Showing papers in "Biochemistry in 1983"
TL;DR: These results corroborate previous static studies, utilizing visible spectroscopy and circular dichroism, which indicate that the formation of an intercalated complex occurs only at GC base pair sites with porphyrins which do not possess axial ligands.
Abstract: The interactions of tetrakis(4-N-methylpyridyl)-porphine (H2TMpyP-4) and its copper(II), nickel(II), zinc(II), cobalt(III), iron(III), and manganese(III) derivatives with several nucleic acids have been investigated. Spectrophotometric titrations of H2TMpyP-4 and Cu(II)TMpyP-4 with the synthetic polymer poly(dG-dC) could be analyzed by a nearest-neighbor exclusion model leading to n approximately equal to two base pairs and equilibrium constants of 7.7 X 10(5) M-1 and 8.0 X 10(5) M-1, respectively. The other metal derivatives [except for the nickel(II) porphyrin] do not provide sufficiently large color changes with poly(dG-dC) to allow analysis. In contrast, all of these porphyrins interact with poly(dA-dT) and DNA. For those porphyrins investigated, the binding profiles are not adequately fit by a nearest-neighbor exclusion model but have profiles suggesting that cooperativity effects are important. Spectral and circular dichroic experiments both suggest base specificity. With calf thymus DNA, the copper(II) and nickel(II) derivatives show prominent negative circular dichroism (CD) features and large red shifts and hypochromicity of the Soret absorption band characteristic of GC specificity, as demonstrated with the synthetic polymer. The other metal derivatives show prominent positive induced visible CD features with small red shifts and hypochromicity of the absorption bands in the Soret region characteristic of AT specificity. Only the metal-free derivative has a conservative CD spectrum suggesting distribution among GC and AT sites.
769 citations
TL;DR: Specific adsorption is demonstrated for serum proteins on immobilized Ni(II) and Fe(III) and ligand-specific desorption (affinity elution) is demonstrated by including in the buffer system certain solutes which are similar to or identical with some particular amino acids found in proteins.
Abstract: Immobilized metal ion affinity adsorption (IMA adsorption) is a collective term that is proposed to include all kinds of adsorptions whereby metal atoms or ions immobilized on a polymer cause or dominate the interaction at the sorption sites. IMA chromatography is one of the most powerful methods available to date for protein fractionation although this is not as yet widely recognized. This study deals with the theoretical aspects of IMA adsorption and its practical applications as exemplified by the various results reported here. The synthesis of iminodiacetate-substituted agarose (IDA-agarose) and tris(carboxymethyl)ethylenediamine-agarose (TED-agarose) is described. Many types of metal ions can easily be immobilized on these gel derivatives to form IMA adsorbents. We have not observed any damage to the proteins during the adsorption-desorption process. After performance of an experiment, the gels can easily be regenerated and can be loaded with the same or a different metal ion for an ensuing experiment. Specific adsorption is demonstrated for serum proteins on immobilized Ni(II) and Fe(III). Ligand-specific desorption (affinity elution) is also demonstrated by including in the buffer system certain solutes which are similar to or identical with some particular amino acids found in proteins. High concentrations of certain salts that affect the structure of water, such as Na2SO4, promote coordinate covalent bonding of proteins by a mechanism that is apparently similar to that found in hydrophobic interactions. Neutral detergents and aquoorganic solvents may be used. This opens up the possibility for the fractionation of membrane components. The IMA-adsorption method could also be expanded to other areas besides protein fractionation.
541 citations
TL;DR: Comparison of the thermodynamics for CCGG, ACCGGp, CCGGUp, and ACCGG up indicates stacking interactions are somewhat more important than pairing interactions in determining the stability of the terminal AU base pairs in ACCGGUp.
Abstract: The thermodynamics of double-helix formation in 1 M NaCl have been measured spectrophotometrically for CCGG, CCGGp, CCGGAp, ACCGGp, CCGGUp, and ACCGGUp. The results indicate additional double-helical stability is conferred by the terminal unpaired bases. The 3' A stabilizes the double helix more than the 5' A or the 3' U. The increased stability is due to a more favorable enthalpy change for double-helix formation. Comparison of the thermodynamics for CCGG, ACCGGp, CCGGUp, and ACCGGUp indicates stacking interactions are somewhat more important than pairing interactions in determining the stability of the terminal AU base pairs in ACCGGUp.
