Gel Filtration Resins Research Articles

Intranuclear Irradiation Inhibits Solid Tumor Growth by Upregulating Caspase8 and Activating Apoptosis.

Radioimmunotherapy (RIT) is a novel and promising cancer treatment method, with ongoing research focusing on RIT antibody selection, radionuclides, treatment options, and benefited patient groups. As we dive into the mechanisms of tumor biology, a deeper exploration of how RIT affects tumor tissue is needed to provide new ways to improve clinical treatment outcome and patient prognosis. We labeled the anti-PD-L1 monoclonal antibody atezolizumab with iodine-131 (131I), separated and purified the labeled mAb with Sephadex G-25 medium gel filtration resin, and tested product stability. We detected the in vivo activity of 131I-PD-L1 mAb by analyzing its in vivo biodistribution and performing SPECT imaging and then set different treatment groups to study the effect of 131I-atezolizumab on the survival of tumor-bearing mice. Western blot, real-time quantitative PCR, and immunohistochemistry were used to detect the expression level of Caspase8 and Nlrp3 in tumor. TUNEL fluorescence staining was used to detect the apoptosis in the tumor. The radiopharmaceutical molecular probe 131I-atezolizumab showed high stability and in vivo biological activity. The treatment regimen adopted had a positive effect on the survival of tumor-bearing mice. 131I internal irradiation upregulated Caspase8 in tumor and ultimately inhibited solid tumor growth by activating apoptosis pathways. We also found a significant increase in the expression of NLRP3, which plays an important role in the pyroptosis pathway, in tumor. In summary, our data demonstrated that radiopharmaceuticals combined with immunotherapy affected tumor tissue by modulating relevant biological pathways, thereby achieving better antitumor effects compared with single therapy and providing new insights for promoting better patient prognosis and combination treatment strategies.

Chromatographic purification of the plasmin-like enzyme from clamworm (Perinereis aibuhitensis Grub) and its fibrinolytic activity by metal ions.

In this study, fibrinolytic protease was isolated and purified from Perinereis aibuhitensis Grub, and the extraction process was optimized. The properties of the enzyme, such as the amino acid composition, thermal stability, optimal temperature, and pH, were investigated. After detoxification, proteins collected from fresh Clamworm (Perinereis aibuhitensis Grub) were concentrated via ammonium sulfate precipitation. The crude protease was purified using gel filtration resin (Sephadex G-100), anion exchange resin (DEAE-Sepharose FF), and hydrophobic resin (Phenyl Sepharose 6FF). The molecular weight of the protease was determined by polyacrylamide gel electrophoresis (SDS-PAGE). The optimum temperature and optimum pH of the protease were determined. The activity of crude protease in the 40-60% salt-out section was the highest, reaching 467.53 U/mg. The optimal process for purifying crude protein involved the application of DEAE-Sepharose FF and Phenyl Sepharose 6FF, which resulted in the isolation of a single protease known as Asp60-D1-P1 with the highest fibrinolytic activity; additionally, the enzyme activity was measured at 3367.76 U/mg. Analysis by Native-PAGE and SDS-PAGE revealed that the molecular weight of Asp60-D1-P1 was 44.5 kDa, which consisted of two subunits with molecular weights of 6.5 and 37.8 kDa, respectively. The optimum temperature for Asp60-D1-P1 was 40°C, and the optimal pH was 8.0.

Cytotoxic, Antiproliferative and Apoptosis-inducing Activity of L-Amino Acid Oxidase from Malaysian Calloselasma rhodostoma on Human Colon Cancer Cells.

