Purification and properties of AMP-deaminase from human uterine smooth muscle

AMP-deaminase from human uterine smooth muscle has been isolated, and properties of the enzyme were characterized. At pH 7.0, and in the presence of 100 mM potassium chloride the enzyme manifests a distinctly sigmoidal type of kinetics, with S0.5 parameter value about 12 mM. 1 mM ATP strongly activates the enzyme, and diminishes the value of S0.5 to 1.2 mM. In contrast to that 2.5 mM orthophosphate slightly inhibits the activity of AMP-deaminase studied and increases the S0.5 to about 14 mM. Similarly to ATP, orthophosphate does not influence the maximum velocity of the reaction. Electrophoresis in the presence of sodium dodecyl sulphate revealed that the molecular weight of human smooth muscle AMP-deaminase subunit is close to 37 kDa.     

Cytotoxic activity toward mouse melanoma following immunization of mice with transfected cells expressing a human melanoma-associated antigen

Summary B78H1 is a mouse melanoma cell line that is weakly antigenic in syngeneic mice. In an attempt to augment their immunogenicity, B78H1 cells were transfected with genomic DNA from a line of human melanoma cells expressing a 96-kDa melanoma-associated antigen (ICAM-1). A selective co-amplification procedure was employed that generated a population of transfected cells (Ui11) that expressed fivefold higher quantities of the melanoma-associated antigen than the cells from which the DNA was obtained. To test the transfected cells' relative capacity to generate a cellular immune response against B78H1 cells, Ui11 cells and B78H1 cells were administered (in parallel) to syngeneic C57BL/6 mice, susceptible to the growth of the melanoma. Each cell line (lethally iradiated beforehand) was injected intraperitoneally at weekly intervals into the mice. After two or three injections, a standard chromium-release assay was employed to detect the presence of cellular immunity toward B78H1 cells. The population of spleen cells from mice immunized with the transfected melanoma cells exhibited higher levels of cytotoxicity toward B78H1 cells than spleen cells from mice immunized with equivalent numbers of nontransfected cells. This observation is consistent with the notion that the transfected human melanoma-associated antigen acted as a second antigen capable of potentiating cellular immune responses against the weakly immunogenic determinants of the mouse melanoma cells. The introduction of genes for foreign antigens into weakly antigenic tumor cells may generate immunogens that can lead to augmented anti-tumor cellular immune responses.     

The Turnover of Rat Cortical α1-Adrenoceptors Is Not Modified by Repeated Electroconvulsive Treatment

The effect of repeated treatment with electroconvulsive shock (ECS) on the turnover of cortical alpha 1-adrenoceptors in rats was measured using the N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)-induced irreversible receptor inactivation method. Repeated treatment with ECS did not affect parameters (the synthesis rate constant r, the degradation rate constant k) of alpha 1-adrenoceptor turnover. Because increase in the density of alpha 1-adrenoceptors in the ECS-treated group disappears later during measurement of turnover, several calculation possibilities were discussed. The present data confirm that repeated treatment with ECS produces a short-lasting up-regulation of cortical alpha 1-adrenoceptors, but does not affect the turnover of this receptor type.     

Growth Enhancement and Developmental Modifications of in Vitro Grown Potato (Solanum tuberosum spp. tuberosum) as Affected by a Nonfluorescent Pseudomonas sp

A plant growth-promoting rhizobacterium, designated Ps JN and isolated from onion roots, was identified as a nonfluorescent Pseudomonas sp. The percentage of similarity of Ps JN to P. gladioli (NCPPB 1891), P. cichorii (NCPPB 943), and P. viridiflava (NCPPB 635), as determined from 135 biochemical and physiological tests was 77, 70, and 66%, respectively. Ps JN persisted through successive generations of in vitro cultured potato plantlets, both as endophytic and epiphytic populations. In vitro inoculated potato (Solanum tuberosum) nodal explants produced plantlets with significant increases in root number (24-196%), root dry weight (44-201%), haulm dry weight (14-151%), and stem length (26-28%) as compared with noninoculated control plants. Bacterization also enhanced leaf hair formation (55-110%), secondary root branching, and total plant lignin content (43%). Other root colonizing bacteria or heat-killed cells of Ps JN had no significant effect on plant growth. Detached leaves from in vitro grown control plants, when exposed to 19°C and 50% relative humidity, lost 55% of their moisture content in 2.5 hours. Moisture loss by leaves of in vitro grown, bacterized plants, as well as greenhouse-acclimated, bacterized plants, and control plants, was less than 20%. Changes in stomatal closure appear to account for this difference.     