467 citations
TL;DR: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified from the breast muscles of 3-week-old chickens and used to raise a specific antiserum in rabbits coupled to an in vitro translation assay to monitor the purification of GAPDH mRNA.
Abstract: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified from the breast muscles of 3-week-old chickens and used to raise a specific antiserum in rabbits. This antiserum was coupled to an in vitro translation assay to monitor the purification of GAPDH mRNA. RNA was isolated from identical breast muscles and consecutively fractionated with several techniques to yield a preparation of GAPDH mRNA which was at least 50% pure. Double-stranded cDNA was made against this purified RNA, inserted into pBR322, and used to transform Escherichia coli. Recombinants were screened by colony filter hybridization with a cDNA probe made against the purified RNA. The hybridization-positive clone with the largest insert, pGAD-28, was then characterized by using pGAD-28-cellulose to select complementary RNA from total poly(A) RNA and then translating the hybridization-selected RNA in vitro. The single translation product was shown to be GAPDH by (1) comigration with pure GAPDH on sodium dodecyl sulfate-polyacrylamide gels, (2) precipitation with specific anti-GAPDH antiserum, (3) cyanylation fingerprinting, and (4) AMP-agarose affinity chromatography. pGAD-28 was mapped with several restriction enzymes and then sequenced by the method of Maxam and Gilbert [Maxam, A. M., & Gilbert, W. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 560]. The 1261-nucleotide insert was found to contain 29 nucleotides of noncoding sequence at the 5' end, the entire coding region, and 230 nucleotides of the 3'-noncoding region including a poly(A) addition signal (AATAAA) and the first five residues of the poly(A) tail.
455 citations
401 citations
TL;DR: Analysis of the purified polypeptide by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels indicates that TGF-beta is composed of two closely related polypePTide chains cross-linked by disulfide bonds.
Abstract: Type beta transforming growth factor (TGF-beta) has been purified 200 000-fold from bovine kidneys. This peptide is characterized by its ability to induce anchorage-dependent normal rat kidney cells to grow in soft agar in the presence of epidermal growth factor (EGF); TGF-beta is not mitogenic for cells grown in monolayer culture. Purified TGF-beta does not compete with EGF for binding to membrane receptors. The concentration of TGF-beta required to elicit a half-maximal response for formation of colonies greater than 3100 micron2 in the soft agar assay is 2-3 pM (55 pg/mL) when assayed in the presence of 0.8 nM EGF (5 ng/mL). The four-step purification procedure which includes chromatography of acid--ethanol tissue extracts on polyacrylamide sizing gels, cation exchange, and two steps of high-pressure liquid chromatography results in a 10% overall yield of colony-forming activity with a recovery of 3-4 micrograms/kg. Amino acid analysis of purified TGF-beta shows 16 half-cystine residues per mole. Analysis of the purified polypeptide by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels indicates that TGF-beta is composed of two closely related polypeptide chains cross-linked by disulfide bonds. In the absence of beta-mercaptoethanol, the colony-forming activity is associated with a single silver-staining band of molecular weight 25 000; in the presence of beta-mercaptoethanol, the TGF-beta is converted to an inactive species that migrates as a single band of molecular weight 12 500-13 000. Sequence analysis indicates that at least the first 15 N-terminal amino acids of the two TGF-beta subunits are identical.
348 citations
323 citations
TL;DR: In inspection of changes in the C-propeptide of the pro alpha 1 (I) chain, it was suggested that there was a highly conserved region around the carbohydrate attachment site similar to the highly Conserved region of 37 amino acids previously found in theC-propezide ofThe pro alpha 2(I) chains.
