Requirement For Divalent Ions Research Articles

Biochemical and Functional Characterization of an Iron-Containing Alcohol Dehydrogenase from Thermococcus barophilus Ch5.

Two iron-containing alcohol dehydrogenases (ADHs) are encoded in the genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba ADH641 and Tba ADH547). In our previous publication, we reported biochemical characteristics and catalytic mechanism of Tba ADH547. Herein, we present evidence that Tba ADH641 possesses two activities for ethanol oxidization and acetaldehyde reduction at high temperature, capable of using NAD(H) and NADP(H) as coenzyme. Biochemical data show that Tba ADH641 possesses optimal reaction temperature, thermostability, divalent ion requirement, and substrate specificity distinct from Tba ADH547 and other iron-containing ADH homologues. However, Tba ADH641 and Tba ADH547 display same optimal reaction pH. Kinetic analyses demonstrate that Tba ADH641 displays higher catalytic efficiency for acetaldehyde reduction than that for ethanol oxidation, which is consistent with Tba ADH547. Mutational data demonstrate that residues D115, K118, E159, D190, and E215 in Tba ADH641, which has not been described to date, are necessary for enzyme activity, thus augmenting our understanding on catalytic mechanism of iron-containing ADH. Overall, our work demonstrates that Tba ADH641 is an iron-containing ADH with novel features, which is distinct from Tba ADH547, thus providing a potential biocatalyst for biotransformation reaction.

Purification and properties of a novel fungal alkaline keratinase from Cunninghamella echinulata

Aim: The purification and characterization of an alkaline keratinase from Cunninghamella echinulata. Materials and Methods: Feather-meal medium was used to isolate and screen the fungi. Acetone precipitation and lectin agarose affinity column were used in the purification. The effect of pH, temperature, metal ions and group modifying chemicals was tested. Results: The purified keratinase is a serine protease with a molecular mass of 33kDa with an optimal pH of 4.5 and 10.0, and an optimum temperature of 30°C and 60oC. A 13.2-fold purification was obtained with affinity methods. Discussion: C.echinulata keratinase was inhibited by PMSF, thus it could be a serine pro- tease. Enzyme was inhibited by group specific reagents like TLCK, IAA, NEM and NAI indicating that serine, cysteine, tyrosine and lysine play an important role in the catalytic activity. There was no effect of metal-chelating agent on enzyme activity indicating that the enzyme is not a metalloenzyme; however, it is a metal-activated enzyme as the activity was enhanced by Mn²+. Inhibitory effects of group specific reagents indicated that the enzyme is a serine protease which does not have any divalent ion requirement. The enzyme isolated also has appreciable activity at two different temperatures and pH values making it a versatile organism for industrial applications.

A preliminary investigation on the growth requirement for monovalent cations, divalent cations and medium ionic strength of marine actinomycete Salinispora

In this paper, we report that three species of Salinispora, S. arenicola, S. tropica, and S. pacifica, require magnesium and calcium, for growth, with S. pacifica having the most stringent growth requirement for these ions. Interaction between these ions in supporting the growth of Salinispora was observed. We also demonstrated that the absolute requirement of sodium to support the growth of Salinispora has not been established as all three species of Salinispora can use either potassium or lithium to replace sodium to support maximum growth. While lithium can replace sodium to support maximum growth of Salinispora, it is more toxic to S. arenicola than S. tropica and S. pacifica, inhibiting the growth of S. arenicola at 189 mM but without effect on the growth of S. tropica and S. pacifica. Using both sodium chloride-based and lithium chloride-based media, we showed that Salinispora has a growth requirement for divalent ions, magnesium and calcium as well as growth requirement for ionic strength (8.29 to 15.2 mS/cm). S. arenicola has a lower growth requirement for ionic strength than S. tropica and S. pacifica.

