Occurrence of 5SrRNA in high molecular weight complexes of aminoacyl-tRNA synthetases in a rat liver supernatant.

The 5SrRNA in the rat liver postmicrosomal supernatant was investigated. Acrylamide gel electrophoresis and Northern blot analysis showed that most of the 5SrRNA was present in the fractions obtained on high molecular weight regions separated by Sephadex G-200 column chromatography of the supernatant, which contained the bulk of the methionyl-tRNA synthetase (Fraction I) and tyrosyl-tRNA synthetase (Fraction II). A high molecular weight complex containing nine aminoacyl-tRNA synthetases [Mirande, M., LeCorre, D., & Waller, J.-P. (1985) Eur. J. Biochem. 147, 281-289] was purified by fractional precipitation with polyethylene glycol 6000, gel filtration on Bio-Gel A-1.5m, and finally tRNA-Sepharose column chromatography, which gave two fractions. Fraction B showed the activities of nine aminoacyl-tRNA synthetases and gave protein bands corresponding to eight previously identified enzymes on SDS-PAGE. Fraction A, eluted with a lower KCl concentration than Fraction B, showed lower activities than fraction B of eight of the aminoacyl-tRNA synthetases, the exception being prolyl-tRNA synthetase. The staining patterns with ethidium bromide of the RNAs after PAGE showed 5SrRNA bands for Fraction A but not for Fraction B. However, Northern blot analysis indicated that 5SrRNA was present in both Fractions A and B. The staining pattern after SDS-PAGE of Fraction A with Coomassie Brilliant Blue showed several protein bands in addition to those observed for Fraction B, one of which, with a staining intensity comparable with those of other bands, was located at the same position as ribosomal protein L5, which is the protein moiety of the 5SrRNA-L5 protein complex of ribosomal 60S subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Algal heme oxygenase from Cyanidium caldarium. Partial purification and fractionation into three required protein components

Enzymatic heme oxygenase activity has been partially purified from extracts of the unicellular red alga Cyanidium caldarium, and the macromolecular components have been separated into three protein fractions, referred to as Fractions I, II, and III, by serial column chromatography through DEAE-cellulose and Reactive Blue 2-Sepharose. Fraction I is retained by DEAE-cellulose at low salt concentration and eluted by 1 M NaCl. Fraction II is retained by Blue Sepharose at low salt concentration and eluted by 1 M NaCl. Fraction III is retained on 2',5'-ADP-agarose and eluted by 1 mM NADPH, while Fraction II is not retained on ADP-agarose. Fractions I-III, have Mr values of 22,000, 38,000, and 37,000, respectively (all +/- 2,000), as determined by Sephadex gel filtration chromatography. In vitro heme oxygenase activity requires the presence of all three fractions, plus substrate, O2, reduced pyridine nucleotide, and another reductant. Ascorbate, isoascorbate, and phenylenediamine serve equally well as the second reductant, but hydroquinone can also be used, with lower activity resulting. Fractions I-III are heat sensitive and inactive by Pronase digestion. Fraction I has a visible absorption spectrum similar to that of ferredoxin and is bleached by dithionite reduction or incubation with p-hydroxymercuribenzoate. Fraction I can be replaced by commercially available ferredoxin derived from the red alga Porphyra umbilicalis, and to a smaller extent, by spinach ferredoxin. Fraction III contains ferredoxin-linked cytochrome c reductase activity and can be partially replaced by spinach ferredoxin-NADP+ oxidoreductase. Reconstituted heme oxygenase and ferredoxin-linked cytochrome c reductase activities are both abolished if Fraction I or III is preincubated with 0.1 mM p-hydroxymercuribenzoate, but heme oxygenase activity is only slightly affected if Fraction II is preincubated with p-hydroxymercuribenzoate. Preincubation of Fraction II with 0.5 mM diethylpyrocarbonate inactivates heme oxygenase in the reconstituted system, and 10 microM mesohemin partially protects this Fraction against diethylpyrocarbonate inactivation. Algal heme oxygenase is inhibited 80% by 2 microM Sn-protoporphyrin even in the presence of 20 microM mesohemin. Fraction II is rate limiting in unfractionated and reconstituted incubation mixtures. None of the three cell fractions could be replaced by bovine spleen microsomal heme oxygenase or NADPH-cytochrome P450 reductase.     