The aim of this study was to investigate the cytotoxic, antiproliferative activity and the induction of apoptosis by L-amino acid oxidase isolated from Calloselasma rhodostoma crude venom (CR-LAAO) on human colon cancer cells. CR-LAAO was purified using three chromatographic steps: molecular exclusion using G-50 gel filtration resin, ion-exchange by MonoQ column and desalted on a G25 column. The purity and identity of the isolated CR-LAAO was confirmed by SDS-PAGE and LC-MS/MS. CR-LAAO demonstrated time- and dose-dependent cytotoxic activity on SW480 (primary human colon cancer cells) and SW620 (metastatic human colon cancer cells) with an EC50 values of 6 μg/ml and 7 μg/ml at 48 hr, respectively. Quantification of apoptotic cells based on morphological features demonstrated significant increase in apoptotic cell population in both SW480 and SW620 cells which peaked at 48 hr. Significant increase in caspase-3 activity and reduction in Bcl-2 levels were demonstrated following CR-LAAO treatment. These data provide evidence on the potential anticancer activity of CR-LAAO from the venom of C. rhodostoma for therapeutic intervention of human colon cancer.

Purification and characterization of malate dehydrogenase from sheep liver (Ovis aries): Application in AST assay diagnostic kit

Malate dehydrogenase enzyme is a major component in aspartate aminotransferase (AST) diagnostic kit that used in diagnosis and monitoring of liver diseases. This study aimed at purification and characterization of MDH enzyme from sheep liver for direct application in preparation of AST kit. Two malate dehydrogenase isoenzymes were resolved from sheep liver by chromatography on DEAE-cellulose column, one major sheep liver MDH (SLMDH) and another minor one. SLMDH was extracted and purified by ammonium sulfate fractionation and chromatographical separation on anion exchanger and gel filtration resins. The purified sheep liver MDH specific activity is 6.7 units / mg protein representing 11.6 purification folds and 27.7 % yield. The molecular mass of SLMDH intact protein is 68 ± 1.6 kDa. SLMDH turned out to be homogeneous and consists of two identical subunits of 34 ± 1.2 kDa each. The SLMDH isoelectric point (pI) value is estimated at pH 6.2 and displayed its optimum activity at pH 9.6 and has Km value of 1.4 mM for NAD and 11 mM for malate. FeCl2, NiCl2 and ZnCl2 inhibited the SLMDH activity. The purified SLMDH is applied in the preparation of AST diagnostic kit that found sensitive and comparable with a commercially available one.

Comparison of Methods for The Purification of Goat Lactoferrin and Antiviral Activity to Human Papillomavirus

Lactoferrin (Lf) has been reported for its multifunctional properties such as antifungal, antibacterial, antiviral, antioxidant and anticancer activities. This protein is an 80 kDa iron-binding protein of the transferrin family. It can be isolated from milk and has been identified from various mammalian secretions. The aims of this study focused on the isolation and purification of Lf from etawa goat colostrum using several methods of chromatography and observing its activities as antiviral human papillomavirus (HPV). Purification was performed by gel filtration chromatography using Sephadex G-75 and G-100, for cation exchange chromatography with carboxymethyl Sephadex C-50, and two-steps chromatography of cation exchange resin carboxymethyl Sephadex C-50 and gel filtration resin Sephadex G-75. All columns were prepared by manual packaging. The native isolated Lf was characterized by SDS-PAGE electrophoresis. Our result showed that the best purified goat lactoferrin (gLf) was by two-steps chromatography with yield of 364 μg/mL and molecular weight 82 kDa. The pure gLf showed anti-HPV effect at concentration of 100 μg/mL after 72 hours incubation by the increased in cycle threshold (Ct) value, from 26 Ct to 36 Ct.

Characterization and Kinetics of Phosphopantothenoylcysteine Synthetase from Enterococcus faecalis