Expression and Characterization of Two Functional Methionine Aminopeptidases from Mycobacterium tuberculosis H37Rv

Methionine aminopeptidase (MetAP) carries out an essential posttranslational modification of nascent proteins by removing the N-terminal methionine and is a potential target for discovering antibacterial agents. To characterize and compare the two MetAPs in Mycobacterium tuberculosis, Rv0734 and Rv2861c genes encoding MetAPs from genome of Mycobacterium tuberculosis H37Rv were cloned and expressed in Escherichia coli. Comparative analysis showed that the two recombinant Mycobacterium tuberculosis MetAPs (MtMetAPs, with 6His-tag being cleaved) have different activities in their substrate specificity, divalent ion requirement, and temperature optima. The temperature for MtMetAP1a and MtMetAP1c were 55 and 37 degrees C, respectively, and MtMetAP1a was found to have good temperature stability. The activities of MtMetAPs were increased by Co2+ ions, but were strongly inhibited by Cu2+, Fe2+, and Ni2+. In addition, the MtMetAP1a and MtMetAP1c activities were stimulated by Mg2+ and Zn2+, respectively. Transcriptional comparative analysis of these two genes revealed that, in both H37Ra and H37Rv, there was approximately a 2-fold decrease of Rv0734 transcripts in 60-day-old stationary phase culture comparing to that in 14-day-old log phase bacilli. On the other hand, the transcription level of Rv2861c in tested mycobacteria in log phase culture was nearly 1.5 times lower than that in stationary phase culture. The result suggests that the two MtMetMAPs may perform important function in different growth phases of Mycobacterium tuberculosis.

Molecular cloning, expression and characterization of three distinctive genes encoding methionine aminopeptidases in cyanobacterium Synechocystis sp. strain PCC6803.

Methionine aminopeptidase, known to be encoded by single genes in prokaryotes, is a cobalt-dependent enzyme that catalyzes the removal of N-terminal methionine residues from nascent polypeptides. Three ORFs encoding putative methionine aminopeptidases from the genome of cyanobacterium Synechocystis sp. strain PCC6803, designated as slr0786 ( map-1), slr0918 ( map-2) and sll0555 ( map-3) were cloned and expressed in Escherichia coli. The purified recombinant proteins encoded by map-1 and map-3 had much higher methionine aminopeptidase activity than the recombinant protein encoded by map-2. Comparative analysis revealed that the three recombinant enzymes differed in their substrate specificity, divalent ion requirement, pH, and temperature optima. The broad activities of the iso-enzymes are discussed in light of the structural similarities with other peptidase families and their levels of specificity in the cell. Potential application of cyanobacterial MetAPs in the production of recombinant proteins used in medicine is proposed. This is the first report of a prokaryote harboring multiple methionine aminopeptidases.

Utilization of a soluble integrin-alkaline phosphatase chimera to characterize integrin alpha 8 beta 1 receptor interactions with tenascin: murine alpha 8 beta 1 binds to the RGD site in tenascin-C fragments, but not to native tenascin-C.

The integrin alpha 8 beta 1 has been reported to bind to fibronectin, vitronectin, and tenascin-C in cell adhesion or neurite outgrowth assays. Here, we describe cDNA cloning of the murine alpha 8 subunit, purification of a recombinant soluble heterodimer consisting of the extracellular domains of the murine alpha 8 and beta1 subunits, and development of a sensitive binding assay using a modified form of this heterodimer fused to alkaline phosphatase (AP). In binding assays, the purified alpha 8 beta 1-AP chimera exhibited the same divalent ion requirements for activation and binding specificity as cell surface alpha 8 beta 1: in the presence of Mn2+ it bound to fibronectin and vitronectin in an RGDS-peptide inhibitable manner. Contrary to previous reports, we found no evidence that alpha 8 beta 1, expressed on K562 cells or as an AP chimera, interacts strongly with native tenascin-C. In binding, adhesion, and spreading assays, significant interactions were observed only to short fragments of tenascin-C containing the third fibronectin type III repeat which contains an RGD sequence. Full length tenascin-C and longer fragments containing this repeat did not appear to serve as ligands, implying that the RGD site in native tenascin-C is a cryptic binding site for this integrin, exposed by removal of adjacent domains. Soluble integrin-AP chimeras should be generally useful for identifying and characterizing integrin interactions with ligands.

