Effect of Ca2+ ions on hydrodynamic properties of pentamer and decamer of C-reactive protein in solution

The molecular mass and sedimentation coefficient of native C-reactive protein in solution were determined by analytical ultracentrifugation in the presence and absence of calcium ions. Pentameric C-reactive protein was shown to be the major macroscopic form of this protein in solution. The removal of calcium ions from solution caused decompaction of the protein accompanied by changes in its hydrodynamic parameters. The sedimentation coefficient s20(0), w of pentameric C-reactive protein in solution containing 2 mM--Ca2+ (6.6S) exceeded that for C-reactive protein in solution containing 2 mM EDTA (6.4S). Analysis of average molecular masses Mw and Mz obtained from sedimentation data demonstrated that the solution of highly purified protein was not homogeneous. As shown by intermolecular crosslinking, the solution also contained the 241-kDa decamer of C-reactive protein (9.5S) as a separate macroscopic form, whose share hardly reached 10% in the presence of 2 mM Ca2+ and increased after removal of calcium ions. The decamers were shown to result from intermolecular association of the pentamers.     

Effect of Calcium Ions on Hydrodynamic Properties of Pentameric and Decameric C-Reactive Protein in Solution

The molecular mass and sedimentation coefficient of native C-reactive protein in solution were determined by analytical ultracentrifugation in the presence and absence of calcium ions. Pentameric C-reactive protein was shown to be the major macroscopic form of this protein in solution. The removal of calcium ions from solution caused decompaction of the protein accompanied by changes in its hydrodynamic parameters. The sedimentation coefficient s ⁰ _20,w of pentameric C-reactive protein in solution containing 2 mM Ca²⁺ (6.6S) exceeded that for C-reactive protein in solution containing 2 mM EDTA (6.4S). Analysis of average molecular masses M _w and M _z obtained from sedimentation data demonstrated that the solution of highly purified protein was not homogeneous. As shown by intermolecular crosslinking, the solution also contained the 241-kDa decamer of C-reactive protein (9.5S) as a separate macroscopic form, whose share hardly reached 10% in the presence of 2 mM Ca²⁺ and increased after removal of calcium ions. The decamers were shown to result from intermolecular association of the pentamers.     

The dnaC protein of Escherichia coli. Purification, physical properties and interaction with dnaB protein.

E.coli dnaC protein was purified to near-homogeneity in using a dnaC complementation assay [S.Wickner, I.Berkower, M.Wright, and J.Hurwitz (1973) Proc. Natl. Acad. Sci. USA 70, 2369-2373]. Purification was achieved by taking advantage of the hydrophobic interaction of dnaC protein with aliphatic and aromatic matrixes and with Brij58 as stabilizing agent. A sedimentation coefficient for the dnaC protein of 2.6 S corresponding to a molecular weight of approximately 26,000 was estimated from glycerol gradient centrifugation. A polypeptide molecular weight of 28,000 was determined by densitometry on a denaturing gel. In the presence of ATP the dnaC protein forms a complex with dnaB protein [S.Wickner and J.Hurwitz (1975) Proc.Natl.Acad.Sci. USA 72, 921-925]. For the dnaB . dnaC complex a sedimentation coefficient of 14.5 S was measured by glycerol gradient centrifugation, indicating a molecular weight of about 400,000. The ratio of the dnaC and dnaB polypeptides in the complex is approximately 1, as determined on a denaturing gel. It is suggested that the complex consists of the dnaB protein hexamer and six dnaC polypeptides amounting to a calculated molecular weight of about 450,000.     

Compact globular structure of Thermus thermophilus ribosomal protein S1 in solution: sedimentation and calorimetric study

Ribosomal protein S1 of Thermus thermophilus overexpressed in Escherichia coli cells has been isolated and subjected to studies by analytical sedimentation and differential scanning microcalorimetry techniques. It has been demonstrated that the protein of 60 kDa sediments at s020,w = 4.6 S and has the diffusion coefficient D020,w = 6.7 x 10(-7) cm2/s in 25 mm HEPES-NaOH buffer, pH 7.5 (similarly to bovine serum albumin of 66 kDa that sediments at s0 20,w = 4.4 S and D020,w =6.0 x 10(-7) cm2/s), indicating its compact globular conformation under these conditions. The microcalorimetry study has shown the presence of a cooperative tertiary structure melting at 90 degrees C, but with several (probably three) independent cooperative domains. In the presence of 100 mm NaCl the protein becomes more asymmetric (s020,w = 3.1 S) but does not lose its cooperativity and thermostability, this suggesting just the weakening of interdomain ionic interactions. The compact globular conformation of protein S1 seems to be most likely within the ribosome.     