The enzyme phosphopantothenoylcysteine synthetase (PPCS) catalyzes the nucleotide-dependent formation of phosphopantothenoylcysteine from (R)-phosphopantothenate and L-cysteine in the biosynthetic pathway leading to the formation of the essential biomolecule, coenzyme A. The Enterococcus faecalis gene coaB encodes a novel monofunctional PPCS which has been cloned into pET23a and expressed in Escherichia coli BL21 AI. The heterologous expression system yielded 30 mg of purified PPCS per liter of cell culture. The purified enzyme chromatographed as a homodimer of 28 kDa subunits on Superdex HR 200 gel filtration resin. The monofunctional protein displayed a nucleotide specificity for cytidine 5'-triphosphate (CTP) analogous to that seen for bifunctional PPCS expressed by most prokaryotes. Kinetic characterization, utilizing initial velocity and product inhibition studies, found the mechanism of PPCS to be Bi Uni Uni Bi Ping-Pong, with the nucleotide CTP binding first and CMP released last. Michaelis constants were 156, 17, and 86 microM for CTP, (R)-phosphopantothenate, and L-cysteine, respectively, and the k(cat) was 2.9 s(-1). [carboxyl-(18)O]Phosphopantothenate was prepared by hydrolysis of methyl pantothenate with Na(18)OH, followed by enzymatic phosphorylation with E. faecalis pantothenate kinase (PanK). The fate of the carboxylate oxygen of labeled phosphopantothenate, during the course of the PPCS-catalyzed reaction with CTP and L-cysteine, was monitored by (31)P NMR spectroscopy. The results show that the carboxylate oxygen of the phosphopantothenate is recovered with the CMP formed during the reaction, indicative of the formation of a phosphopantothenoyl cytidylate catalytic intermediate, which is consistent with the kinetic mechanism.

A Network-based Analysis of Polyanion-binding Proteins Utilizing Yeast Protein Arrays

The high affinity of certain cellular polyanions for many proteins (polyanion-binding proteins (PABPs)) has been demonstrated previously. It has been hypothesized that such polyanions may be involved in protein structure stabilization, stimulation of folding through chaperone-like activity, and intra- and extracellular protein transport as well as intracellular organization. The purpose of the proteomics studies reported here was to seek evidence for the idea that the nonspecific but high affinity interactions of PABPs with polyanions have a functional role in intracellular processes. Utilizing yeast protein arrays and five biotinylated cellular polyanion probes (actin, tubulin, heparin, heparan sulfate, and DNA), we identified proteins that interact with these probes and analyzed their structural and amino acid sequence requirements as well as their predicted functions in the yeast proteome. We also provide evidence for the existence of a network-like system for PABPs and their potential roles as critical hubs in intracellular behavior. This investigation takes a first step toward achieving a better understanding of the nature of polyanion-protein interactions within cells and introduces an alternative way of thinking about intracellular organization.

Identification of Mn2+-binding Aspartates from α, β, and γ Subunits of Human NAD-dependent Isocitrate Dehydrogenase

The human NAD-dependent isocitrate dehydrogenase (IDH), with three types of subunits present in the ratio of 2alpha:1beta:1gamma, requires a divalent metal ion to catalyze the oxidative decarboxylation of isocitrate. With the aim of identifying ligands of the enzyme-bound Mn(2+), we mutated aspartates on the alpha, beta, or gamma subunits. Mutagenesis target sites were based on crystal structures of metal-isocitrate complexes of Escherichia coli and pig mitochondrial NADP-IDH and sequence alignments. Aspartates replaced by asparagine or cysteine were 206, 230, and 234 of the alpha subunit and those corresponding to alpha-Asp-206: 217 of the beta subunit and 215 of the gamma subunit. Each expressed, purified mutant enzyme has two wild-type subunits and one subunit with a single mutation. Specific activities of WT, alpha-D206N, alpha-D230C, alpha-D234C, beta-D217N, and gamma-D215N enzymes are 22, 29, 1.4, 0.2, 7.3 and 3.7 micromol of NADH/min/mg, respectively, whereas alpha-D230N and alpha-D234N enzymes showed no activity. The K(m,Mn(2+)) for alpha-D230C and gamma-D215N are increased 32- and 100-fold, respectively, along with elevations in K(m,isocitrate). The K(m,NAD) of alpha-D230C is increased 16-fold, whereas that of beta-D217N is elevated 10-fold. For all the mutants K(m,isocitrate) is decreased by ADP, indicating that these aspartates are not needed for normal ADP activation. This study demonstrates that alpha-Asp-230 and alpha-Asp-234 are critical for catalytic activity, but alpha-Asp-206 is not needed; alpha-Asp-230 and gamma-Asp-215 may interact directly with the Mn(2+); and alpha-Asp-230 and beta-Asp-217 contribute to the affinity of the enzyme for NAD. These results suggest that the active sites of the human NAD-IDH are shared between alpha and gamma subunits and between alpha and beta subunits.