Regulation of cyclic AMP phosphodiesterase activity by particulate protein tyrosine kinase and phosphotyrosine phosphatase activities sensitive to sodium orthovanadate.

Sodium orthovanadate (vanadate) stimulated cAMP phosphodiesterase (PDE) and protein tyrosine kinase (PTK) activities and inhibited the phosphotyrosine phosphatase (PTPase) activity in the particulate of isolated rat fat pads. Okadaic acid never showed any increase in the PDE activity up to 1 microM. Amiloride inhibited in part both stimulations of PDE and PTK activities by vanadate. The particulate PTK activity had an optimal divalent ion requirement of 15 mM Mg+2+2 mM Mn+2 in the assay medium and was not inhibited by 1 mM N-ethylmaleimide, suggesting it to be a different type from the insulin receptor and cytosolic PTK activities. The PDE, PTK, and PTPase active fractions were separated from the solubilized particulate fraction on a DEAE-Sephacel column. PDE activity was increased by the addition of the PTK active fraction. A further increase was observed by using the PTK active fraction pretreated with 1 mM vanadate. In contrast, the addition of PTPase active fraction decreased the PDE activity. This decrease disappeared by using the PTPase active fraction pretreated with 1 mM vanadate. These results suggest that the PDE activity is in part regulated through a process involving the particulate PTK and PTPase activities sensitive to vanadate.

A mechanism for divalent cation regulation of beta 3-integrins.

Integrins alpha IIb beta 3 and alpha V beta 3 mediate numerous cell-matrix and cell-cell contacts. Both integrins contain multiple divalent cation-binding motifs that regulate ligand binding. Here, we elucidate a major difference in the regulation of alpha IIb beta 3 and alpha V beta 3 by divalent ions. Fibrinogen binding to alpha IIb beta 3 in Ca(2+)-containing buffer is rapid, with an apparent association rate constant (k1app) of 8.2 x 10(5) M-1 s-1, but Ca2+ does not support association between fibrinogen and alpha V beta 3. Interestingly, Mn2+ supports fibrinogen binding to both integrins, albeit with a relatively slow association rate (k1app = 10(4) M-1 s-1). This influence of divalent ions on ligand association rates accounts for the opposite divalent ion requirements for platelet aggregation and tumor cell adhesion to fibrinogen. Furthermore, the regulation of fibrinogen binding to alpha V beta 3 is complex when both Ca2+ and Mn2+ are present. Physiological concentrations of Ca2+ completely ablated adhesion. Kinetic analysis demonstrated that Ca2+ is a mixed-type inhibitor of Mn(2+)-supported fibrinogen binding to alpha V beta 3. Consequently, the data presented here suggest a mechanism in which two separate cation-binding sites regulate ligand binding to beta 3-integrins.

Substrate specificity of iodothyronine 5′-deiodinase in rat liver homogenates and its requirements of divalent cations [formula omitted

Studies were carried out to compare the 5′-deiodination reactions of thyroxine (T 4) and 3,3′ −5′-triiodothyronine (rT 3) in 2.5% rat liver homogenates. The 5′-deiodinase activity was assayed by the 3,5,3′-triiodothyronine (T 3) produced from T 4 or by 125I-rT 3. Under our experimental conditions, the two 5′-monodeiodination reactions resulted in similar apparent K Ms: 1.5 μM for T 4 and 1.1 μM for rT 3. However, the apparent V max values of T 4 and rT 3 deiodination reactions were, respectively, 0.91 and 222 pmol/mg protein/min. Both reactions were stimulated by thiol reagents but only rT 3 deiodination showed complete thiol dependence. The inhibitory effect of 6-propyl-2-thiouracil on the 5′-deiodination of rT 3 was at least 50 fold greater than that of T 4. The divalent ion requirement of the deiodination system was tested with CaCl 2, MgCl 2, and ZnCl 2 at a range of concentrations. Zinc ion appeared to be a potent inhibitor in both T 4 and rT 3 deiodination systems. Only the 5′-deiodination of rT 3 was inhibited slightly by low concentrations of calcium and magnesium ions. Our results suggest that based on their apparently distinct regulation mechanisms, the 5′-monodiodination of T 4 and rT 3 in rat liver homogenates is likely mediated by more than one enzyme, despite the similarity of observed K Ms.