Conversion of Anhydride to Thiolactone in Biotin Synthesis

This study was conducted to identify the sourness-suppressing peptides in cooked pork and to clarify the mechanism of sour taste suppression by the peptides. An extract prepared from pork loins vacuum-cooked at 60 °C for 6 hours after conditioning at 4 °C for 20 days was separated into three fractions: under MW 500 (Fraction I), MW 500–1,000 (Fraction II), and over MW 1,000 (Fraction III). The Fraction I content was largest. As judged by sensory evaluation, the addition of Fraction II was capable of suppressing stronger sourness than the other fractions. Fraction II also enhanced umami and saltiness. Three peptides (APPPPAEVHEVV, APPPPAEVHEVVE, and APPPPAEVHEVHEEVH) in Fraction II increased greatly during conditioning. A common peptide, APPPPAEVHEV, in the amino acid sequences of the three peptides suppressed the sour taste. The mechanism of sourness suppression by the peptide was concluded to comprise inhibition of the binding of sour taste substances to the membranes of the tongue.     

Thermodynamics of calmodulin trapping by Ca2+/calmodulin-dependent protein kinase II: subpicomolar Kd determined using competition titration calorimetry

Calmodulin (CaM) trapping by Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a phenomenon whereby the affinity of CaM for CaMKII increases >1000-fold following CaMKII autophosphorylation. The molecular basis of this effect is not entirely understood. Binding of CaM to the phosphorylated and the unphosphorylated states of CaMKII is well mimicked by the interaction of CaM with two different length peptides taken from the CaM-binding region of CaMKII, peptides we refer to as the long and intermediate peptides. To better understand the conformational change accompanying CaM trapping, we have used isothermal titration calorimetry (ITC) to compare the binding thermodynamics of CaM to these peptides as well as to a shorter CaMKII-based peptide. Calorimetric analysis revealed that the enthalpy, rather than the entropy, distinguished binding of these three peptides. Furthermore, the heat capacity change was found to be similar for the long and intermediate peptides but smaller in magnitude for the short peptide. Direct titration of CaM with peptide provided the Kd value for the short peptide (Kd = 5.9 +/- 2.4 microM), but a novel, two-phased competitive binding strategy was necessary to ascertain the affinities of the intermediate (Kd = 0.17 +/- 0.06 nM) and long (Kd = 0.07 +/- 0.04 pM) peptides. To our knowledge, the Kd for the long peptide is the most potent measured to date using ITC. Together, the findings reported here support a model whereby the final conformational change accompanying CaM trapping buries little additional surface area but does involve formation of new hydrogen bonds and van der Waals contacts that contribute to formation of the high-affinity, CaM-trapped state.     

Sourness-suppressing peptides in cooked pork loins

This study was conducted to identify the sourness-suppressing peptides in cooked pork and to clarify the mechanism of sour taste suppression by the peptides. An extract prepared from pork loins vacuum-cooked at 60 degrees C for 6 hours after conditioning at 4 degrees C for 20 days was separated into three fractions: under MW 500 (Fraction I), MW 500-1,000 (Fraction II), and over MW 1,000 (Fraction III). The Fraction I content was largest. As judged by sensory evaluation, the addition of Fraction II was capable of suppressing stronger sourness than the other fractions. Fraction II also enhanced umami and saltiness. Three peptides (APPPPAEVHEVV, APPPPAEVHEVVE, and APPPPAEVHEVHEEVH) in Fraction II increased greatly during conditioning. A common peptide, APPPPAEVHEV, in the amino acid sequences of the three peptides suppressed the sour taste. The mechanism of sourness suppression by the peptide was concluded to comprise inhibition of the binding of sour taste substances to the membranes of the tongue.     

