Salt-stable binding of the large T protein to DNA in polyoma virus chromatin.

Nucleoprotein complexes extracted from the nuclei of mouse cells lytically infected with polyoma virus contain an ATPase activity which appears to correspond to that of the viral large T protein, as it exhibits the same characteristic properties; in particular, the activity is extensively inhibited by polyclonal antibodies from animals bearing polyoma tumors (anti-T antigen antibodies) and by monoclonal antibodies against large T. Significant amounts of DNA were immunoprecipitated by adding these antibodies to the nucleoprotein complex, suggesting that the protein is tightly bound to DNA in the viral chromatin. Since one of the monoclonal antibodies quantitatively immunoprecipitated the pulse-labeled replicative intermediates, we conclude that some large T protein remains physically associated with the DNA throughout its replication cycle. After exposure to salt concentrations higher than 1 M KCl, about half of the large T-specific ATPase activity was still observed to co-sediment with 21S form I viral DNA. The observations that the sedimentation coefficient of the salt-stable complexes was shifted to 16S after a limited endonucleolytic digestion, and that both the viral DNA and the ATPase activity were co-precipitated in the presence of polyethylene glycol at high ionic strength, further demonstrated that the protein is engaged in an unusually stable complex with DNA in the viral chromatin.     

Effect of active immunization against 17 beta-estradiol on cytosolic estrogen receptors in the rat uterus

Following active immunization of female rats against estradiol-17 beta, the amount of specific binding sites for estrogen decreased in uterine cytosol as a function of antiserum titres. They were undetected when antibodies titres were higher than 1/2000. Moreover, a binding protein specific for estradiol-17 beta appeared. Estradiol binding was not displaced with an excess of unlabeled DES nor precipitated with protamine sulfate. The sedimentation coefficient of the hormone-protein complex (7-8 S) was not modified in medium of high ionic strength (0.4 M KCl). That protein represented antibodies to Estradiol-17 beta which could be precipitated with antiserum to rat IgG.     

The denaturation-renaturation of chicken-muscle triosephosphate isomerase in guanidinium chloride

1. The process of denaturation of the chicken muscle dimeric enzyme triosephosphate isomerase on addition of guanidinium chloride has been studied at pH 7.6, the pH at which the recovery of activity is optimal (100%) on removal of denaturant. Determinations of the sedimentation coefficient, intrinsic viscosity, molecular weight (by sedimentation equilibrium studies) and the absorption coefficient at 280 nm in various concentrations of guanidinium chloride concurred in showing a single, sharp transition at about 0.7 M guanidinium chloride at a protein concentration 1-5 mg/ml from the native enzyme to the dissociated, unfolded chains of the monomer. Relative fluorescent intensity measurements revealed a single transition at about 0.4 M guanidinium chloride at enzyme concentrations of about 0.05 mg/ml. 2. The process of denaturation in different guanidinium chloride concentrations was first order with respect to enzyme and about sixth order with respect to denaturant. 3. The rate of attainment of equilibrium during the renaturation obeyed second-order/first-order reversible kinetics. It was concluded that the rate-determining step in renaturation at pH 7.6 must be the association of two subunits.     

Ribonucleic acid is a component of the oligomeric, transformed mouse AtT-20 cell glucocorticoid receptor