Phosphorylation of the Human Fhit Tumor Suppressor on Tyrosine 114 in Escherichia coli and Unexpected Steady State Kinetics of the Phosphorylated Forms

The human tumor suppressor Fhit is a homodimeric histidine triad (HIT) protein of 147 amino acids which has Ap(3)A hydrolase activity. We have recently discovered that Fhit is phosphorylated in vivo and is phosphorylated in vitro by Src kinase [Pekarsky, Y., Garrison, P. N., Palamarchuk, A., Zanesi, N., Aqeilan, R. I., Huebner, K., Barnes, L. D., and Croce, C. M. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 3775-3779]. Now we have coexpressed Fhit with the elk tyrosine kinase in Escherichia coli to generate phosphorylated forms of Fhit. Unphosphorylated Fhit, Fhit phosphorylated on one subunit, and Fhit phosphorylated on both subunits were purified to apparent homogeneity by column chromatography on anion-exchange and gel filtration resins. MALDI-TOF and HPLC-ESI tandem mass spectrometry of intact Fhit and proteolytic peptides of Fhit demonstrated that Fhit is phosphorylated on Y(114) on either one or both subunits. Monophosphorylated Fhit exhibited monophasic kinetics with K(m) and k(cat) values approximately 2- and approximately 7-fold lower, respectively, than the corresponding values for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic kinetics. One site had K(m) and k(cat) values approximately 2- and approximately 140-fold lower, respectively, than the corresponding values for unphosphorylated Fhit. The second site had a K(m) approximately 60-fold higher and a k(cat) approximately 6-fold lower than the corresponding values for unphosphorylated Fhit. The unexpected kinetic patterns for the phosphorylated forms suggest the system may be enzymologically novel. The decreases in the values of K(m) and k(cat) for the phosphorylated forms in comparison to those of unphosphorylated Fhit favor the formation and lifetime of the Fhit-Ap(3)A complex, which may enhance the tumor suppressor activity of Fhit.

Cell-wall polysaccharide modifications during postharvest ripening of papaya fruit (Carica papaya)

Abstract Modifications of cell-wall polysaccharides and flesh firmness, as a measure of texture, were evaluated during postharvest ripening of papaya fruit (Carica papaya). Cell-wall material (CWM) was isolated from pericarp at different ripening stages. Three pectic and one hemicellulosic fractions, as well as a cellulosic residue, were obtained from CWM. A water soluble fraction contained pectins presumably not cell wall associated. Imidazole and sodium carbonate soluble fractions contained pectins with low degree of branching from middle lamella and profusely branched structures with neutral sugar chains arising from primary cell walls, respectively. Solubilization and concomitant depolymerization of both kinds of pectin was observed. Loss of galactose at two phases of ripening was detected, one at the beginning, and the other, with a simultaneous release of rhamnose, at the onset of the postharvest period. Polysaccharide analysis of 4 M sodium hydroxide soluble fraction was compatible with a xyloglucan type and a mannose containing polymer. Depolymerization of this hemicellulosic fraction was not evident during ripening. A continuous depolymerization of water-extracted pectins was evident. For this fraction, a polymer containing galacturonic acid and covalently associated rhamnose units, i.e., a rhamnogalacturonan type structure that lost acidic monomeric units during ripening, appeared beyond the exclusion limit of gel filtration resin. Cellulose residue exhibited decreasing quantities of galacturonic acid and non-glucose monosaccharides during ripening indicating that association between polysaccharides from matrix and microfibrilar phases may be involved in the softening process. Methyl esterification decreased for middle lamella and primary cell wall pectins while it increased steadily for water soluble pectins. Finally, a scheme of suggested temporal events undergone by cell-wall polysaccharides and the possible enzymes involved along with firmness variation during postharvest ripening of papaya fruit is presented.