Characterization and distribution of the hemagglutinins produced by Vibrio cholerae

Examination of the distribution of cell-associated and soluble hemagglutinins (HA) produced by Vibrio cholerae revealed the existence of four different HAs. A cell-associated mannose-sensitive HA (MSHA) was produced only by the El Tor biotype. This was evident with all El Tor strains examined. It appears to be responsible for the HA biotyping differentiation of El Tor from classical biotype V. cholerae. The MSHA had no apparent divalent ion requirement; it was inhibited by D-mannose and D-fructose; and it was active on all human (A, B, O) and all chicken erythrocytes tested. Spontaneous MSHA- mutants of El Tor strains were selected by cosedimentation of MSHA+ parent bacteria with erythrocytes. An L-fucose-sensitive HA was detected transiently in early log-phase growth with two of the four classical strains examined and with MSHA- mutants of El Tor biotype strains 3083, 26-3, and 17. MSHA- mutants also expressed another cell-associated HA in late log-phase cultures. A "soluble" HA was detected in late log-phase cultures of all strains tested. This HA was not inhibitable by any sugars tested; it required CA2+ ions for maximum activity; and it was active on some chicken erythrocytes but not others.

Studies on the intercellular adhesion of rat and chicken hepatocytes. Conditions for stimulation by liver plasma membranes.

A new assay was developed for measuring the stimulation of intercellular adhesion of rat hepatocytes by rat liver plasma membranes. Aggregates formed in the presence of the membranes are separated from single cells by filtration, and the number of cells in the aggregates is determined by their lactate dehydrogenase content. The formation of aggregates was specifically stimulated by rat liver plasma membranes and the rate of aggregate formation was proportional to the quantity of added membranes. The effects of divalent cations on the initial rates of rat and chicken hepatocytes adhesion were also examined. Optimal rates of adhesion of rat hepatocytes were obtained in the presence of physiological levels of Mg2+, and adhesion was inhibited by Ca2+, whereas optimal rates of chicken hepatocyte adhesion were obtained in the presence of physiological levels of Ca2+. Plasma membrane stimulation of hepatocyte adhesion also showed the same divalent ion requirements with the respective cell types. The stimulatory activity in the rat plasma membranes determined by the filter assay was found to be sensitive both to trypsin digestion and to mild periodate oxidation. The activity was also completely resistant to vigorous reductive alkylation. These results taken together suggest that the rat plasma membrane stimulatory activity is associated with a glycoprotein(s).

Mise en évidence, études cinétiques et solubilisation de l'adénylate cyclase particulaire de Nocardia restricta

An adenylate cyclase (EC r.6.1.1.) was found in cell-free extracts of several Nocardia species. The enzyme from Nocardia restricta has been specially studied. It is a membrane enzyme which exhibits a strong specific activity, one hundred times greater than that of mammals. It has an optimal pH of 8.5 in Tris buffer and an absolute requirement for divalent ions, Mg++ or Mn++ (Mn++ ions are the most efficient). The kinetic properties of this adenylate cyclase are similar to those that could be expected of an allosteric enzyme having, as a substrate, the ATP-Mg++ complex and, as an activator, free Mn++ ions. Ca++ ions are activators: they set up the maximum velocity without modification of the KM. GTP is a competitive inhibitor (KI = 5.10(-5) M). Fluoride ions have no detectable effect on activity. Non-ionic detergents, Lubrol WX and Triton X 100, are inhibitors of the enzyme which has been partially solubilized by repeated freezing and thawing, following by brief ultra-sonic treatment. Catalytic sites are not modified after the solubilization, but cooperative effects between moles of substrate ATP-Mn++ are diminished: the KM becomes smaller and the sigmoidal shape of the curve v = f (ATP-Mn++) is attenuated.

Purification and properties of adductor muscle phosphofructokinase from the oyster, Crassostrea virginica. The aerobic/anaerobic transition: role of arginine phosphate in enzyme control.