Isolation and Partial Characterization of the Major Seed Protein from Nicotiana tabacum,and Accumulation during Development

During the seed development of Nicotiana tabacum, appreciable accumulation of the soluble protein fraction started to occur at around the 6th day after anthesis and finally reached 12% on the basis of dry weight when seed maturation was accomplished. In the soluble fraction of mature seeds, four protein fractions were observed on analytical ultracentrifugation, and the protein having a sedimentation coefficient of 11.7S was the major one. The 11.7S protein was isolated and SDS-polyacrylamide gel electrophoresis indicated that the protein consisted of at least five subunits with molecular weights of 49,000, 31,000, 29,000, 21,000 and 19,000. The 11.7S protein was rich in glutamic acid or glutamine and arginine, and the presence of carbohydrate was confirmed.

During development, all of the five subunits started to appear during the period between the 12th and 15th day after anthesis.     

Eukaryotic initiation factor 5 from calf liver is a single polypeptide chain protein of Mr = 62,000

Eukaryotic initiation factor 5 (eIF-5), which specifically catalyzes the joining of a 60 S ribosomal subunit to a 40 S initiation complex to form a functional 80 S initiation complex, has been purified from ribosomal salt wash proteins of calf liver. The purified factor exhibits only one polypeptide band of Mr = 62,000 following electrophoresis in 10% polyacrylamide gels in the presence of sodium dodecyl sulfate. The native protein has a sedimentation coefficient of 4.2 S and a Stokes radius of 33 A which is consistent with eIF-5 being a monomeric protein of Mr = 58,000-62,000. Less pure preparations of eIF-5 elute in gel filtration columns with an apparent Mr of 160,000-180,000 presumably due to association of eIF-5 with other high molecular weight proteins since eIF-5 activity present in such preparations can also be shown by gel electrophoretic separation under denaturing conditions to be associated with a 62,000-dalton protein. Furthermore, eIF-5 purified from calf liver extracts with or without a number of protease inhibitors is indistinguishable with regard to molecular weight and final specific activity of purified preparations. The purified factor catalyzes the hydrolysis of GTP present in 40 S initiation complexes in the absence of 60 S ribosomal subunits. The presence of 60 S ribosomal subunits neither stimulates nor inhibits the hydrolysis of GTP. However, the factor cannot mediate 40 S or 40 + 60 S ribosome-dependent hydrolysis of GTP in the absence of Met-tRNAf or other components required for 40 S initiation complex formation. It can be calculated that 1 pmol of eIF-5 protein can catalyze the formation of at least 10 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.     

Binding sites of iron transferrin on rat reticulocytes. Inhibition by specific antibodies

Rat ventral prostate contains an acidic protein which can bind spermine selectively. The relative binding affinities of various aliphatic amines for the protein are, in decreasing order, spermine greater than thermine greater than greater than putrecine greater than 1,10-diaminodecane, cadaverine and 1,12-diaminododecane. The binding protein has an isoelectric point at pH 4.3 and a sedimentation coefficient of 3 S. Its molecular weight is approx. 30 000. Histones and nuclear chromatin preparations of the prostate can interact with the binding protein. The spermine-binding activity of the purified prostate protein can be inactivated by treatment with intestinal alkaline phosphatases. The phosphatase treated preparation can then be reactivated by beef heart protein kinase in the presence of cyclic AMP and ATP. The spermine-binding activity of the prostate cytosol protein fraction decreases after castration, but increases very rapidly after the castrated rats are injected with 5alpha-dihydrotestosterone. This finding raises the possibility that, in the postate, certain androgen actions may be dependent on the androgen-induced increase in the acidic protein binding of polyamines and their translocation to a functional cellular site such as nuclear chromatin. In the prostate cytosol, spermine also binds to 4-S tRNAs and to a unique RNA which has a sedimentation coefficient of 1.5 S.     