Abstract: Nucleotide sequences were determined for two cloned cDNAs encoding for over three-fourths of the pro alpha 1 (I) chain of type I procollagen from man. Comparison with previously published data on amino acid sequences of the alpha 1 (I) chain of type I collagen made it possible to examine mutations in the transcribed products of the gene which have occurred during the evolution of man, calf, rat, mouse, and chick. Comparison of the nucleotide sequences with the corresponding sequences of cDNAs from chick [Fuller, F., & Boedtker, H. (1981) Biochemistry 20, 996] and with cDNAs for the pro alpha 2(I) chain from man [Bernard, M.P., Myers, J. C., Chu, M.-L., Ramirez, F., Eikenberry, E. F., & Prockop, D. J. (1983) Biochemistry 22, 1139] demonstrated that selective pressure during evolution for 250 million or more years acted more strongly on the structure of the pro alpha 1 (I) chain than on the pro alpha 2(I) chain. To improve the reliability of the comparison, the nucleotide sequences were examined with a modification of previous procedures for evaluating mutations in replacement sites and silent sites. The corrected divergence for replacement sites between the alpha 1 (I) chains was 6 +/- 0.8% whereas it was 15 +/- 1.9% for the alpha 2(I) chains. The C-propeptide domain of the pro alpha 1 (I) chain was also highly conserved with a corrected divergence at replacement sites of 5 +/- 0.9%, a value that was not distinguishable from the value previously found for the C-propeptide of the pro alpha 2(I) chain. Therefore, a large part of the structure of both C-propeptides appears to be under selective pressure. Inspection of changes in the C-propeptide of the pro alpha 1 (I) chain suggested that there was a highly conserved region around the carbohydrate attachment site similar to the highly conserved region of 37 amino acids previously found in the C-propeptide of the pro alpha 2(I) chain. Two statistical tests, however, were unable to confirm nonrandom distribution of changes in the C-propeptide of the pro alpha 1(I) chain. The same tests established the presence of a nonrandom distribution in nucleotide changes of the C-propeptide of the pro alpha 2(I) chain. The 3'-noncoding region of the cDNA for pro alpha 1(I) of human type I procollagen showed no homology with the same region in the chick.(ABSTRACT TRUNCATED AT 400 WORDS)
308 citations
TL;DR: Thermodynamic binding constants for gallium complexation at the two specific metal binding sites of human serum transferrin at pH 7.4 and 5 mM NaHCO3 have been determined by UV difference spectroscopy and are discussed in relation to the thermodynamics of transferrin binding of Fe3+.
Abstract: Gallium-67 is widely used as an imaging agent for tumors and inflammatory abscesses. It is well established that Ga3+ travels through the circulatory system bound to the serum iron transport protein transferrin and that this protein binding is an essential step in tumor localization. However, there have been conflicting reports on the magnitude of the gallium-transferrin binding constants. Therefore, thermodynamic binding constants for gallium complexation at the two specific metal binding sites of human serum transferrin at pH 7.4 and 5 mM NaHCO3 have been determined by UV difference spectroscopy. The conditional constants calculated for 27 mM NaHCO3 are log K1 = 20.3 and log K2 = 19.3. These results are discussed in relation to the thermodynamics of transferrin binding of Fe3+ and to previous reports on gallium binding. The strength of transferrin complexation is also compared to that of a series of low molecular weight ligands by using calculated pM values (pM = -log [Ga-(H2O)6]) to express the effective binding strength at pH 7.4.
300 citations
297 citations
TL;DR: The DNA sequences of a complementary deoxyribonucleic acid (cDNA) and a portion of the gene coding for human prothrombin have been determined and it is proposed that the leader sequence consists of a signal sequence and a pro sequence for the mature protein that circulates in plasma.
Abstract: The DNA sequences of a complementary deoxyribonucleic acid (cDNA) and a portion of the gene coding for human prothrombin have been determined. The cDNA was 2005 base pairs in length and was found to code for part of a leader sequence of 36 amino acids, 579 amino acids present in the mature protein, a stop codon, a noncoding region of 97 base pairs, and a poly(A) tail of 27 base pairs. It is proposed that the leader sequence consists of a signal sequence and a pro sequence for the mature protein that circulates in plasma. The 10 glutamic acid residues that are present in the amino-terminal region of prothrombin and are converted to gamma-carboxyglutamic acid in the mature protein are coded by only the GAG codon. The cDNA for prothrombin was also employed as a probe for screening a human fetal liver genomic DNA library. One of the strongly positive phage containing a human DNA insert of 5 kilobases was mapped with restriction endonucleases and sequenced. This DNA contained approximately half of the gene for human prothrombin and included six introns and five exons coding for amino acid residues 144-448. The two largest intervening sequences in the genomic DNA contained two copies each of AluI repetitive DNA.