The glucocorticoid receptor from mouse AtT-20 pituitary tumor cells exists in three forms. The largest form is an untransformed (non-DNA-binding), oligomeric species (9.1 S, 8.3 nm, Mr 319 000). Two transformed (DNA-binding) forms can be generated. One is an oligomeric protein (5.2 S, 6-8.3 nm, Mr 132 000-182 000), while the other is the monomeric, hormone-binding subunit (3.8 S, 6 nm, Mr 96 000). The composition of the oligomeric, transformed receptor and its relationship to the monomeric protein were examined. The 3.8S monomer can be isolated from DEAE-cellulose (0.12 M step elution) in a form that continues to sediment at about 3.8 S on molybdate-containing sucrose gradients and at about 4.2 S on molybdate-free gradients. Addition of a non-hormone-binding component isolated from the same DEAE-cellulose column (0.5 M KCl step) can apparently interact with the 3.8-4.2 S monomer, increasing its sedimentation coefficient to 5.2 S (on molybdate-containing gradients) or 6.6 S (on low-salt, molybdate-free gradients). This factor is a macromolecule (nondialyzable) and is heat-stable (100 degrees C, 20 min). A dose-dependent shift to the higher sedimentation coefficient is observed when increasing quantities of the 0.5 M step material are added to the receptor monomer. This activity is abolished when the 0.5 M step material is treated with ribonuclease A. Further, when RNA is purified from the 0.5 M step by phenol/chloroform extraction, its ability to increase the S value of the monomer is retained. Ribonuclease treatment of the untransformed, 9.1S, oligomeric complex does not cause a significant decrease in sedimentation rate, while the same treatment of the 5.2S, oligomeric, transformed receptor (obtained after Sephadex G-25 transformation) causes a decrease in sedimentation rate to about 3.8 S. The addition of bovine liver mRNA and rRNA does not cause a shift in sedimentation rate of the receptor monomer to a discrete, higher sedimenting receptor form. However, the addition of total rabbit liver tRNA or three distinct tRNA species causes a shift in sedimentation to a similar, but not identical, form as that with the 0.5 M step material. We propose that the 5.2S, oligomeric transformed glucocorticoid receptor is composed of one monomeric hormone-binding, protein subunit (Mr 96 000) and a low molecular weight RNA (Mr 36 000). This interaction may be important for the role of the receptor in regulating gene expression.     

The inactivation of the bovine J blood group substance by the periodate ion

1. Treatment of J-positive (Jcs) bovine erythrocytes with periodate (0.25 mmol/l final concentration, 1 hour, room temperature) has no effect on the J activity. Higher periodate concentrations cause spontaneous haemolyses. 2. Treatment of the lipids extracted from (and containing all J activity of) Jcs erythrocytes with periodate leads to a decrease of J activity even with lower periodate concentrations. 3. Treatment of the stroma prepared from Jcs erythrocytes with periodate demonstrated the relative stability of the J antigen up to 0.25 mmol/l periodate. At the same time the sialic acid concentration of stroma is reduced to about 13% of the initial concentration. 4. Desialylation of Jcs erythrocytes or Jcs stroma with sialidase does not affect the J activity thus confirming previous findings. On the other hand, the J activity of desialylated Jcs stroma is much more susceptible to periodate. 5. It is concluded that membrane-bound sialic acid shields the membrane-bound J antigen from being attacked by periodate.     

Developmental changes and acetylcholinesterase activity in the metamorphosing brain of Tenebrio molitor: Correlation to ecdysteroid titers

The brain of Tenebrio molitor exhibited marked fluctuations in acetylcholinesterase (AChE) activity throughout metamorphosis. This was true AChE activity, since it was inhibited by high substrate concentrations and by 10 μM of the specific AChE inhibitor BW284C51 [(1,5-bis'4-allyldimethylammoniumphenyl)-pentan-3-one dibromide] but not by iso-OMPA (tetraisopropylpyrophosphoramide), a cholinesterase (but not AChE) inhibitor. The histochemical AChE activity was localized in the neuropile and the nuclear envelope of neurons and glial cells. The enzyme extracted from brains with 1% Triton X-100 and 1 M NaCl sedimented as a single peak in a sucrose density gradient, with a sedimentation coefficient of 5.4S. This single AChE sedimentation peak was mainly due to an amphiphilic dimeric form. AChE activity per brain increased in newly ecdysed pupa. AChE activity per milligram of protein exhibited a peak in the mid-pupa which could be correlated to the increase in ecdysteroid titers. © 1994 Wiley-Liss, Inc.     