Calcineurin B- and calmodulin-binding preferences identified with phage-displayed peptide libraries

Calcineurin B (CnB) and calmodulin (CaM) are two structurally similar but functionally distinct 'EF-hand' Ca2+-binding proteins. CnB is the regulatory subunit of the CaM-stimulated protein phosphatase, calcineurin. CaM is a unique multifunctional protein that interacts with and modulates the activity of many target proteins. CnB and CaM are both required for the full activation of the phosphatase activity of calcineurin and are not interchangeable. The two proteins recognize distinct binding sites on calcineurin A subunit (CnA) and perform different functions. Phage-displayed peptide libraries (pIII and pVIII libraries) were screened with CnB and CaM to isolate peptides that could then be compared to determine if there were binding preferences of the two proteins. The Ca2+-dependent binding of phage-displayed peptides to CnB and CaM is specifically blocked by synthetic peptides derived from the CnB-binding domain of CnA and the CaM-binding domain of myosin light chain kinase respectively. Both CnB- and CaM-binding peptides have a high content of tryptophan and leucine, but CnB-binding peptides are more hydrophobic than CaM-binding peptides. CnB-binding peptides are negatively charged with clusters of hydrophobic residues rich in phenylalanine, whereas the CaM-binding peptides are positively charged and often contain an Arg/Lys-Trp motif. The binding preferences identified with peptide libraries are consistent with the features of the CnB-binding domains of all CnA isoforms and the CaM-binding domains of CaM targets.     

Schistosoma mansoni: antigenic heterogeneity of excretions and secretions

The excretory-secretory antigens of adult Schistosoma mansoni were obtained by in vitro cultivation of worms in a chemically-defined medium. The protein output in this system was low, 0.2–0.4 μg of proteinworm/48 hr. The composition of the crude culture antigen (CA) was approximately 80% protein, 15% carbohydrate and 5% nucleic acid. Disc gel electrophoresis of CA revealed the presence of at least 15 protein components, many with carbohydrate moieties. Three major fractions were obtained by gel filtration on Sephadex G-200. Fraction I contained the bulk of the glycoprotein material. Immunoelectrophoresis of CA with hyperimmune rabbit serum indicated the presence of at least 6 antigens, most of which eluted in Fraction II. Serum from infected mice and monkeys, but not from rabbits and rats, reacted with CA and its fractions, especially Fraction II, on immunodiffusion analysis. Comparison of CA with other adult worm extracts by immunodiffusion techniques showed that most of the excretory-secretory antigens could be obtained by either freezing and thawing or by extraction with 3 M KCl. The P.K.-type activity of CA was considerably greater than that of a lipid-free adult worm antigen. Both Fractions I and II had the P.K.-type activity. An antigen capable of eliciting macrophage migration inhibition factor from infected rat lymphocytes was detected in CA, although the lymphocyte toxicity of CA was high at concentrations above 10 μg/ml.     

The specificity of proteinases from Streptomyces griseus (pronase)

Purification of pronase by ion-exchange chromatography gave four proteolytically active fractions. Fraction A(2) contained an endopeptidase that attacks poly l-valine. Fraction B contained an endopeptidase, an aminopeptidase and carboxypeptidases. The activities against hippuryl-l-arginine and hippuryl-l-phenylalanine could be inhibited to a considerable extent by di-isopropyl phosphorofluoridate and by EDTA. Fraction C contained an endopeptidase resembling bovine trypsin. The pure enzyme was completely inactivated by di-isopropyl phosphorofluoridate and pancreatic trypsin inhibitor and to about 90% by other naturally occurring trypsin inhibitors. Fraction D contained an apparently homogeneous endopeptidase, inhibited by diisopropyl phosphorofluoridate, that adsorbed to and hydrolysed elastin. The activity of all these fractions was tested qualitatively against a wide range of small peptides and synthetic substrates.     