TL;DR: There is an inherent specificity for the incorporation of adenine nucleotides opposite AP sites in this in vitro system and insofar as the model system reflects in vivo mutational events, the data suggest that depurination should produce transversions and depyrimidination should producing transitions.
Abstract: M13 DNA containing 20-30 apurinic/apyrimidinic (AP) sites per intact circular molecule was prepared by growing phage on an ung- dut- Escherichia coli mutant and treating the DNA with uracil N-glycosylase. AP sites obstruct in vitro DNA synthesis catalyzed by E. coli pol I. The position at which termination of synthesis occurs was determined for four enzymes. T4 DNA polymerase terminates one nucleotide before putative AP sites. DNA pol I, AMV reverse transcriptase, and DNA polymerase alpha terminate synthesis either before or at the site of an AP lesion depending on the particular sequence. We determined the identity of the nucleotide inserted opposite an AP site by synthesizing up to the lesion in a first-stage reaction using T4 DNA polymerase and then determining elongation in a second stage. Purines are inserted opposite AP sites more readily than pyrimidines, and dATP is more efficient than dGTP in promoting such elongation. The DNA-dependent conversion of dNTP to dNMP was determined in mixtures of all four dNTP's by using AP DNA. The production of dAMP from dATP occurs most readily. We conclude that there is an inherent specificity for the incorporation of adenine nucleotides opposite AP sites in this in vitro system. Insofar as the model system reflects in vivo mutational events, our data suggest that depurination should produce transversions and depyrimidination should produce transitions.
TL;DR: Human "enkephalinase" can be classified as a neutral metalloendopeptidase with a broad substrate specificity and compared to that of homogeneous human kidney converting enzyme, which inactivated bradykinin by release of the C-terminal dipeptide but were inhibited differentially by specific inhibitors.
Abstract: After extracting converting enzyme from a membrane fraction of homogenized human kidney, "enkephalinase" activity was solubilized with Triton X-100. Ion-exchange chromatography resolved two peaks of the "enkephalinase" activity, both of which cleaved Leu5-enkephalin at the Gly3-Phe4 bond. The major "enkephalinase" form was purified 1140-fold to homogeneity with a 14% yield. This homogeneous "enkephalinase" had a specific activity of 46 mumol min-1 mg-1 with Leu5-enkephalin as substrate. The purified enzyme, in addition to hydrolyzing Leu5-enkephalin, cleaved synthetic substrates with protected N- and C-terminal ends. On the basis of the specificity of the enzyme and its inhibition by chelating agents, human "enkephalinase" can be classified as a neutral metalloendopeptidase with a broad substrate specificity. The activity of this neutral endopeptidase with several biologically active peptides was compared to that of homogeneous human kidney converting enzyme. Both enzymes inactivated bradykinin by release of the C-terminal dipeptide but were inhibited differentially by specific inhibitors. Comparison of hydrolysis of bradykinin with that of its protected C-terminal peptide indicated that the neutral endopeptidase is more active toward the larger substrate than is converting enzyme. Although the neutral endopeptidase did not convert angiotensin I to II, it did hydrolyze angiotensin I at Pro7-Phe8 and inactivate angiotensin II by cleavage at the Tyr4-Ile5 bond.
TL;DR: The correlation noted between inhibitor Ki and substrate Km/kcat is the most extensive one of this type, providing strong evidence that the phosphonamidates are transition- state analogues and not simply multisubstrate ground-state analogues.
Abstract: Six phosphorus-containing peptide analogues of the form Cbz-NHCH2PO2--L-Leu-Y (Y = D-Ala, NH2, Gly, L-Phe, L-Ala, L-Leu) have been prepared and evaluated as inhibitors of thermolysin. The Ki values for these compounds range from 1.7 microM to 9.1 nM and correlate well with the Km/kcat values for the corresponding peptide substrates [Morihara, K., & Tsuzuki, H. (1970) Eur. J. Biochem. 15, 374-380] but not with the Km values alone. The correlation noted between inhibitor Ki and substrate Km/kcat is the most extensive one of this type, providing strong evidence that the phosphonamidates are transition-state analogues and not simply multisubstrate ground-state analogues. Cbz-NH2CH2PO2--L-Leu-L-Leu (Ki = 9.1 nM) is the most potent inhibitor yet reported for thermolysin.