Phosphofructokinase from oyster (Crassostrea virginica) adductor muscle occurs in a single electrophorectic form at an activity of 8.1 mumol of product formed per minute per gram wet weight. The enzyme was purified to homogeneity by a novel method involving extraction in dilute ethanol and subsequent precipitation with polyethylene glycol. Oyster adductor phosphofructokinase has a molecular weight of 3400000 +/- 20000 as measured by Sephadex gel chromatography. Mg2+ or Mn2+ can satisfy the divalent ion requirement while ATP, GTP, or ITP can serve as phosphate donors for the reaction. Oyster adductor phosphofructokinase displays hyperbolic saturation kinetics with respect to all substrates (fructose 6-phosphate, ATP, and Mg2+) at either pH 7.9 OR PH 6.8. The Michaelis constant for fructose 6 phosphate at pH 6.8, the cellular pH of anoxic oyster tissues, is 3.5 mM. In the presence of AMP, by far the most potent activator and deinhibitor of the enzyme, this drops to 0.70 mM. Many traditional effectors of phosphofructokinase including citrate, NAD(P)H,Ca2+, fructose 1,6-bisphosphate, 3-phosphoglycerate, ADP, and phosphoenolpyruvate do not alter enzyme activity when tested at their physiological concentrations. Monovalent ions (K +, NH4+) are activators of the enzyme. ATP and arginine phosphate are the only compounds found to inhibit the adductor enzyme. The inhibitory action of both can be reversed by physiological concentrations of AMP(0.2- 1.0mM) and to a lesser extent by high concentrations of Pi (20 mM) and adenosine 3' :5'-monophosphate (0.1 mM). The two inhibitors exhibit very different pH versus inhibition profiles. The Ki (ATP) decreases from 5.0 mM to 1.3 mM as the pH decreases from 7.9 to 6.8, whereas the Ki for arginine phosphate increases from 1.3 mM to 4.5 mM for the same pH drop. Of all compounds tested, only AMP, within its physiological range, activated adductor phosphofructokinase significantly at low pH values. The kinetic data support the proposal that arginine phosphate, not ATP or citrate, is the most likely regulator of adductor phosphofructokinase in vivo under aerobic, high tissue pH, conditions. In anoxia, the depletion of arginine phosphate reserves and the increase in AMP concentrations in the tissue, coupled with the increase in the Ki for arginine phosphate brought about by low pH conditions, serves to activate phosphofructokinase to aid maintenance of anaerobic energy production.

Fructose-1, 6-bisphosphatase of an extreme thermophile.

D-Fructose-1,6-bisphosphatase [D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11] from Flavobacterium thermophilum HB8, an extremely thermophilic bacterium, was partially purified. The preparation was free from non-specific phosphatase activity and its specific activity was 8 times that of the crude extract. The enzyme specifically hydrolyzed the C-l phosphate ester bond of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. The enzyme was similar to the E. coli fructose 1,6-bisphosphatase as regards optimum pH (8.1), requirement for divalent ions, and Km for D-fructose 1,6-bisphosphate (2×10−5M). The substrate, D-fructose 1,6-bisphosphate, was inhibitory to the enzyme at high concentrations. The enzyme was activated by phosphoenolpyruvate, which released the substrate inhibition. AMP inhibited the enzyme activity, emphasizing the substrate inhibition. Inactivation by AMP was recovered to some extent by the addition of phosphoenolpyruvate. The enzyme was heat resistant and both the catalytic and allosteric properties of the enzyme remained unchanged after treatment at high temperatures, for instance, at 70°C for 1 hr. The role of phosphoenolpyruvate in the regulation of glycolysis-gluco-neogenesis in the bacterium is discussed.