Evaluation of gel filtration resins for the removal of PCR-inhibitory substances from soils and sediments

A variety of gel filtration resins (Sephadex G200 and G150; Sepharose 6B, 4B and 2B; Bio-Gel P100, P200; and Toyopearl HW 55, HW 65, and HW 75) were evaluated for their efficacy in removing PCR-inhibitory substances from feedlot soil DNA crude extracts using gravity-flow disposable columns. Sepharose resins demonstrated the best properties for DNA purification when compared to other gel filtration resins, and Sepharose 2B was the most efficient purification resin based upon flow rate and the elution of DNA and humic acids from the columns. A method for purifying large solution volumes of DNA extract economically was also developed using low-cost disposable Disposaflex columns. Crude DNA extracts of cattle feedlot soil and aquifer sediment impacted by animal and human wastes were easily purified using the Disposaflex column method regardless of whether a gentle chemical lysis or a bead mill homogenization DNA extraction method was employed.

Identification of a transcription factor IIIA-interacting protein.

Transcription factor IIIA (TFIIIA) activates 5S ribosomal RNA gene transcription in eukaryotes. The protein from vertebrates has nine contiguous Cys(2)His(2)zinc fingers which function in nucleic acid binding, and a C-terminal region involved in transcription activation. In order to identify protein partners for TFIIIA, yeast two-hybrid screens were performed using the C-terminal region of Xenopus TFIIIA as an attractor and a rat cDNA library as a source of potential partners. A cDNA clone was identified which produced a protein in yeast that interacted with Xenopus TFIIIA but not with yeast TFIIIA. This rat clone was sequenced and the primary structure of the human homolog (termed TFIIIA-intP for TFIIIA-interacting protein) was determined from expressed sequence tags. In vitro interaction of recombinant human TFIIIA-intP with recombinant Xenopus TFIIIA was demonstrated by immuno-precipitation of the complex using anti-TFIIIA-intP antibody. Interaction of rat TFIIIA with rat TFIIIA-intP was indicated by co-chromatography of the two proteins on DEAE-5PW following fractionation of a rat liver extract on cation, anion and gel filtration resins. In a HeLa cell nuclear extract, recombinant TFIIIA-intP was able to stimulate TFIIIA-dependent transcription of the Xenopus 5S ribosomal RNA gene but not TFIIIA-independent transcription of the human adenovirus VA RNA gene.

Reaction of common bean callus to culture filtrate of Colletotrichum lindemuthianum: Differences in the composition and toxic activity of fungal culture filtrates

Culture filtrate of Colletotrichum lindemuthianum caused dark brown lesions on the lower surface veins of bean leaves. This phenomenon was used as a bioassay to study the production of toxic fungal metabolites. Calli from anthracnose-susceptible bean cultivars 'Collacia', 'Andecha' and 'Seronda' were sensitive to a 12.5% solution of race 38 filtrate or to a 25% solution of race 7 filtrate. In contrast, calli from anthracnose-resistant bean genotypes A 247, TU, PI 207262, 'Collacia' × 'Tu', 'Collacia' × AB 136 and 'Collacia' × PI 207262 did not develop browning. Culture filtrates were passed through an ionic-exchange resin and a gel filtration resin. Toxic activity of fractions from two races of the fungal pathogen was different, although in both races slight necrosis was produced by the same nine fractions. Pathogenicity could be related with common substances and toxicity could be identified with differential compounds.

Purification and Characterization of an α1,2-L-Fucosyltransferase, Which Modifies the Cytosolic Protein FP21, from the Cytosol of Dictyostelium