TL;DR: The interaction energy of the side chain of Cys-35 with the substrate is not fully realized in the enzyme-substrate complex but is used preferentially to stabilize the transition state.
Abstract: Oligodeoxynucleotide-directed mutagenesis has been used on the gene of tyrosyl-tRNA synthetase from Bacillus stearothermophilus to produce mutant enzymes altered at the adenosine 5'-triphosphate (ATP) binding site. Deliberate attempts were made to alter rather than destroy enzymic activity so that kinetic measurements may be made to identify the subtle roles of the enzyme-substrate interactions in catalysis. Cys-35, the -SH group of which is involved in binding the 3'-OH of the ribose ring of ATP, has been mutated to a serine residue [Winter, G., Fersht, A. R., Wilkinson, A. J., Zoller, M., & Smith, M. (1982) Nature (London) 299, 756-758] or glycine residue. The mutant enzymes are less active than the wild type, and the reduction in activity can be attributed to a decrease in the value of kcat and an increase in KM. Thus, the interaction energy of the side chain of Cys-35 with the substrate is not fully realized in the enzyme-substrate complex but is used preferentially to stabilize the transition state. Relative to its absence in the Gly-35 mutant, the side chain of Cys-35 is calculated to stabilize the transition state for pyrophosphate exchange by 1.2 kcal/mol and the transition state for aminoacylation by 1.0 kcal/mol.
TL;DR: The denatured peptide displayed an electrophoretic microheterogeneity which may be ascribed to variations in bulky carbohydrate substituents and an extremely high free mobility which is inferred to result from binding of unusually large amounts of NaDodSO4.
Abstract: Preparations of the tetrodotoxin (TTX) and saxitoxin binding protein isolated from the electroplax of Electrophorus electricus are of high specific activity (greater than or equal to 2000 pmol of TTX binding sites/mg of protein) and appear to be homogeneous in that they contain only the large polypeptide previously identified to make up part of the voltage-sensitive sodium channel [Agnew, W. S., Moore, A. C., Levinson, S. R., & Raftery, M. S. (1980) Biochem. Biophys. Res. Commun. 92, 860-866]. This permits the inference that the TTX binding site, thought to be associated with the mouth of the ion channel, is located on this peptide. This peptide presumably corresponds to the large peptide, designated the alpha-peptide subunit, of the synaptosomal sodium channel [Hartshorne, R. P., & Catterall, W. A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4620-4624]. No convincing evidence for lower molecular weight peptides has yet been found for the electroplax protein. A rapid and convenient method is described for preparation of milligram quantities of the pure, sodium dodecyl sulfate (NaDodSO4) denatured form of the peptide, and its amino acid and carbohydrate compositions are reported. The peptide behaved anomalously on NaDodSO4-polyacrylamide gels. It was demonstrated that the molecular weight cannot be accurately quantified by this method but that the true value likely exceeds the value of 260 000 reported previously. The denatured peptide displayed an electrophoretic microheterogeneity which may be ascribed to variations in bulky carbohydrate substituents and an extremely high free mobility which is inferred to result from binding of unusually large amounts of NaDodSO4.
TL;DR: Comparisons with previously published data on homologous cDNAs from chick embryos revealed three different classes of conservation of nucleotide sequence which have no apparent effect on the structure of the protein: a preference for U on the third base position of codons for glycine, proline, and alanine; a high degree ofucleotide conservation in the 51 amino acid highly conserved region of the C-propeptide.