Arginine residues at the active site of avian liver phosphoenolpyruvate carboxykinase

The presence of arginine at the active site of avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification followed by a characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione all irreversibly inhibit the enzyme with second-order rate constants of 3.42 M-1 min-1, 3.13 M-1 min-1 and 0.313 M-1 min-1, respectively. The substrates phosphoenolpyruvate, IDP, and the activator Mn2+ offer little to modest protection from inhibition. Either CO2 or CO2 in the presence of any of the other substrates elicited potent protection against modification. Protection by CO2 against modification by phenylglyoxal or 1,2-cyclohexanedione gave a biphasic pattern. Rapid loss in activity to 40-60% occurred, followed by a very slow loss. Kinetics of inhibition suggest that the modification of arginine is specific and leads to loss of enzymatic activity. Substrate protection studies indicate an arginine residue(s) at the CO2 site of phosphoenolpyruvate carboxykinase. Apparently no arginine residues are at the binding site of the phosphate-containing substrates. Partially inactive (40-60% activity) enzyme, formed in the presence of CO2, has a slight change of its kinetic constants, and no alteration of its binding parameters or secondary structure as demonstrated by kinetic, proton relaxation rate, and circular dichroism studies. Labeling of enzyme with [(7-)14C]phenylglyoxal in the presence of CO2 (40-60% activity) showed 2 mol of phenylglyoxal/enzyme or 1 arginine or cysteine residue modified. Labeling of phosphoenolpyruvate carboxykinase in the absence of CO2 yielded 6 mol of label/enzyme. Labeling results indicate that avian phosphoenolpyruvate carboxykinase has 2 or 3 reactive arginine residues out of a total of 52 and only 1 or 2 are located at the active site and are involved in CO2 binding and activation.     

Caldesmon weakens the bonding between myosin heads and actin in ghost fibers

Earlier studies using polarized microphotometry have shown that caldesmon inhibits the alterations in structure and flexibility of actin in ghost fibers that take place upon the binding of myosin heads (Ga?azkiewicz et al. (1987) Biochim. Biophys. Acta 916, 368-375). The present investigations, performed with an IAEDANS label attached to myosin subfragment 1 (S-1), revealed that this inhibition results from the weakening of the binding between myosin heads and actin as indicated by the caldesmon-induced increase in the random movement of S-1. Parallel experiments with actin labeled at Cys-374 demonstrated that this effect of caldesmon is transmitted to the C-terminus of the actin molecule resulting in a conformational adjustment in this region of the molecule.     

Partial purification and characterization of bapa-ase fromVigna unguiculata (L.) walp

α-N-Benzoyl-DL-arginine-p-nitroanilide (BAPA) hydrolytic enzyme was partially purified from cotyledons of mature dry seeds ofVigna unguiculata (L.) Walp, cv. Seridó. Extracts of a finely ground meal ofVigna seeds were obtained with 0.02 M phosphate buffer (KH₂PO₄/ /Na₂HPO₄) pH 7.6. A protein fraction was obtained from the extracts by ammonium sulfate precipitation (25 to 50% saturation). This protein fraction was subjected to chromatography on DEAE-cellulose. A separated fraction of the cellulose column presented one main component and two minor bands as seen on polyacrylamide gels. The main component of the separated fraction gave a positive reaction with BAPA and acetyl-DL-phenylalanine-β-naphthyl ester (APNE). This fraction shows a molecular mass of 60 000 by gel filtration on Sephadex G-100, pH 7.6. The BAPA-ase activity was stable at pH values of 7.0 to 9.0 and no decrease in activity was observed by heating at 40 °C up to 40 min. The activity was not affected by PMSF, EDTA, cysteine,p- CMB, and IAC but was inhibited by NEM and strongly inhibited by TLCK. Leucine-p-nitroanilide (LPA) hydrolytic enzyme properties were also determined. This activity was inhibited by PMSF,p- CMB, and NEM.     