Juvenile-hormone-binding protein from the hemolymph of Galleria mellonella (L). Isolation and characterization

A juvenile-hormone-binding protein (JHBP) has been isolated from Galleria mellonella hemolymph by gel filtration, phosphocellulose chromatography, and by chromatofocusing. The isolated protein is homogeneous as judged by column chromatography and gel electrophoresis in the presence and absence of denaturing agent. It has a relative molecular mass of 32,000, Stokes radius 2.4 nm, sedimentation coefficient of 2.3 S, molar absorption coefficient at 280 nm epsilon = 2.34 X 10(4) M-1 cm-1, and is composed of a single polypeptide chain. Chromatofocusing analysis (pI 8.6) and isoelectric focusing (pI 8.1) indicate that the JHBP is an alkaline protein. Its amino acid composition and fluorescence absorption spectra indicate that the protein does not contain tryptophan residues. The protein exhibits one class of binding sites for juvenile hormone (JH), 0.8 per molecule, with the following dissociation constants: JH I, 8.5 X 10(-8) M; JH II, 7.2 X 10(-8) M; JH III, 47 X 10(-8) M. The JHBP binds (10R, 11S)-JH II enantiomer with 2.3-times higher affinity then (10S, 11R)-JH II enantiomer. The pH optimum of binding is 7.0.     

Selective polyamine-binding proteins spermine binding by an androgen-sensitive phosphoprotein

Rat ventral prostate contains an acidic protein which can bind spermine selectively. The relative binding affinities of various aliphatic amines for the protein are, in decreasing order, spermine > thermine >> spermidine > putrecine > 1,10-diaminodecane, cadaverine and 1,12-diaminododecane. The binding protein has an isoelectric point at pH 4.3 and a sedimentation coefficient of 3 S. Its molecular weight is approx. 30 000. Histones and nuclear chromatin preparations of the prostate can interact with the binding protein.The spermine-binding activity of the purified prostate protein can be inactivated by treatment with intestinal alkaline phosphatases. The phosphatase-treated preparation can then be reactivated by beef heart protein kinase in the presence of cyclic AMP and ATP.The spermine-binding activity of the prostate cytosol protein fraction decreases after castraction, but increases very rapidly after the castrated rats are injected with 5α-dihydrotesterone. This finding raises the possibility that, in the prostate, certain androgen actions may be dependent on the androgen-induced increase in the acidic protein binding of polyamines and their translocation to a functional cellular site such as nuclear chromatin.In the prostate cytosol, spermine also binds to 4-S tRNAs and to a unique RNA which has a sedimentation coefficient of 1.5 S.     

Studies on the Regulation, Subunit Structure, and some Properties of NAD-Specific Glutamate Dehydrogenase of Neurospora

Some studies on the induction of NAD-specific glutamate dehydrogenase(GDH) of Neurospora are presented. A method for purification,yielding a homogeneous protein, is reported. NAD-GDH is proposedto be a hexamer, with a molecular weight of 320000±7000,sedimentation coefficient of 14.7 S, and a Stokes' radius of(60.9±0.4) x 10^–8 cm. The protomer resulting fromguanidine hydrochloride treatment is catalytically inactive,with an apparent sedimentation coefficient of 4.1 S. NAD-GDHis a very unstable enzyme, the inactivation being time- andprotein-concentration-dependent. It is stabilized by high concentrationsof potassium phosphate buffer and by the presence of sulphydrylgroups in the environment.     

The binding of ribosomal protein S4 does not change the gross conformation of the 16 S RNA.

The binding of ribosomal protein S4 to the 16 S RNA does not result in a large shape or conformational change in the 16 S RNA under the conditions of reconstitution. The sedimentation coefficient, frictional coefficient ratio, and effective hydrodynamic radius of the 16 S RNA.protein S4 complex are very similar to those obtained for the 16 S RNA free in solution. Only subtle conformational differences were obtained in the comparison of the complex and free 16 S RNA by circular dichroism. Thus, extensive organization of the 16 S RNA by ribosomal protein S4 is not a step in the process of self-assembly of the 30 S subunit.     