Abstract: Nucleotide sequences were determined for cloned cDNAs encoding for more than half of the pro alpha 2 chain of type I procollagen from man. Comparisons with previously published data on homologous cDNAs from chick embryos made it possible to examine evolution of the gene in two species which have diverged for 250-300 million years. The amino acid sequence of the alpha-chain domain supported previous indications that there is a strong selective pressure to maintain glycine as every third amino acid and to maintain a prescribed distribution of charged amino acids. However, there is little apparent selective pressure on other amino acids. The amino acid sequence of the C-propeptide domain showed less divergence than the alpha-chain domain. The 5' end or N terminus of the human C-propeptide, however, contained an insert of 12 bases coding for 4 amino acids not found in the chick C-propeptide. About 100 amino acid residues from the N terminus, two residues found in the chick sequence were missing from the human. In the second half of the C-propeptide, there was complete conservation of a 37 amino acid sequence and conservation of 50 out of 51 amino acids in the same region, an observation which suggested that the region serves some special purpose such as directing the association of one pro alpha 2(I) C-propeptide with two pro alpha 1(I) C-propeptides so as to produce the heteropolymeric structure of type I procollagen. In addition, comparison of human and chick DNAs for pro alpha 2(I) revealed three different classes of conservation of nucleotide sequence which have no apparent effect on the structure of the protein: a preference for U on the third base position of codons for glycine, proline, and alanine; a high degree of nucleotide conservation in the 51 amino acid highly conserved region of the C-propeptide; a high degree of nucleotide conservation in the 3'-noncoding region. These three classes of nucleotide conservation may reflect unusual features of collagen genes, such as their high GC content or their highly repetitive coding sequences.
TL;DR: Castanospermine proved to be a potent inhibitor of glycoprotein processing by virtue of the fact that it inhibits glucosidase I, and influenza virus produced in the presence of this alkaloid were fully infective and apparently produced in similar amounts to that of control cells, as determined by plaque counts.
Abstract: Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is a plant alkaloid that inhibits alpha- and beta-glucosidase in fibroblast extracts [Saul, R., Chambers, J. P., Molyneux, R. J., & Elbein, A. D. (1983) Arch. Biochem. Biophys. 221, 593-597]. In the present study, castanospermine also proved to be a potent inhibitor of glycoprotein processing by virtue of the fact that it inhibits glucosidase I. Thus, when influenza virus was raised in the presence of castanospermine, at 10 micrograms/mL or higher, 80-90% of the viral glycopeptides were susceptible to the action of endoglucosaminidase H, whereas in the normal virus 70% of the glycopeptides are resistant to this enzyme. The major oligosaccharide released by endoglucosaminidase H from castanospermine-grown virus migrated like a hexose10GlcNac on a calibrated Bio-Gel P-4 column. This oligosaccharide was characterized as a Glc 3 Man 7 GlcNAc on the basis of various enzymatic treatments, as well as by methylation analysis of the [2-3H]-mannose-labeled or [6-3H]galactose-labeled oligosaccharide. The presence of three glucose residues in the oligosaccharide was also confirmed by periodate oxidation studies of the [6-3H]galactose-labeled hexose10GlcNAc. Castanospermine did not inhibit the incorporation of [3H]leucine or [14C]alanine into protein in MDCK cells at levels as high as 50 micrograms/mL. In addition, influenza virus produced in the presence of this alkaloid were fully infective and apparently produced in similar amounts to that of control cells, as determined by plaque counts. Castanospermine did, however, cause considerable changes in cell surface properties, since MDCK cells grown in 10 micrograms/mL castanospermine were able to bind twice as much [3H]concanavalin A as were control cells.
TL;DR: The nucleotide sequence of the merA gene from the mercury-resistance transposon Tn501 is determined and the structure of the gene product, mercuric reductase, is predicted and it is shown that codons with C or G at the third position are preferentially utilized.
Abstract: We have determined the nucleotide sequence of the merA gene from the mercury-resistance transposon Tn501 and have predicted the structure of the gene product, mercuric reductase. The DNA sequence predicts a polypeptide of Mr 58 660, the primary structure of which shows strong homologies to glutathione reductase and lipoamide dehydrogenase, but mercuric reductase contains as additional N-terminal region that may form a separate domain. The implications of these comparisons for the tertiary structure and mechanism of mercuric reductase are discussed. The DNA sequence presented here has an overall G+C content of 65.1 mol%, typical of the bulk DNA of Pseudomonas aeruginosa from which Tn501 was originally isolated. Analysis of the codon usage in the merA gene shows that codons with C or G at the third position are preferentially utilized.
TL;DR: Chemical cleavages of the cross-linked products followed by peptide mappings revealed that two distinct segments of S1 heavy chain spanning the 18K-20K region and the 27K-35K region from its C terminus participated in cross-linking with actin.