Specificity of a serum peptidase hydrolyzing thyroliberin at pyroglutamyl-histidine bone

The substrate specificity of a serum enzyme which degrades thyroliberin (less than Glu-His-Pro-NH2) into pyroglutamic acid and His-Pro-NH2 has been investigated and compared with that of the pyroglutamyl aminopeptidase from calf liver. The latter enzyme has a broad specificity, causing rapid degradation of thyroliberin, pyroglutamyl beta-naphthylamide and luliberin. In contrast, the serum enzyme causes rapid stereospecific cleavage only of the pyroglutamyl-histidine bond of thyroliberin and closely related peptides. Compounds such as less than Glu-Ala, less than Glu-His and pyroglutamyl beta-naphthylamide, which are known substrates of the pyroglutamyl aminopeptidases (such as the liver enzyme), are not substrates of the serum enzyme, and inhibit it only poorly. Pyroglutamyl-containing peptides such as luliberin and neurotensin and thyroliberin analogues such as LLD-thyroliberin, less than Glu-His-Pro-NHCH3, less than Glu-His-Pro-Gly-NH2 and less than Glu-Phe-Pro-NH2 inhibit effectively the degradation of thyroliberin by the serum enzyme, but are not hydrolyzed by this enzyme. The high specificity of the serum enzyme implies a physiological function.     

Bacterial mutagenesis and host cell DNA damage by chemical carcinogens in the Salmonella/hepatocyte system

Coincubation of isolated and intact rat hepatocytes and Salmonella typhimurium, (Salmonella/hepatocyte system) strain TA 98 was employed to determine both bacterial mutagenicity and DNA damage in the hepatocytes as measured by alkaline elution, following treatment with 2-acetylaminofluorene (AAF), 2-aminofluorene (AF) and N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Both the mutagenicity and the rate of DNA elution were dose-dependent for all three compounds. N-OH-AAF was 5 times more mutagenic and caused 80–100 times more DNA damage in the hepatocytes than AAF and AF when compared on a molar basis. The Salmonella/hepatocyte system may provide a more comprehensive evaluation of the potential genotoxic effect of chemicals than the currently used microbial mutagenesis sytems.     

The DNA polymerase beta reaction with ultraviolet-irradiated DNA incised by correndonuclease

Covalently closed circular Col E1 DNA was ultraviolet-irradiated with a dose of 60 J/m2, thus introducing about 3.2 pyrimidine dimers per DNA molecule. Treatment of irradiated Col E1 DNA with Micrococcus luteus correndonuclease resulted, in the vicinity of pyrimidine dimers, in an average of 3.3 incisions per DNA molecule, and converted DNA to the open circular form. Incised Col E1 DNA stimulated no reaction with calf thymus DNA polymerase alpha but was recognized as a template by DNA polymerase beta. The latter enzyme incorporated about 1.6 molecules of dTMP (corresponding to 6 molecules od dNMP) per one correndonuclease incision. The length of the DNA polymerase beta product was comparable to the anticipated length of the DNA region within which the hydrogen bonds were disrupted owing to dimer formation. The enzyme required Mg(2)=nd four dNTPs for reaction and was resistant to N-ethylmaleimide or p-mercuribenzoate. The average numbers of deoxynucleotides incorporated per one DNAase I incision or per one nonspecific break, measured in control samples, were equal, amounting to 0.3 dTMP molecule. This value corresponded to 1.2 dNMP molecule; in our opinion, this reflects contaminating nuclease activity of the system used. The present results testify to the ability of DNA polymerase beta to repair synthesis by the "patch and cut' mechanism.