Enzymatic Hydrolysis of Uridine Diphosphate-N-acetyl-d-galactosamine and Uridine Diphosphate-N-acetyl-d-glucosamine by Normal Cells, and Blocks in This Hydrolysis in Transformed Cells and Their Revertants

Abstract The two sugar nucleotides, UDP-N-acetyl-d-galactosamine and UDP-N-acetyl-d-glucosamine, are enzymatically hydrolyzed by normal hamster cell extracts to yield free N-acetyl-d-galactosamine and N-acetyl-d-glucosamine, respectively. A nucleotide pyrophosphatase activity splits the sugar nucleotides to UMP and the appropriate N-acetyl-d-hexosamine-1-phosphate, and another enzymatic step with orthophosphoric monoester phosphohydrolase activity then hydrolyzes the phosphorylated intermediate sugar to free N-acetyl-d-hexosamine. The two enzymatic activities differ in their divalent ion requirement and optimal pH and show the same hydrolytic activity on both sugar nucleotides. Different lines of hamster transformed cells and their revertants showed a block in either one or the other enzyme. Cell lines transformed by Rous sarcoma virus or Simian virus 40 were blocked in the nucleotide pyrophosphatase activity, whereas cell lines transformed by polyoma virus or after treatment with the chemical carcinogen dimethylnitrosamine were blocked in the second phosphohydrolase step. Variants from the polyoma- and dimethylnitrosamine-transformed cells, which had reverted in the biological properties characteristic of transformation, showed the same enzymatic block as their parental transformed cells. The block in enzyme activities may thus be due to a common genetic change in transformed cells and their revertants that is not found in the normal cells.

The Biosynthesis of Trehalose in Moniliformis dubius (Acanthocephala)

The occurrence, distribution, and synthesis of the nonreducing disaccharide, trehalose, were investigated in the tissues of female Moniliformis dubius, an acanthocephalan parasite of the rat. It was found that, although the sugar was present in large quantity in the body fluid and acanthors in all stages of development, the enzymes responsible for synthesis could not be detected. A survey of the trehalose concentration in whole worms of various ages showed the pool levels in unstarved worms to be high and rather constant regardless of infection age. The enzymes of trehalose synthesis were located in the body wall of adult female worms. The pathway was found to resemble closely that described in yeast and insects; the enzyme has a specific requirement for uridine diphosphoglucose and glucose-6-phosphate as cosubstrates. There was an absolute requirement for divalent ions and trehalose inhibited its own synthesis. The formation of trehalose-6-phosphate, although never demonstrated, was presumed to occur. The biosynthesis of trehalose [1-(a-D-glucopyranosyl)-a-D-glucopyranoside] was first demonstrated in yeast (Cabib and Leloir, 1958). These workers showed that the pathways involved two enzymes: (1) Trehalose phosphate synthetase: UDPGt + Glucose-6-P -> UDP + Trehalose-6-P (2) Trehalose-6-phosphatase: Trehalose-6-P + H20 -> Trehalose + Pi The biosynthesis of trehalose was subsequently shown to proceed by a similar pathway in insect fat body (Candy and Kilby, 1961); this same reaction was later found to occur in other systems (Goldman and Lornitzo, 1962; Received for publication 20 May 1971. * This work supported by grants from the NIH (GM12263, 2 T01 AI00106) and the Robert A. Welch Foundation (C-239). t Present address: Department of Medical Zoology, Division of Communicable Diseases and Immunology, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, D. C. 20012. t The following abbreviations are used: ATP for adenosine-5'-triphosphate, UDPG for uridine diphosphoglucose, GDPG for guanosine diphosphoglucose, ADPG for adenosine diphosphoglucose, CDPG for cytidine diphosphoglucose, TDPG for thymidine diphosphoglucose, EDTA for ethylene-diaminetetraacetic acid, Tris for Tris(hydroxymethyl) amino-methane, TM for Tris-maleate buffer, and KRT for Tris-buffered Krebs-Ringer's saline, pH 7.4. Roth and Sussman, 1966). Elbein (1967) demonstrated that GDPG was involved in the synthesis of trehalose-phosphate in Streptomyces hygroscopicus: GDPG + Glucose-6-P -> Trehalose-6-P GDP. This reaction has subsequently been shown to occur in a number of other species of Streptomyces (Elbein, 1968). More recently Liu et al. (1969) demonstrated that several species of Mycobacterium have the capacity to synthesize the disaccharide phosphate using either UDPG or GDPG as a precursor. It was futher shown that GDPG is the normal glucosyl donor for the synthesis of trehalose in Mycobacterium smegmatis, but in the presence of a polynucleotide fraction, the enzyme can be modified to utilize UDPG. Fairbairn (1958) reported trehalose to be present in 71 species of invertebrates representing the major phyla. One of the species examined was Moniliformis dubius, which was found to contain trehalose in a concentration of 2.3% of total tissue solids. Fairbairn speculated that trehalose would eventually be accepted as a carbohydrate occurring widely in invertebrates. Fairbairn and Passey (1957) examined the occurrence and distribution of trehalose and glycogen in the eggs of Ascaris suum. The egg glycogen was almost entirely concentrated in the embryo proper, while most of the trehalose was confined to the perivitelline