Abstract: When the rigor complex of actin and myosin subfragment 1 (S1) was treated with a zero-length cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, covalently linked complexes of actin and S1 heavy chain with apparent molecular weights of 165,000 and 175,000 were generated. Measurements of the molar ratio of actin to S1 heavy chain in the 165K and 175K products showed that they were 1:1 complexes of actin and S1 heavy chain. Chemical cleavages of the cross-linked products followed by peptide mappings revealed that two distinct segments of S1 heavy chain spanning the 18K-20K region and the 27K-35K region from its C terminus participated in cross-linking with actin. Cross-linking of actin to the former site generated the 165K peptide while the latter site was responsible for generating the 175K peptide.
TL;DR: Two glutathione S-transferase activities have been identified and purified from etiolated corn tissue, and both catalyze the formation of a glutathion-herbicide conjugate in vitro when the herbicide alachlor is used as a substrate.
Abstract: Two glutathione S-transferase (GST) activities have been identified and purified from etiolated corn tissue. The first, designated GST I enzyme, is constitutively present in corn tissue, and the second, designated GST II enzyme, is present only in tissue which has been treated with chemical antidotes which protect corn against chloroacetanilide herbicides. The total activity constitutes approximately 2% of the soluble protein in these tissues. The native forms of these enzymes have molecular weights of approximately 50 000 as determined by Sephadex G-100 chromatography. On sodium dodecyl sulfate-polyacrylamide gels, GST I enzyme migrates primarily as a single band of molecular weight 29 000, and GST II enzyme migrates as primarily two bands of molecular weight 29 000 and 27 000. Both enzymes catalyze the formation of a glutathione-herbicide conjugate in vitro when the herbicide alachlor is used as a substrate. This conjugation results in elimination of the biological activity of the herbicide.
TL;DR: Experimental thermodynamic data for the binding of cytidine 3'-phosphate to ribonuclease A demonstrate apparent enthalpy-entropy compensation when pH and ionic strength are varied.
Abstract: General thermodynamic models are presented that can account for the existence of heat capacity changes and compensation between the enthalpy and entropy changes in protein-ligand interactions. The models involve the coupling between some type of transition in the state of the protein (or ligand) and the binding process. The coupled transition may be a proton dissociation, the binding of a second ligand, a change in the degree of aggregation, or a conformational change in either the protein or ligand. Both mandatory coupling and nonmandatory coupling between the binding process and the transition are considered. The model is also extended to include a multistate transition of the protein. Computer simulations show that apparently linear compensation plots (plots of delta H degrees vs. delta S degrees) with a slope approximately equal to the experimental temperature are to be expected for the binding of a ligand to a protein when such coupled reactions exist. Also heat capacity changes, which may be either positive or negative, are to be expected to accompany the reaction. Experimental thermodynamic data for the binding of cytidine 3'-phosphate to ribonuclease A are presented. These data demonstrate apparent enthalpy-entropy compensation when pH and ionic strength are varied. A negative heat capacity change, ranging from -145 (at mu = 1.0 M) to -225 cal/(mol X deg) (at mu = 0.05 M), is also observed for this protein-ligand interaction. The apparent compensation and heat capacity change data are interpreted according to the models presented.
TL;DR: 3H-PAF receptor binding was demonstrated with purified plasma membrane from several smooth muscles and from polymorphonuclear leukocytes but not from presumably PAF nonresponsive cells such as erythrocytes and alveolar macrophages.
Abstract: By using tritiated 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (3H-PAF), we have directly identified its specific binding sites on rabbit platelet plasma membranes. The equilibrium dissociation constant for 3H-PAF is 1.36 (+/- 0.05) X 10(-9) M at 0 degrees C. The number of binding sites is 1.61 (+/- 0.34) X 10(12)/mg of membrane, which corresponds to approximately 150-300 receptors/platelet (depending on membrane vesicle orientation). Binding of 3H-PAF to rabbit platelet plasma membrane is rapid (t1/2 less than 5 min at 0 degrees C) and reversible. For a series of PAF analogues, their affinity for the receptor sites parallels with their relative potency to induce platelet aggregation. PAF can cause contraction of smooth muscle of heart, parenchymal strip, trachea, and ileum. Specific PAF receptor binding was demonstrated with purified plasma membrane from several smooth muscles and from polymorphonuclear leukocytes but not from presumably PAF nonresponsive cells such as erythrocytes and alveolar macrophages. It is likely that the interaction of PAF with these binding sites initiates the specific responses of platelets, polymorphonuclear leukocytes, and smooth muscles.