Nuclear envelope glycoprotein with poly(A) polymerase activity of rat liver: isolation, characterization, and immunohistochemical localization

A protein with poly(A) polymerase activity has been identified and isolated from hepatic nuclear envelopes of rats to near homogeneity. The ability of the enzyme to bind to concanavalin A-agarose and to be eluted from the column with methyl alpha-D-mannopyranoside (0.2 M) as well as the inhibitory effects of alpha-mannosidase suggested that it was a glycoprotein. Poly(A) polymerase has an absolute requirement for a divalent cation, ATP, and an oligonucleotide primer. The enzyme activity with Mn2+ was about 20-fold higher than that with Mg2+. Several known inhibitors adversely affected poly(A) polymerase activity. The enzyme has a molecular weight of 64,000 when analyzed by polyacrylamide gel electrophoresis under denaturing conditions and has a sedimentation coefficient of 4.5 S. Immunohistochemical studies using polyclonal antibodies raised against the purified enzyme revealed that the antigen was localized in the nuclear membranes.     

Sedimentation velocity of sodium hyaluronate-lysozyme mixtures

A study of the sedimentation behaviour of lysozyme in sodium hyaluronate (Na-HA) solution and of the Na-HA medium itself, has been carried out to determine whether the strongly basic enzyme lysozyme forms complexes with Na-HA at physiological ionic strength. At typical physiological salt concentration, 0.146 m NaCl, and also in 0.100 M NaCl, lysozyme sedimentation in an Na-HA solution can be adequately described as independent sedimentation of a slightly associated protein through a three-dimensional network acting partially as a macromolecular sieve. The s_20,w of lysozyme when determined in 0.146 M NaCl, indicated partial aggregation of the enzyme at this salt concentration. Decreases in sedimentation coefficients of lysozyme with increase in Na-HA concentration show a pronounced sieving effect by the equality of observed sedimentation coefficient of lysozyme and Na-HA at higher Na-HA concentrations, but typically individual sedimentation coefficients when the macromolecular mixture was diluted approximately ten-fold.     

Salt-induced structural changes in nucleosomes

Ehrlich ascites tumor (EAT) nucleosomes treated with increasing NaCl concentrations were analyzed by sucrose density gradient centrifugation. Two events were found to take place in the course of the salt treatment: a) increasing amounts of nucleosomes dissociated into free DNA and protein in the interval 0.6M–1.5M NaCl, and b) the sedimentation coefficient of the nucleosomes decreased from 11S to 8S in the interval 0.6M-1M NaCl. This decrease was not caused by loss of protein and was fully reversible upon slow and gradual lowering of the ionic strength. This shows that before dissociation of the protein core from DNA, nucleosomes undergo a structural transition. The electron microscopic observations revealed that it consisted in detachment of the ends of nucleosomal DNA from the protein core. It is suggested that an arginine-rich domain in the protein core exists, which holds more tightly the central part of the nucleosomal DNA, while its ends are relatively loosely bound to lysine-rich domains.     

Evidence for similar conformational changes in α2-macroglobulin on reaction with primary amines or proteolytic enzymes

Reactions of alpha(2)-macroglobulin (alpha(2)M) with primary amines (ammonium chloride, methylammonium chloride and ethylammonium chloride) or proteolytic enzymes (trypsin, chymotrypsin and thrombin) resulted in changes of the absorption, fluorescence and circular-dichroism spectra and of the sedimentation coefficient of the inhibitor. All physico-chemical changes caused by the inactivation of alpha(2)M by the amines were identical with, or highly similar to, those induced by the formation of the enzyme-inhibitor complexes. This suggests that similar conformational changes of the inhibitor occur in the two types of reactions. The frictional ratio, calculated from the increase in sedimentation coefficient, decreased from 1.67 for untreated alpha(2)M to 1.57 for the amine- or proteinase-treated inhibitor. This change is due to a decrease in either asymmetry or hydration of the protein, resulting in a slightly smaller hydrodynamic volume. The circular-dichroism analyses indicated that the reaction of alpha(2)M with either amines or proteinases is accompanied by a loss of the small amount (about 5%) of alpha-helix of the untreated protein. The changes of u.v. absorption and fluorescence suggested that about one out of the eight to ten tryptophan residues of each alpha(2)M subunit is buried as a result of the conformational change. All spectroscopic and hydrodynamic changes that were observed are compatible with a spatial rearrangement of the subunits of alpha(2)M, as implicated by the ;trap' hypothesis for the mechanism of inhibition of proteinases. However, a conformational change involving a decrease in the hydrodynamic volume of each subunit cannot be excluded.     