A novel fucosyltransferase (cFTase) activity has been enriched over 10(6)-fold from the cytosolic compartment of Dictyostelium based on transfer of [3H]fucose from GDP-[3H]fucose to Galbeta1,3 GlcNAc beta-paranitrophenyl (paranitrophenyl-lacto-N-bioside or pNP-LNB). The activity behaved as a single component during purification over DEAE-, phenyl-, Reactive Blue-4-, GDP-adipate-, GDP-hexanolamine-, and Superdex gel filtration resins. The purified activity possessed an apparent Mr of 95 X 10(3), was Mg2+-dependent with a neutral pH optimum, and exhibited a Km for GDP-fucose of 0.34 microM, a Km for pNP-LNB of 0.6 mM, and a Vmax for pN-P-LNB of 620 nmol/min/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the Superdex elution profile identified a polypeptide with an apparent Mr of 85 X 10(3), which coeluted with the cFTase activity and could be specifically photolabeled with the donor substrate inhibitor GDP-hexanolaminyl-azido-125I-salicylate. Based on substrate analogue studies, exoglycosidase digestions, and co-chromatography with fucosylated standards, the product of the reaction with pNP-LNB was Fucalpha1, 2Galbeta1,3GIcNAcbeta-pNP. The cFTase preferred substrates with a Galbeta1,3linkage, and thus its acceptor substrate specificity resembles the human Secretor-type alpha1,2- FTase. Afucosyl isoforms of the FP21 glycoprotein, GP21-I and GP21-II, were purified from the cytosol of a Dictyostelium mutant and found to be substrates for the cFTase, which exhibited an apparent K(m) of 0.21 microM and an apparent V(max) of 460 nmol/min/mg protein toward GP21-II. The highly purified cFTase was inhibited by the reaction products Fucalpha1,2Galbeta1,3GlcNAcbeta-pNP and FP21-II. FP21-I and recombinant FP21 were not inhibitory, suggesting that acceptor substrate specificity is based primarily on carbohydrate recognition. A cytosolic location for this step of FP21 glycosylation is implied by the isolation of the cFTase from the cytosolic fraction, its high affinity for its substrates, and its failure to be detected in crude membrane preparations.

Unexpected interaction of some anti-TNP hybridoma antibodies with Superose HPLC gel filtration resins

Five different hybridoma antibodies (the isotypes IgG1, IgG2a and IgG2b), reactive with TNP, showed increased elution times when gel filtered on a Superose-12 HPLC column corresponding to apparent molecular weights ranging from 54 kDa to 120 kDa compared to the normal of 150 kDa. One of the antibodies (C1901-B4) was studied in detail showing that the unusual gel filtration behaviour was localized to the Fab part of the molecule. SDS-PAGE analysis showed that the intact antibodies had normal molecular weights. Gel filtration on a Toyosoda G-3000SW HPLC column and Sephadex G-150 as well as Sepharose 6B generally showed normal elution times. These results support the hypothesis that the retardation on the Superose gel is probably due to aromatic interactions between amino acid residues supposedly exposed in the hypervariable region (i.e., the antibody combining site) of the antibody, and the gel matrix which is rich in ether O-atoms created during the manufacturing process. If this hypothesis is correct one might expect such interactions between Superose resins and antibodies of many different specificities in which aromatic amino acids are exposed in the combining sites.

Identification and characterization of large, complex forms of chloroplast translational initiation factor 2 from Euglena gracilis.

Chromatography of partially purified preparations of Euglena gracilis chloroplast initiation factor 2 (IF-2chl) on gel filtration resins indicates that this factor is present in high molecular mass forms ranging from 200 to 700 kDa. The higher molecular weight complexes can be separated from the 200,000 Mr form of this factor by chromatography on DEAE-cellulose. Further purification indicates that the majority of the IF-2chl is present as dimeric, tetrameric, and probably hexameric complexes of polypeptides of 97,000-110,000 in molecular weight. In addition, one form consisting of subunits of about 200,000 Mr has been detected. All of these species are active in promoting fMet-tRNA binding to chloroplast 30 S subunits in a message-dependent reaction. Initiation complex formation promoted by IF-2chl requires the presence of GTP. Similar levels of binding are obtained when GTP is replaced by a nonhydrolyzable analog suggesting that IF-2chl is acting stoichiometrically rather than catalytically under the conditions used. The activity of this factor is stimulated by the presence of either Escherichia coli or chloroplast IF-3. None of the forms of IF-2chl detected is active on E. coli ribosomes.