TL;DR: A new calmodulin (CaM) binding protein, designated P-57, has been purified to apparent homogeneity from bovine cerebral cortex membranes and appears to have novel binding properties for cal modulin distinct from all other calmod insulin binding proteins described thus far.
Abstract: A new calmodulin (CaM) binding protein, designated P-57, has been purified to apparent homogeneity from bovine cerebral cortex membranes. In contrast to other calmodulin binding proteins, P-57 has higher affinity for calmodulin in the absence of bound Ca2+ than in its presence. The protein was purified by DEAE-Sephacel chromatography and two CaM-Sepharose affinity column steps. The first CaM-Sepharose column was run in the presence of Ca2+; the second was run in the presence of chelator in excess of Ca2+. P-57 was adsorbed by CaM-Sepharose only in the absence of bound Ca2+ and was eluted from the second column by buffers containing Ca2+. Sodium dodecyl sulfate (SDS)-polyacrylamide gels of the purified protein showed only one band at Mr 57 000. The major form of the protein on Bio-Gel A-1.5m and native polyacrylamide gradient gel electrophoresis ran with an apparent Stokes radius of 41 A. Photoaffinity labeling of P-57 with azido[125I]calmodulin yielded one cross-linked product on SDS gels with an Mr of 70 000. This interaction occurred only when excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was present and was inhibited by the presence of Ca2+ in excess of chelator. It appears that P-57 has novel binding properties for calmodulin distinct from all other calmodulin binding proteins described thus far.
TL;DR: The interaction between phage R17 coat protein and its RNA binding site for translational repression was studied as an example of a sequence-specific RNA--protein interaction.
Abstract: The interaction between phage R17 coat protein and its RNA binding site for translational repression was studied as an example of a sequence-specific RNA--protein interaction. Nuclease protection and selection experiments define the binding site to about 20 contiguous nucleotides which form a hairpin. A nitrocellulose filter retention assay is used to show that the binding between the coat protein and a synthetic 21-nucleotide RNA fragment conforms to a simple bimolecular reaction. Unit stoichiometry and a Kd of about 1 nM are obtained at 2 degrees C in buffer containing 0.19 M salt. The interaction is highly sequence specific since a variety of RNAs failed to compete with the 21-nucleotide fragment for coat protein binding.
TL;DR: The DNA binding sites for the antitumor, antiviral, antibiotics chromomycin, mithramycin, and olivomycin on 70 base pairs of heterogeneous DNA have been determined by using the (methidiumpropyl-EDTA)iron(II) [MPE x Fe(II)] DNA cleavage inhibition pattern technique.
Abstract: The DNA binding sites for the antitumor, antiviral, antibiotics chromomycin, mithramycin, and olivomycin on 70 base pairs of heterogeneous DNA have been determined by using the (methidiumpropyl-EDTA)iron(II) [MPE x Fe(II)] DNA cleavage inhibition pattern technique Two DNA restriction fragments 117 and 168 base pairs in length containing the lactose operon promoter-operator region were prepared with complementary strands labeled with 32P at the 3' end MPE x Fe(II) was allowed to partially cleave the restriction fragment preequilibrated with either chromomycin, mithramycin, or olivomycin in the presence of Mg2+ The preferred binding sites for chromomycin, mithramycin, and olivomycin in the presence of Mg2+ appear to be a minimum of 3 base pairs in size containing at least 2 contiguous dG x dC base pairs Many binding sites are similar for the three antibiotics; chromomycin and olivomycin binding sites are nearly identical The number of sites protected from MPE x Fe(II) cleavage increases as the concentration of drug is raised For chromomycin/Mg2+, the preferred sites on the 70 base pairs of DNA examined are (in decreasing affinity) 3'-GGG, CGA greater than CCG, GCC greater than CGA, CCT greater than CTG-5' The sequence 3'-CGA-5' has different affinities, indicating the importance of either flanking sequences or a nearly bound drug