RNA extracts with polyadenylic acid sequences transfer specific sensitivity for a low molecular weight antigen (MW 486).

RNA prepared from the lymphoid tissues of guinea pigs specifically sensitized to mono(p-azobenzene-arsonate)-N-chloroacetyl-l-tyrosine (ARSNAT) (MW = 486) was fractionated by oligo(dT)-cellulose affinity chromatography. Two fractions designated as I and II were eluted from the column. Fraction II, binding to the column and containing polyadenylic acid sequences, transferred ARSNAT or keyhole-limpet-hemocyanin (KLH) sensitivity to nonsensitized guinea pig peritoneal exudate cells (GP-PEC) in 14 experiments. Fraction I was unable to transfer KLH or ARSNAT sensitivity to GP-PEC. The amount of Fraction II needed to transfer ARSNAT sensitivity was 10 times less than previously reported. Synthetic nonlymphoid cell poly(A) tested in this system failed to transfer ARSNAT or KLH sensitivity to GP-PEC. Both Fractions I and II were inactivated by ribonuclease. The results suggest a possible messenger function for the poly (A)-containing RNA fractions.     

Mycobacterium tuberculosis phagosomes exhibit altered calmodulin-dependent signal transduction: contribution to inhibition of phagosome-lysosome fusion and intracellular survival in human macrophages

Mycobacterium tuberculosis successfully parasitizes macrophages by disrupting the maturation of its phagosome, creating an intracellular compartment with endosomal rather than lysosomal characteristics. We have recently demonstrated that live M. tuberculosis infect human macrophages in the absence of an increase in cytosolic Ca(2+) ([Ca(2+)](c)), which correlates with inhibition of phagosome-lysosome fusion and intracellular viability. In contrast, killed M. tuberculosis induces an elevation in [Ca(2+)](c) that is coupled to phagosome-lysosome fusion. We tested the hypothesis that defective activation of the Ca(2+)-dependent effector proteins calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) contributes to the intracellular pathogenesis of tuberculosis. Phagosomes containing live M. tuberculosis exhibited decreased levels of CaM and the activated form of CaMKII compared with phagosomes encompassing killed tubercle bacilli. Furthermore, ionophore-induced elevations in [Ca(2+)](c) resulted in recruitment of CaM and activation of CaMKII on phagosomes containing live M. tuberculosis. Specific inhibitors of CaM or CaMKII blocked Ca(2+) ionophore-induced phagosomal maturation and enhanced the bacilli's intracellular viability. These results demonstrate a novel role for CaM and CaMKII in the regulation of phagosome-lysosome fusion and suggest that defective activation of these Ca(2+)-activated signaling components contributes to the successful parasitism of human macrophages by M. tuberculosis.     

Computational design of calmodulin mutants with up to 900-fold increase in binding specificity

Calmodulin (CaM) is a ubiquitous second messenger protein that regulates a variety of structurally and functionally diverse targets in response to changes in Ca²⁺ concentration. CaM-dependent protein kinase II (CaMKII) and calcineurin (CaN) are the prominent CaM targets that play an opposing role in many cellular functions including synaptic regulation. Since CaMKII and CaN compete for the available Ca²⁺/CaM, the differential affinity of these enzymes for CaM is crucial for achieving a balance in Ca²⁺ signaling. We used the computational protein design approach to modify CaM binding specificity for these two targets. Starting from the X-ray structure of CaM in complex with the CaM-binding domain of CaMKII, we optimized CaM interactions with CaMKII by introducing mutations into the CaM sequence. CaM optimization was performed with a protein design program, ORBIT, using a modified energy function that emphasized intermolecular interactions in the sequence selection procedure. Several CaM variants were experimentally constructed and tested for binding to the CaMKII and CaN peptides using the surface plasmon resonance technique. Most of our CaM mutants demonstrated small increase in affinity for the CaMKII peptide and substantial decrease in affinity for the CaN peptide compared to that of wild-type CaM. Our best CaM design exhibited an about 900-fold increase in binding specificity towards the CaMKII peptide, becoming the highest specificity switch achieved in any protein-protein interface through the computational protein design approach. Our results show that computational redesign of protein-protein interfaces becomes a reliable method for altering protein binding affinity and specificity.     

