Schwartz, J., and Carter-Su, C. (1988) Biochemistry 27, 326-334). In the present study, we investigated the possibility that a tyrosine kinase is specifically associated with the GH receptor. GH-receptor complexes were first partially purified from GH-treated 3T3-F442A fibroblasts, a GH-responsive cell, by immunoprecipitation using anti-GH antiserum. 35S-Labeled proteins of Mr = 105,000-125,000 were observed in the immunoprecipitate from GH-treated cells labeled metabolically with 35S-amino-acids. These proteins were not observed in immunoprecipitates from cells not exposed to GH or when non-immune serum replaced the anti-GH antiserum, consistent with the proteins being GH receptors. GH receptors appeared to be phosphorylated, as evidenced by the presence of 32P-labeled bands, comigrating with the 105-125 kDa 35S-labeled proteins, in the immunoprecipitate of GH-treated cells labeled metabolically with [32P]Pi. When partially purified GH receptor preparation was incubated with [gamma-32P]ATP (7-15 microM) for 10 min at 30 degrees C in the presence of MnCl2, a protein of Mr = 121,000 was phosphorylated exclusively on tyrosyl residues. As expected for the GH receptor, this protein was not observed in immunoprecipitates when cells had not been treated with GH nor when non-immune serum replaced the anti-GH antiserum. GH-receptor complexes were also purified to near homogeneity by sequential immunoprecipitation with phosphotyrosyl-binding antibody followed by anti-GH antiserum. When cells were labeled metabolically with 35S-amino acids, the 35S label migrated almost exclusively as an Mr = 105,000-125,000 protein. This protein also incorporated 32P into tyrosyl residues when incubated in solution with [gamma-32P]ATP. These results show that highly purified GH receptor preparations undergo tyrosyl phosphorylation, suggesting that either the GH receptor itself is a tyrosine kinase or is tightly associated with a tyrosine kinase.

Nonenzymatic phosphorylation of tyrosine and serine by ATP is catalyzed by manganese but not magnesium.

Many cellular and retroviral protein-tyrosine kinases display either a requirement or a preference for manganese over magnesium for maximal activity. We have observed that peptides and proteins are non-enzymatically phosphorylated at tyrosine and serine by ATP when heated in the presence of MnCl2 at neutral pH. The extent of the reaction is negligible below 50 degrees C but increases rapidly at higher temperatures. The reaction proceeds in the presence of sodium dodecyl sulfate but is blocked by EDTA. No reaction is observed in the absence of Mn2+, even if Mg2+ is present. Manganese therefore acts as a catalyst for the non-enzymatic reaction, but magnesium does not. We propose that the preference for manganese shown by many protein tyrosine kinases is due at least in part to the intrinsic ability of Mn2+ to catalyze the transfer of phosphate from ATP to a phosphate acceptor such as tyrosine. The nonenzymatic phosphorylation reaction also offers a new synthetic pathway for the preparation of radiolabeled peptides containing phosphotyrosine and phosphoserine.

Ehrlich ascites tumor cell UDP-Gal:N-acetyl-D-glucosamine beta(1,4)-galactosyltransferase. Purification, characterization, and topography of the acceptor-binding site.

A UDP-Gal:N-acetylglucosamine beta(1,4)-galactosyltransferase which catalyzes the synthesis of beta-D-Gal(1,4)-D-GlcNAc units has been purified 17,560-fold from Ehrlich tumor cells to apparent electrophoretic homogeneity. The enzyme appears to be a monomeric protein with Mr = 56,000-58,000. Enzymatic activity requires the presence of MnCl2, is stimulated by detergent, and exhibits a pH optimum at 6.9. The Km values for GlcNAc and UDP-Gal are 1.89 and 0.046 mM, respectively. The Ehrlich cell beta-galactosyltransferase acts efficiently on glycoproteins and glycolipids terminating in GlcNAc, but is inactive toward glycoconjugates possessing terminal GalNAc units. The oligosaccharides beta-D-GlcNAc(1,3)-D-Gal and beta-D-GlcNAc(1,3)[beta-D-GlcNAc(1,6)]-D-Gal are good acceptors for the beta-galactosyltransferase from Ehrlich cells, suggesting that the enzyme may participate in the biosynthesis of i/I structures. In addition, other linear and branched sugars presenting GlcNAc residues at their nonreducing termini also act as acceptors for the enzyme. The activity of Ehrlich cell beta-galactosyltransferase both in the presence and absence of alpha-lactalbumin has been studied using a series of derivatives of Glc and GlcNAc which were substituted at various positions of the pyranose ring. This study has provided a map of the molecular contacts necessary for enzymatic activity in the presence and in the absence of alpha-lactalbumin.