Defective assembly of the mitochondrial ribosomes in yeast cells grown in the presence of mitochondrial protein synthesis inhibitors

The involvement of mitochondrial protein synthesis in the assembly of the mitochondrial ribosomes was investigated by studying the extent to which the assembly process can proceed in the presence of mitochondrial protein synthesis inhibitors erythromycin and chloramphenicol. Yeast cells grown in the presence of erythromycin (2 mg/ml) do not appear to contain any detectable amounts of the mitochondrial small (37 S) ribosomal subunit. Instead, a ribonucleoparticle with a sedimentation coefficient of 30 S was observed; this particle could be shown to be related to the mitochondrial small ribosomal subunit by two-dimensional gel electrophoretic analysis of its protein components. Since the var1 protein is the only mitochondrial translation product known to be associated with the mitochondrial ribosome, our results suggest that this protein is essential for the assembly of the mature small subunit, and that the var1 protein enters the pathway for the assembly of the small subunit at a late step. In at least one strain of yeast the accumulation of the 30-S particle appears to be very sensitive to catabolite repression. When yeast cells are grown in the presence of chloramphenicol instead of erythromycin, assembly of the small subunit appears to be only partially inhibited, and the presence of the 30-S particle could not be clearly demonstrated. This observation is consistent with the fact that in yeast, chloramphenicol inhibits mitochondrial protein synthesis by about 95% only and that the synthesis of the var1 protein appears to be the least sensitive to this inhibition.     

Physical characteristics of ribosomal protein S4 from Escherichia coli

A hydrodynamic study of protein S4 from Escherichia coli 30 S ribosomal subunits indicates that this protein is moderately asymmetric. A sedimentation coefficient of 1.69 S and a diffusion coefficient of 7.58 X 10(-7) cm2/s suggest that S4 has an axial ratio of about 5:1 using a prolate ellipsoidal model. This structure should give a radius of gyration of about 29-30 A from small-angle neutron or small-angle x-ray scattering studies. This study has utilized quasi-elastic light scattering as an analytical tool to obtain a diffusion coefficient as well as a method to monitor sample quality. Using quasi-elastic light scattering in this manner allows an assessment of problems associated with protein purity which may be responsible for the many disparate results reported for ribosomal proteins and especially protein S4.     

Properties of a purified nuclear topoisomerase from L1210 cells

A nuclear type I topoisomerase from mouse leukemia L1210 cells has been partially purified and characterized. The sedimentation coefficient of the enzyme by velocity sedimentation is 4.3 S, consistent with a globular protein of 68 kDa. Enzyme activity is stimulated 20-fold in the presence of magnesium over that achieved in KCl alone. The enzyme is completely inhibited in the presence of the berenil congeners HOE 13548 and 15030 while berenil itself caused only partial inhibition at concentrations below 200 micrograms/ml. An acid soluble protein of 30 kDa (by SDS-polyacrylamide gel electrophoresis) co-purified with the topoisomerase but could be separated by precipitation in a low salt buffer. This protein, as well as a protein of similar characteristics, histone H1, stimulated topoisomerase activity over a narrow concentration range. The role of topoisomerase in the DNA strand scission observed in L1210 cells following exposure to intercalating agents remains conjectural as the purified enzyme did not produce nicks in plasmid DNA in the presence of adriamycin.     

Phosphorylation of the androgen receptor by a nuclear cAMP-independent protein kinase

The androgen receptor was purified from rat ventral prostate. The purified receptor migrated as a single band of mol. wt. 87000 on SDS-polyacrylamide gels, had a kd for R-1881 (17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one) binding as 6 nM, and sedimentation coefficient of 4.5 S. Phosphorylation of the purified receptor was studied by incubating it with [gamma-32P]ATP in the presence of several purified protein kinases including cAMP-dependent protein kinase, and four cAMP-independent protein kinases (which were active towards substrates such as phosvitin and casein). Phosphorylation of the 87000 mol. wt. androgen receptor protein occurred only in the presence of a nuclear cAMP-independent protein kinase (of the N2 type). No auto-phosphorylation of the receptor was detected. The results indicate that the androgen receptor is a phosphoprotein. Further, phosphorylation of the androgen receptor by only a specific nuclear cAMP-independent protein kinase may be important in determining the dynamics of its function.     

Chickpea seed proteins: Modification during germination

Changes in the proteins of chickpea during a 12-day germination period are reported using techniques of gel filtration, DEAE-cellulose chromatography, polyacrylamide gel (PAG) electrophoresis and ultracentrifugation. In the ultracentrifuge, the total proteins of dormant seeds resolve into 3 components which have the sedimentation coefficients of 2.2 S, 6.9 S and 10.3 S respectively. On germination, the presence of fractions of lower sedimentation coefficient indicates possible degradation of these components; in the early stages, the degradation rate of the 7 S fraction is higher, while the 10 S fraction is broken down faster in the later stages. Gel filtration experiments indicate the possibility of degradation of high polymer into intermediary products. Increase in the relative mobility of protein components on PAG and elution constant on DEAE-cellulose chromatographs indicates an increase in the net negative charge of the protein fractions. The accumulation of subunits of the proteins is negligible during the germination period.     