Conformational frustration in calmodulin–target recognition

Calmodulin (CaM) is a primary calcium (Ca²⁺)‐signaling protein that specifically recognizes and activates highly diverse target proteins. We explored the molecular basis of target recognition of CaM with peptides representing the CaM‐binding domains from two Ca²⁺‐CaM‐dependent kinases, CaMKI and CaMKII, by employing experimentally constrained molecular simulations. Detailed binding route analysis revealed that the two CaM target peptides, although similar in length and net charge, follow distinct routes that lead to a higher binding frustration in the CaM–CaMKII complex than in the CaM–CaMKI complex. We discovered that the molecular origin of the binding frustration is caused by intermolecular contacts formed with the C‐domain of CaM that need to be broken before the formation of intermolecular contacts with the N‐domain of CaM. We argue that the binding frustration is important for determining the kinetics of the recognition process of proteins involving large structural fluctuations. Copyright © 2015 John Wiley & Sons, Ltd.     

Apurinic DNA endonucleases from Drosophila melanogaster embryos

Summary Apurinic DNA endonuclease activity from Drosophila melanogaster embryos was resolved into two separable forms by phosphocellulose chromatography, one which flowed through the column (Fraction I) and the other which was retained and eluted at approximately 200 mM potassium phosphate (Fraction II). Both fractions, purified further by glycerol gradient sedimentation, were found to introduce nicks into DNA that were specific for and equal in number to the alkali-labile sites in depurinated DNA. They had similar apparent K_m values for apurinic sites (0.7 nM apurinic sites for Fraction I and 0.8 nM for Fraction II), but differed with respect to optimal pH, Mg⁺⁺ requirement and sensitivity to EDTA.     

Improving a natural CaMKII inhibitor by random and rational design

BACKGROUND: CaM-KIIN has evolved to inhibit stimulated and autonomous activity of the Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) efficiently, selectively, and potently (IC50 ～100 nM). The CN class of peptides, derived from the inhibitory region of CaM-KIIN, provides powerful new tools to study CaMKII functions. The goal of this study was to identify the residues required for CaMKII inhibition, and to assess if artificial mutations could further improve the potency achieved during evolution. METHODOLOGY/PRINCIPAL FINDINGS: First, the minimal region with full inhibitory potency was identified (CN19) by determining the effect of truncated peptides on CaMKII activity in biochemical assays. Then, individual residues of CN19 were mutated. Most individual Ala substitutions decreased potency of CaMKII inhibition, however, P3A, K13A, and R14A increased potency. Importantly, this initial Ala scan suggested a specific interaction of the region around R11 with the CaMKII substrate binding site, which was exploited for further rational mutagenesis to generate an optimized pseudo-substrate sequence. Indeed, the potency of the optimized peptide CN19o was >250fold improved (IC50 <0.4 nM), and CN19o has characteristics of a tight-binding inhibitor. The selectivity for CaMKII versus CaMKI was similarly improved (to almost 100,000fold for CN19o). A phospho-mimetic S12D mutation decreased potency, indicating potential for regulation by cellular signaling. Consistent with importance of this residue in inhibition, most other S12 mutations also significantly decreased potency, however, mutation to V or Q did not. CONLUSIONS/SIGNIFICANCE: These results provide improved research tools for studying CaMKII function, and indicate that evolution fine-tuned CaM-KIIN not for maximal potency of CaMKII inhibition, but for lower potency that may be optimal for dynamic regulation of signal transduction.     