Properties of chromatin from liver and from a chemically induced minimal deviation hepatoma of the rat

Chromatin and DNA from a minimal deviation rat hepatoma and from rat liver were prepared and various physicochemical and template properties studied. More RNA and total protein but similar amounts of histone were associated with hepatoma chromatin than with liver chromatin. The spectral and thermal denaturation properties of the two chromatins are nearly the same, as is the case for DNA from the two sources. The kinetics of incorporation of labeled nucleoside triphosphates and the divalent ion requirements of DNA-directed RNA polymerase with hepatoma chromatin template are similar to those published for liver chromatin. The base compositions of the in vitro transcribed RNA from hepatoma and liver chromatins were similar to each other and (A + U)-rich. The RNA products from the two DNA templates also had similar compositions but were more (A + U)-rich than chromatin products. The relation of this work to the study of RNA populations in liver and hepatoma is briefly discussed.

Properties of Aldolase from Francisella tularensis

The aldolase of Francisella tularensis resembles Class II aldolases in its requirement for divalent ions and its inactivation by metal chelating agents. Cysteine and other reducing agents stimulated the activity of the enzyme.

Ability of nonenzymic nitration or acetylation of E. coli glutamine synthetase to produce effects analogous to enzymic adenylylation.

Treatment of unadenylylated glutamine synthetase from Escherichia coli with tetranitromethane or with N-acetylimidazole produces alterations in catalytic parameters that are similar to alterations caused by the physiologically important process of adenylylation. All three modification reactions lead to a change in divalent ion requirement for biosynthetic activity; the unmodified enzyme requires Mg(2+) for activity, whereas the modified enzymes exhibit increased activity with Mn(2+). The gamma-glutamyl transferase activity of the modified enzyme is more sensitive to feedback inhibition by tryptophan, histidine, CTP, and AMP, and to inhibition by Mg(2+) or to inactivation by 5 M urea. Finally, the pH optimum for the unmodified enzyme is 7.9, while the modified enzymes are more active at pH 6.8. Since treatment of the enzyme with N-acetylimidazole results in a decrease in absorbancy at 278 mmu and treatment with tetranitromethane causes an increase in absorbancy at 428 mmu, the effects of these reagents are probably due to modification of certain tyrosyl groups on the enzyme. However, other evidence indicates that the tyrosyl residues which are susceptible to adenylylation in the adenylyltransferase-catalyzed reaction are not involved in the acetylation or nitration reactions.

Comparison of the Effects of Divalent Ion Chelating Agents on Complement-Mediated Hemolysis in Rabbit and Human Serum

Abstract The chelating agent, magnesium ethylene-glycol-diamino-ethyl-tetraacetic acid (MgEGTA), inhibits EAC′ hemolysis when human serum is used as a complement source, but does not inhibit rabbit platelet injury which occurs in the presence of antigen-antibody reactions unrelated to platelet antigens. This difference has been assumed to indicate that the mechanism of complement action in producing this type of immunologically-induced platelet injury differs from that subserving EAC′ hemolysis. The present studies indicate that when rabbit serum rather than human serum is used as a source of complement, EAC′ hemolysis is not inhibited by MgEGTA. It is concluded that rabbit complement and human complement differ with respect to divalent ion requirement. The explanation for this difference may be that the concentration of calcium ion required for maintaining the rabbit C′1 molecule in an assembled state is lower than the concentration required for normal human C′1 activity.