Protein kinases of the chick oviduct: a study of the cytoplasmic and nuclear enzymes.

Subcellular fractionation of oviduct tissue from estrogen-treated chicks indicated that the bulk of the protein kinase activity of this tissue is located in the cytoplasmic and nuclear fractions, DEAE-cellulose chromatography of cytosol revealed a major peak of cAMP stimulatable activity eluting at 0.2 M KCl. This peak was further characterized and found to exhibit properties consistent with cytoplasmic cAMP dependent protein kinases isolated from other tissues; it had a Km for ATP of 2 X 10(-5) M, preferred basic proteins such as histones, as substrate, and had a M of 165 000. Addition of 10(-6) M cAMP caused the holoenzyme to dissociate into cAMP binding regulatory subunit and a protein kinase catalytic subunit. Extraction of purified oviduct nuclei with 0.3 M KCl released greater than 80% of the kinase activity in this fraction. Upon elution from phospho-cellulose, the nuclear extract was resolved into two equal peaks of kinase activity (designated I and II). Peak I had a sedimentation coefficient of 3S and a Km for ATP of 13 muM. while peak II had a sedimentation coefficient of 6S and a Km for ATP of 9 muM. Both enzymes preferred alpha-casein as a substrate over phosvitin or whole histone, although they exhibited different salt-activity profiles. The cytoplasmic and nuclear enzymes were well separated on phospho-cellulose and this resin was used to quantitate the amount of cAMP dependent histone kinase activity in the nucleus and the amount of casein kinase activity in the cytosol. Protein kinase activity in nuclei from estrogen-stimulated chicks was found to be 40% greater than hormone-withdrawn animals. This increase in activity was not due to translocation of the cytoplasmic protein kinase in response to hormone, but to an increase in nuclear (casein) kinase activity. During the course of this work, we observed small but significant amounts of cAMP binding activity very tightly bound to the nuclear fraction. Solubilization of the binding activity by sonication in high salt allowed comparison studies to be performed which indicated that the nuclear binding protein is identical with the cytoplasmic cAMP binding regulatory subunit. The possible role of the nuclear binding activity is discussed.     

The renaturation of reduced chymotrypsinogen A in guanidine HCl. Refolding versus aggregation

The refolding and reoxidation of fully reduced and denatured chymotrypsinogen A have been studied in the presence of low concentrations of guanidine HCl or urea. Renaturation yields of 60 to 70% were observed when the reoxidation was facilitated by mixtures of reduced and oxidized glutathione. Refolding occurred within a narrow range of denaturant concentration (1.0 to 1.3 M guanidine HCl and 2 M urea) in which the native protein was shown to be stable, and the reduced protein was shown to regain the correct disulfide pairing. Renatured chymotrypsinogen is indistinguishable from the native zymogen in chromatographic behavior, potential chymotryptic activity, sedimentation coefficient, and spectral properties. The kinetics of renaturation were determined. Some of the protein species obtained at various times of renaturation were characterized as incorrectly oxidized molecules which could be renatured by thiol-catalyzed interchange of disulfide bonds.     

The inactivation of the bovine J blood group substance by the periodate ion

• 1 Treatment of J-positive (JR) bovine erythrocytes with periodate (0.25 mmol/1 final concentration, 1 hour, room temperature) has no effect on the J activity. Higher periodate concentrations cause spontaneous haemolyses.
• 2 Treatment of the lipids extracted from (and containing all J activity of) J^cs erythrocytes with periodate leads to a decrease of J activity even with lower periodate concentrations.
• 3 Treatment of the stroma prepared from J^cs erythrocytes with periodate demonstrated the relative stability of the J antigen up to 0.25 mmol/l periodate. At the same time the sialic acid concentration of stroma is reduced to about 13 % of the initial concentration.
• 4 Desialylation of J^cs erythrocytes or J^cs stroma with sialidase does not affect the J activity thus confirming previous findings. On the other hand, the J activity of desialylated J^cs stroma is much more susceptible to periodate.
• 5 It is concluded that membrane-bound sialic acid shields the membrane-bound J antigen from being attacked by periodate.