Evidence for the presence of an endogenous cytosolic protein inhibitor of intestinal fucosyltransferase activities

Soluble endogenous inhibitory activities for glycoprotein: alpha (1-2) and alpha (1-3) fucosyltransferases are demonstrated in rat small intestinal cytosol. These inhibitors are retained on DEAE-cellulose and are eluted as two fractions A and B. Fraction B is non dialyzable, heat stable and pronase-resistant and consists probably of poly-nucleotides. Fraction A is also non-dialyzable, but is thermolabile and pronase-sensitive, suggesting that it contains proteins. The inhibition of fucosyltransferase activity by fraction A is competitive for GDP-fucose and non-competitive for the glycoprotein substrate. Inhibition is not due to interfering enzymatic activities (glycosyl-nucleotide pyrophosphatases, glycosidases or proteases) and is reversible. This protein inhibitor, with a molecular weight of 60,000, is found only in the intestine and the pancreas and appears to be different from the previously reported inhibitors of brain glycolipid glycosyltransferases.     

Analysis of cancer-associated colonic mucin by ion-exchange chromatography: evidence for a mucin species of lower molecular charge and weight in cancer

Cancer-associated mucins in the colon are antigenically distinct and glycosylated differently from their normal counterparts. Mucin-rich glycoconjugate preparations were made from nine non-neoplastic colons, seven colon cancers, and two different xenografts from mucin-producing human colon cancer cell lines, and radiolabeled with 3H. The preparation was applied to a DEAE-cellulose ion-exchange column, and eluted with a discontinuous ascending NaCl gradient resulting in seven discrete fractions or 'species'. Over half of the 3H-labeled glycoconjugates from specimens of non-neoplastic colonic epithelium eluted in fraction V (eluted with 0.25 NaCl). Significantly less of the 3H-labeled glycoconjugates from specimens of colon cancer eluted in fraction V (34%, P less than 0.0005), and there were significant increases in glycoconjugates eluted in fractions IV (P less than 0.008), III (P less than 0.0005), and II (P less than 0.028). Additional samples were prepared without the radiolabeling procedures, chromatographed on a DEAE-cellulose ion-exchange column, and analyzed for monosaccharide content. Each of the fractions contained the monosaccharides expected in mucin-type glycoproteins, but only sialic acid was differentially expressed in the seven fractions or 'species', occurring principally in the more charged species. However, differences in sialic acid content were not sufficient to explain the differences in retention on the ion-exchange column, nor were differences in O-acetylation of the mucins. Mucin-type glycoconjugates from colon cancers are relatively less charged than those from the normal colon, and elute at lower ionic strengths. Of interest, cancer-associated mucins appear to be of lower molecular weight than their normal counterparts. Additional studies of oligosaccharide and apomucin structure will be required to explain the molecular basis of these differences in charge.     

Cytoplasmic 3H-arginine polypeptides of Ehrlich ascites tumour cells

Summary Low molecular weight ninhydrin positive peptide fractions of the Ehrlich tumour cell cytoplasm were isolated and characterized. After preliminary gel filtration of the cytoplasm on Sephadex G-25 column, the peptide mixture was fractionated on cationic exchanger SP-Sephadex C-25 column and eluted with increasing pH gradient. Five peaks were obtained. Only the first peak contained sugar component. All five peptides were studied with respect to molecular weight, isoelectric point and electrophoretic homogeneity. The cytoplasm of Ehrlich tumour cells contains one peptide of acidic (pI - 5.0), two slightly basic (pI - 7.7 and pI - 7.7) and two strongly basic nature (pI - 8.7 and pI - 8.9). Molecular weights varied from 8 500 to 18 500 daltons. The origin of these peptides is briefly discussed.     