Effects of Ca2+ and Zn2+ on trifluoperazine-S100 proteins interactions: induced circular dichroism and fluorescence spectra

Interactions of trifluoperazine (TFP) with S100 proteins, EF-hand type Ca2+-binding proteins, in the presence of Ca2+ and Zn2+ were studied with induced circular dichroism (CD) and fluorescence spectra. The positive CD bands of TFP were induced at around 265 nm by adding either S100a or S100a0 protein in the presence of Ca2+. No CD band of TFP was, however, induced by adding S100b protein in the presence of Ca2+. Addition of Zn2+ to the TFP/S100 protein solutions did not induce any CD band at all. The fluorescence intensity of 2-p-toluidinylnaphthalene 6-sulfonate (TNS) bound to S100a or S100a0 protein decreased by adding TFP in the presence of Ca2+, while that bound to S100b protein decreased by adding TFP in the presence of Zn2+, indicating that TFP binds to S100a protein and S100a0 protein in a Ca2+-dependent manner and to S100b protein in a Zn2+-dependent manner. From these results together with other experimental findings it was suggested that (1) TFP binds to S100a protein and S100a0 protein in the presence of Ca2+, with half-saturation points of 18 and 3 microM, respectively, (2) TFP binds to S100b protein only in the presence of Zn2+, (3) alpha-subunit of S100 protein binds to TFP specifically in a Ca2+-dependent manner and beta-subunit in a Zn2+-dependent manner.     

Limited proteolysis of the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The hydrogenase from Paracoccus denitrificans, which is an intrinsic membrane protein, has been solubilised from membranes by Triton X-100. The partial specific volume of the solubilised protein has been determined using sucrose density gradient centrifugation in H2O and 2H2O. The values of the specific volumes of hydrogenase, measured in the presence or absence of Triton X-100, are 0.73 and 0.74 ml . g-1, respectively, indicating that hydrogenase binds much less than one micelle of Triton X-100. The sedimentation coefficient of hydrogenase is increased from 10.4 S to 15.9 S on removal of detergent. The Stokes' radius of hydrogenase, determined by gel filtration on Sepharose 6B, is 5.5 nm in the presence of Triton X-100 compared to 6.7 nm in the absence of detergent. The apparent molecular weight therefore increases from 242,500 to 466,000 on removal of detergent. In the presence of urea and sodium dodecylsulphate, the hydrogenase has an apparent molecular weight of 63,000. The enzyme therefore behaves as a non-covalently linked tetramer in the presence of Triton X-100. Removal of Triton X-100 results in association of tetramers to form octamers.     

Poly a blocks in the nuclear ribonucleoprotein complexes,containing pre-mRNA

Poly A was found in nuclear particles containing pre-mRNA. It was shown that during the isolation of 30S particles from rat liver or Ehrlich ascites carcinoma nuclei, all poly A is detached from the particles containing pre-mRNA and is found in the form of RNP with a sedimentation coefficient of about 14S. When RNP polyparticles are isolated in the presence of RNase inhibitor poly A is distributed among the particles of higher molecular weights.Since the sedimentation properties and buoyant density of the poly A-containing particles are different from the 30S particles it was suggested that the poly A fragments are bound not with informofers, but with another kind of protein.     

Isolation and characterization of the Escherichia coli mutL gene product

The Escherichia coli mutL gene product has been purified to near homogeneity from an overproducing clone. The mutL locus encodes a polypeptide of 70,000 daltons as determined by denaturing gel electrophoresis. The native molecular weight of MutL protein as calculated from the sedimentation coefficient of 5.5 S and Stokes radius of 61 A is 139,000 daltons, indicating that MutL exists as a dimer in solution. In addition to its ability to complement methyl-directed DNA mismatch repair in mutL-deficient cell-free extracts, DNase I protection experiments demonstrate that the purified MutL protein interacts with the MutS-heteroduplex DNA complex in the presence of ATP.