     

The subunit structure of Pseudomonas cytochrome oxidase.

Pseudomonas cytochrome oxidase (EC 1.9.3.2) is composed of two subunits. Each subunit has a molecular weight of approx. 63000 and, according to the iron determination, contains two hemes. Cytochrome oxidase was subjected to various dissociation procedures to determine the stability of the dimeric structure. Progressive succinylation of 14 to 68% of the lysine residues of the enzyme increases the amount of the protein appearing in the subunit form (S20,W approximately 4 S) from 18 to 92%. At a high degree of succinylation a component with a sedimentation coefficient of approx. 2 S appears. The subunits with sedimentation coefficients of approx. 4 S and 2 S are also formed when the pH is below 4 or above 11. The same molecular weight (63000) was found for these two components in sodium dodecylsulphate electrophoresis. No dissociation of cytochrome oxidase was observed in salt solutions like 3 M NaC1 and 1 M Na2SO4, or in 6 M urea. The slight decrease in the sedimentation coefficients in NaC1 solutions is partly explained by preferential hydratation of the protein.     

Analysis of the effect of periodate oxidation and phenol extraction on the antitumour properties of C. parvum

Summary The antitumour activity of C. parvum and its relationship to spleen weight increase have been analysed in a tumour model using syngeneic M₄ fibrosarcoma cells. It was found that treatment of C. parvum with low concentrations of periodate (2 mM) produced a minor decrease in the ability to abrogate tumour growth, following intratumour injection, although the capacity to increase spleen weight after IP injection was abolished. Higher concentrations of periodate (20 mM) produced complete inactivation of the bacteria. A phenol extract of C. parvum was weakly active in the intratumour test but amounts up to 1 mg injected IP were unable to increase the spleen weight. The data support the hypothesis that a surface carbohydrate is responsible for the increase in spleen weight. It is suggested that the antitumour activity may reside in a molecule that also reacts with periodate but at a slower rate, perhaps because of a more complex structure or a different location in the cell, for instance in the cytoplasm. The phenol extract was soluble in chloroform and at least two major components were glycolipids, so it is conceivable that a molecule of this type, which could be degraded by periodate, might be responsible for the antitumour activity of C. parvum. The precise biochemical nature of the active component is, as yet, undetermined.     

The effect of ribonuclease on rat-liver ribosomes

1. Rat-liver ribosomes lose about 50% of their amino acid-incorporating activity when preincubated with ribonuclease. 2. This preincubation results also in loss of about 50% of the original protein content and 75% of the RNA. 3. Ribosomes sedimented by ultracentrifugation, after preincubation with ribonuclease, show negligible contamination by crystalline enzyme. 4. Washing of ribosomes treated with ribonuclease releases further protein, restoring the original RNA/protein ratio. 5. The washed particle is again capable of promoting amino acid incorporation. 6. Examination of ribosomes treated with ribonuclease in the analytical ultracentrifuge reveals destruction of ribosomes, disappearance of dimers and a decrease in the sedimentation coefficient of monomers. 7. Washed ribosomes consist of even smaller particles with a sedimentation coefficient 60s.     

Purification and properties of the membrane-bound form of acetylcholinesterase from chicken brain. Evidence for two distinct polypeptide chains

The major molecular form of acetylcholinesterase (AChE) from chicken brain is a membrane-bound glycoprotein with an apparent sedimentation coefficient of 11.4 S. Analysis of the purified protein by gel filtration, velocity sedimentation, and sodium dodecyl sulfate-gel electrophoresis shows that the solubilized enzyme is a globular tetramer with an apparent Mr = 420,000. This membrane-bound form of AChE is hydrophobic and readily aggregates in the absence of detergent. These aggregates are concentration-dependent, relatively stable in the presence of high salt concentrations, yet readily dissociate upon addition of detergent to the 11.4 S form, indicating that the interactions are hydrophobic. Polyclonal and monoclonal antibodies raised against chicken brain AChE purified by ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis precipitate AChE enzyme activity. However, these antibodies do not cross-react with the enzyme from chicken muscle which preferentially hydrolyses butyrylcholine. Immunoprecipitation of isotopically labeled enzyme molecules from tissue cultured brain cells and analysis by sodium dodecyl sulfate-gel electrophoresis shows that AChE consists of two polypeptide chains with apparent Mr = 105,000 (alpha) and 100,000 (beta) in a 1:1 ratio. Immunoblotting of brain AChE with either the polyclonal or monoclonal antibodies indicates that the alpha and beta chains share antigenic determinants. Furthermore, both polypeptide chains can be labeled with [3H]diisopropyl fluorophosphate, indicating that they each contain a catalytic site. This is the first indication that globular forms of AChE may consist of multiple polypeptide chains.     