Dual mechanism of a natural CaMKII inhibitor

Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of cellular Ca(2+) signaling. Several inhibitors are commonly used to study CaMKII function, but these inhibitors all lack specificity. CaM-KIIN is a natural, specific CaMKII inhibitor protein. CN21 (derived from CaM-KIIN amino acids 43-63) showed full specificity and potency of CaMKII inhibition. CNs completely blocked Ca(2+)-stimulated and autonomous substrate phosphorylation by CaMKII and autophosphorylation at T305. However, T286 autophosphorylation (the autophosphorylation generating autonomous activity) was only mildly affected. Two mechanisms can explain this unusual differential inhibitor effect. First, CNs inhibited activity by interacting with the CaMKII T-site (and thereby also interfered with NMDA-type glutamate receptor binding to the T-site). Because of this, the CaMKII region surrounding T286 competed with CNs for T-site interaction, whereas other substrates did not. Second, the intersubunit T286 autophosphorylation requires CaM binding both to the "kinase" and the "substrate" subunit. CNs dramatically decreased CaM dissociation, thus facilitating the ability of CaM to make T286 accessible for phosphorylation. Tat-fusion made CN21 cell penetrating, as demonstrated by a strong inhibition of filopodia motility in neurons and insulin secrection from isolated Langerhans' islets. These results reveal the inhibitory mechanism of CaM-KIIN and establish a powerful new tool for dissecting CaMKII function.     

Ubiquitylation of proliferating cell nuclear antigen and recruitment of human DNA polymerase eta

This study investigated the requirement for ubiquitylation of PCNA at lysine 164 during polymerase eta-dependent translesion synthesis (TLS) of site-specific cis-syn cyclobutane thymine dimers (T (wedge)T). The in vitro assay recapitulated origin-dependent initiation, fork assembly, and semiconservative, bidirectional replication of double-stranded circular DNA substrates. A phosphocellulose column was used to fractionate HeLa cell extracts into two fractions; flow-through column fraction I (CFI) contained endogenous PCNA, RPA, ubiquitin-activating enzyme E1, and ubiquitin conjugase Rad6, and eluted column fraction II (CFII) included pol delta, pol eta, and RFC. CFII supplemented with purified recombinant RPA and PCNA (wild type or K164R, in which lysine was replaced with arginine) was competent for DNA replication and TLS. K164R-PCNA complemented CFII for these activities to the same extent and efficiency as wild-type PCNA. CFII mixed with CFI (endogenous PCNA, E1, Rad6) exhibited enhanced DNA replication activity, but the same TLS efficiency determined with the purified proteins. These results demonstrate that PCNA ubiquitylation at K164 of PCNA is not required in vitro for pol eta to gain access to replication complexes at forks stalled by T (wedge)T and to catalyze TLS across this dimer.     

Affinity isolation of heat-shock and other calmodulin-binding proteins following hyperthermia

The interaction of calmodulin (CaM) with heat-shock and other binding proteins was studied in rat adenocarcinoma cells. Cells were equilibrium-labeled for 48 h prior to heating for 1 h at 43 degrees C, or pulse-labeled for 2 h at 37 degrees C after heating, to monitor the effect of heat on the affinity of CaM-binding proteins synthesized under these conditions. A CaM antagonist shown to sensitize to heat killing, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], was used in competition assays to help monitor any changes in affinity. We found that heating tended to reduce the CaM-binding of proteins synthesized before heating relative to their 37 degrees C controls and proteins synthesized after heating tended to have increased binding relative to their respective controls. Members of the heat-shock protein (hsp) 90-, 70-, and 26-kDa families were among the proteins that bound to CaM and were eluted by W-7. The peak elution fractions for the hsp's and other cellular proteins varied, but hsp-70 eluted in the early fractions. The hsp-70 family was also found to be among a number of W-7-binding proteins. We conclude that the assumption that CaM antagonists potentiate killing of heated cells solely by competing nonspecifically for CaM-binding protein sites on CaM does not explain the process completely. These antagonists could also act by competing for CaM-binding sites with specific proteins whose interaction with CaM is important for survival following heating, or by directly binding to other proteins whose function is important for survival and inhibiting their activity. We do not have sufficient data to discern the predominant mechanism among these possibilities, but we believe all are likely to occur in heated cells and speculate that inhibition of the functions of the hsp-70 family is important in several of these antagonist actions.