Separation of isozymic forms of type II calcium/calmodulin-dependent protein kinase using cation-exchange chromatography

Three distinct isozymes of a type II calcium/calmodulin-dependent protein kinase (CaM kinase II) from rat forebrain cytosol were separated using S-Sepharose cation-exchange resin. About 90% of the applied kinase activity was recovered in three protein peaks. Each isozymic form of the kinase was purified 200–300 fold by chromatography on S-Sepharose, calmodulin-affinity and gel filtration resins. All 3 forms of CaM kinase II had apparent molecular masses of 650–700 kDa, but contained variable proportions of 50 kDa and 58–60 kDa subunits. The molar ratios of the 50 kDa/58–60 kDa kinase subunits in each holoenzyme were determined by protein staining and [ 125I]calmodulin-binding studies and were approximately: 6/1, 3/1 and 1/1. The isozyme containing a 3/1 ratio of subunits corresponds to the predominant form of CaM kinase II in forebrain representing 70–80% of the total activity in cytosol, whereas the other forms each represent 5–10% of the total cytosolic activity. The substrate specificities and time courses of substrate phosphorylation for the isozymes were comparable. These studies provide a basis to examine regional, subcellular, and developmental differences in the isozymic forms of CaM kinase II which may subserve different neuronal functions.

Differentiation increases type II calmodulin-dependent protein kinase in the neuroblastoma/glioma cell line 108CC15 (NG108-15)

A type II calcium/calmodulin-dependent protein kinase (CaM kinase II) was purified approximately 300-fold from cultured neuroblastoma/glioma (NG108) cell homogenate. The purification of the kinase, which used a combination of differential centrifugation and chromatography on cation-exchange, calmodulin-affinity, and gel-filtration resins, was monitored by the ability of the kinase to phosphorylate the high-molecular-weight microtubule-associated protein 2 (MAP-2). The kinase was compared with authentic CaM kinase II purified from rat brain cytosol. Based upon holoenzyme molecular weight, subunit composition and molecular weight, calcium-dependent calmodulin-binding to subunits, calcium/calmodulin-dependent autophosphorylation of subunits, substrate specificity, apparent km's for ATP and calmodulin, phosphopeptide maps of subunits, time course, and heat lability, the kinase was identified as a type II calcium/calmodulin-dependent protein kinase. When cellular differentiation was induced under specific conditions of cell culture, a significant increase in the apparent activity and amount of the kinase per mg protein was observed relative to control cells. These studies suggest that there is an increase in CaM kinase II expression during cellular differentiation, which may relate to the concurrent development of electrical excitability, synaptogenesis, and elaboration of cytoskeletal elements. Thus, the NG108 cell should provide a useful model to study the physiological functions of CaM kinase II.

Both positional isomers of aminoacyl-tRNA's are bound by elongation factor Tu.

Six purified Escherichia coli and yeast tRNA's were converted to positionally defined tRNA's terminating in 2'- and 3'-deoxyadenosine; the modified (amino-acyl) tRNA's were compared for their abilities to bind to elongation factor Tu (EF-Tu) in the presence both of GTP and guanylylimidodiphosphate (GMP-P(NH)P). Formation of aminoacyl-tRNA . EF-Tu . guanine nucleotide ternary complexes was monitored by gel filtration on Sephadex G-100 and Ultrogel ACA 44 columns and also by measurement of the ability of the factor to diminish the rate of chemical hydrolysis of the aminoacyl-tRNA's. The apparent positional specificity of the factor was found to be affected substantially both by the choice of guanine nucleotide and gel filtration resin utilized, but not in any systematic fashion. Likewise, assay of ternary complex formation by diminution of the rate of chemical deacylation failed to reveal any consistent positional preference from one isoacceptor to another. It is worthy of note that each modified aminoacyl-tRNA tested did form a ternary complex with EF-Tu under each of the experimental conditions used for assay, but that in each case the difference in affinity of the factor for isomeric aminoacyl-tRNA's was less than that between either of the modified aminoacyl-tRNA's and the corresponding unmodified species. On the basis of the experiments performed, we conclude that (i) EF-Tu has remarkable conformation flexibility, possibly reflecting its physiological role in recognizing 20 tRNA isoacceptors and (ii) the factor has no obvious preference for a single positional isomer of aminoacyl-tRNA and it is not clear that any preference that might exist could be established convincingly using tRNA's terminating in 2'- and 3'-deoxyadenosine.