Excitation-contraction coupling in non-spiking smooth muscle in the gill ofAplysia

1. Excitation-contraction coupling (E-C Coupling) was investigated in the non-spiking longitudinal efferent vein muscle (LEVM) from theAplysia gill. The sucrose-gap method was used to study the E-C coupling in response to acetylcholine (ACh) and to elevated external potassium (Ko). 2. LEVMs from mature and oldAplysia were used in this study. The response to high Ko and ACh was not significantly different in the two groups. 3. Depolarization increased directly with the concentration of either Ko (50 to 500 mM) or ACh (1 μM to 1 mM). It preceded the development of tension, which increased with depolarization. These stimulants did not evoke action potentials. 4. In the presence of MnCl2 (10–20 mM) for 30 min, ACh-contracture was reduced to near zero; in contrast K-contracture was only reduced to 60% of the control level; and depolarization was unaffected. In a nominally Ca-free medium for 30 min, ACh-contracture was reduced to zero, whereas K-concentration persisted at 16% of the control level. Hence, ACh and high Ko apparently mobilize different Ca stores to initiate E-C coupling: In the non-spiking LEVM, ACh-contracture primarily involves influx of Ca, whereas K-contracture appears to rely on primarily cellular Ca. The results are contrasted to those in a spiking molluscan muscle. 5. We propose that mobilizing both stores of calcium to initiate E-C coupling insures synchronized contraction of LEVM fibers. The similarity of activation in vitro, by Ko and ACh, and in situ, by neuronal firing, is shown and discussed.

In vitro augmentation of natural killer cell activity by manganese chloride

The in vitro cultivation of murine spleen cells with MnCl2 resulted in the enhancement of natural killer (NK) cell activity as measured in a 4-h 51Cr-release assay. Optimal enhancement of NK activity was observed at concentrations of 10-20 micrograms MnCl2/culture (72-144 microM Mn2+). Enhancement of NK activity by MnCl2 was not associated with any changes in the number or viability of cells following culture. The addition of antiasialo GM1 antibody and complement to spleen cell cultures completely abrogated the enhancement of NK activity by MnCl2. The enhancement of NK activity by MnCl2 in vitro was accompanied by interferon induction. The addition of rabbit antimouse interferon to spleen cells cultured with MnCl2 reduced NK activity. NK activity in cultures treated with MnCl2 was also reduced upon removal of plastic adherent cells. However, restoration of enhanced NK activity by addition of adherent cells to nonadherent cells in the presence of MnCl2 was not observed. Similar effects of NK activity were observed with polyinosinic-polycytidylic acid (Poly I X C), a known interferon inducer and NK enhancer. The results demonstrate that murine splenic NK activity is enhanced in vitro by MnCl2 and that this enhancement may be mediated by interferon induction. The results also suggest that in vitro enhancement of NK activity by MnCl2, as with Poly I X C, may require participation of an adherent cell population for NK augmentation.

Production of pea lectin in Escherichia coli.

In order to explore the molecular basis for the glycopeptide specificity of legume lectins, we have developed an experimental system in which specific amino acid alterations can be introduced into the carbohydrate binding site of pea lectin. This system is based on the production of pea lectin in Escherichia coli. The plasmid coding for the lectin was constructed from two lectin cDNA sequences isolated from Pisum sativum seeds (Higgins, T. J. V., Chandler, P. M., Zurawski, G., Button, S. C., and Spencer, D. (1983) J. Biol. Chem. 258, 9544-9549) and an expression vector based on the gene for the outer membrane lipoprotein of E. coli (Nakamura, K., and Inouye, M. (1982) EMBO J. 1, 771-775). The lectin is produced as a single polypeptide chain and forms insoluble aggregates in E. coli cells (2-5 mg/liter). Functional lectin is recovered by solubilization of the aggregates in guanidinium hydrochloride, renaturation in the presence of MnCl2 and CaCl2, and affinity purification on Sephadex. This procedure yields a homogeneous 28,000-dalton protein. Comparison of the recombinant lectin with natural pea lectin in an inhibition of hemagglutination assay demonstrated that there is no detectable difference in the carbohydrate binding properties of the two lectins.