An ultracentrifugal study of the self-association of canine apolipoprotein A-I in solution.

The sedimentation behavior of canine apolipoprotein (apo) A-I in 0.02 M EDTA, pH 8.6, was studied as a function of protein concentration by the techniques of sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge. At concentrations of less than 1 g/liter, apo-A-I exhibited a monomodal sedimentation pattern, with apparent sedimentation coefficients which varied from 2.3 to 3.5 S with increasing protein concentrations. Above 1.5 g/liter, apo-A-I had two well resolved peaks with s20,w values of 4.15 S and 5.75 S. The proportion of the 5.75 S component increased with increasing apo-A-I concentrations, with a concomitant decrease of the 4.15 S component. By sedimentation equilibrium ultracentrifugation with both the conventional and meniscus-depletion methods, the apparent weight-average molecular weight of apo-A-I was found to be concentration-dependent. At a protein concentration of 5.25 g/liter, an apparent weight average molecular weight of 138,000 was determined, indicating that molecular species larger than a tetramer (monomer molecular weight = 28,000) were present in solution. When analyzed in terms of a reversible self-associating system, the experimental data could best be described according to a monomer-dimer-tetramer-octamer model, as previously reported from human apo-A-I (Vitello, L. B., and Scanu, A. M. (1975) J. Biol. Chem. 251, 1131-1136). The equilibrium constants were: K2 = 4.5 liters/g, K4 = 470 liters3/g3, and K8 = 41,600 liters7/g7, respectively.     

Salt-dependent interconversion of inner histone oligomers.

The inner histone complex, extracted from chicken erythrocyte chromatin in 2 M NaCL AT pH 7.4, has been characterized by sedimentation equilibrium and sedimentation velocity. High speed sedimentation equilibrium studies indicate that in 2 M NaCl the inner histones are a weakly associating system with contributions from species ranging in molecular weight from dimer to octamer. The appearance of a single boundary (3.8S at 2 M NaCl) in sedimentation velocity studies conducted over a wide range of protein concentrations and ionic conditions indicates that the various histone oligomers present are in rapid equilibrium with one another. At higher salts the equilibrium is shifted to favor higher molecular weight species; in 4 M NaCl essentially all of the histone is octameric at protein concentrations above 0.2 mg/ml. The facile interconversion of histone oligomers suggests that small alterations in histone-histone interactions may be responsible for changes in nucleosome conformations during various biological processes.     

Reaction of yeast phosphofructokinase with succinic and maleic anhydride.

Modification of yeast phosphofructokinase by succinic and maleic anhydride influences the catalytic activity and the allosteric behaviour of the enzyme. Depending on the degree of succinylation and maleinylation a decrease of maximum activity, an increase of the apparent affinity for fructose-6-phosphate, a decrease of the Hill-coefficient and a diminution of ATP-inhibition are observed. Up to about 40% of the lysyl residues could be succinylated without dissociation of the hexameric protein, however with a decrease of the enzyme activity. More extensive succinylation or maleinylation causes a dissociation into subunits. The sedimentation coefficient is lowered from 20 S to about 3 S. The molecular weight of the smallest dissociation product was determined to 50 000 (+/- 10 000) by the sedimentation equilibrium method. The number of bound succinyl groups, as determined from radioactivity incorporation, exceeds the content of lysyl groups of the enzyme, indicating that the modifying reagent is also reacting with other amino acid residues.