High Concentrations Of Ammonium Sulfate Research Articles

An investigation of the role of Glu-842, Glu-844 and His-846 in the function of the cytoplasmic domain of the epidermal growth factor receptor.

Activation of several protein kinases is mediated, at least in part, by phosphorylation of conserved Thr or Tyr residues located in a variable loop region, near the active site. In certain kinases, this activation loop also controls access of peptide substrates to the active site. In the corresponding region of the epidermal growth factor (EGF) receptor, a potential phosphorylation site, Tyr-845, does not appear to have a major regulatory role. In order to find out whether this variable loop can modulate the peptide phosphorylation and self-phosphorylation activities of the EGF receptor kinase, we investigated the role of residues around Tyr-845, using site-directed mutagenesis. Multiple sequence alignment showed that residues Glu-842, Glu-844 and His-846 are conserved or nearly conserved in eight members of the EGF receptor family. Mutants Glu-842-->Ser, Glu-844-->Gln and His-846-->Ala were expressed in the baculovirus/insect cell system, purified to near-homogeneity and characterized with respect to their peptide phosphorylation and self-phosphorylation activities. All three mutants were active, and these changes did not affect ATP binding directly. However, all mutations increased the Km(app.) for peptide substrates and MnATP in peptide phosphorylation reactions. The Vmax. for the phosphorylation of peptide RREELQDDYEDD was unaltered, but the Vmax. for self-phosphorylation (with variable [MnATP]) decreased 4-, 2- and 7-fold for mutants Glu-842-->Ser, Glu-844-->Gln and His-846-->Ala respectively, compared with the wild-type. These results suggest that binding of this peptide restored an optimal conformation at the active site that might be impaired by the mutations. A study of the dependence of initial rates of self-phosphorylation on cytoplasmic domain concentration showed that the order of reaction increased with the progress of self-phosphorylation. Both pre-phosphorylation and high concentrations of ammonium sulphate restored maximal or near-maximal levels of self-phosphorylation in the mutants, possibly through compensating conformational changes. A plausible homology model, based on the cyclic AMP-dependent protein kinase catalytic subunit, accommodated the sequence Glu-841-Glu-Lys-Glu as an insertion in the peptide binding loop at the edge of the active site cleft. The model suggests that Glu-844 and His-846 may participate in H-bonding interactions, thus stabilizing the active site region, while Glu-842 does not appear to interact with regions of the catalytic core.

Further characterization of the interaction between the Epstein-Barr virus DNA polymerase catalytic subunit and its accessory subunit with regard to the 3'-to-5' exonucleolytic activity and stability of initiation complex at primer terminus.

The Epstein-Barr virus (EBV) DNA polymerase catalytic subunit, BALF5 gene product, possesses an intrinsic 3'-to 5' proofreading exonuclease activity in addition to 5'-to-3' DNA polymerase activity (T. Tsurumi, A. Kobayashi, K. Tamai, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:4651-4658, 1993). The exonuclease hydrolyzed both double-and single-stranded DNA substrates with 3'-to-5' directionality, releasing deoxyribonucleoside 5'-monophosphates. The double-strand exonucleolytic activity catalyzed by the BALF5 polymerase catalytic subunit was very sensitive to high ionic strength, whereas the single-strand exonucleolytic activity was moderately resistant. The addition of the BMRF1 polymerase accessory subunit to the reaction enhanced the double-strand exonucleolytic activity in the presence of high concentrations of ammonium sulfate (fourfold stimulation at 75 mM ammonium sulfate). Optimal stimulation was obtained when the molar ratio of BMRF1 protein to BALF5 protein was 2 and higher, identical to the values required for reconstituting the optimum DNA polymerizing activity (T. Tsurumi, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:7648-7653, 1993). Furthermore, product size analyses revealed that the polymerase catalytic subunit alone excised a few nucleotides from the 3' termini of the primer hybridized to template DNA and that the addition of the BMFR1 polymerase accessory subunit stimulated the nucleotide excision several times. In contrast, the hydrolysis of single-stranded DNA by the BALF5 protein was not affected by the addition of the BMRF1 polymerase accessory subunit at all. These observations suggest that the BMRF1 polymerase accessory subunit forms a complex with the BALF5 polymerase catalytic subunit to stabilize the interaction of the holoenzyme complex with the 3'-OH end of the primer on the template DNA during exonucleolysis. On the other hand, challenger DNA experiments revealed that the BALF5 polymerase catalytic subunit alone stably binds to the primer terminus in a stationary state, whereas the reconstituted polymerase holoenzyme is unstable. The instability of the initiation complex of the EBV DNA polymerase would allow the rapid removal of the EBV DNA polymerase holoenzyme from the lagging strand after it has replicated up to the previous Okazaki fragment. This feature of the EBV DNA polymerase holoenzyme in a stationary state is in marked contrast to the moving holoenzyme complex tightly bound to the primer end during polymerization and exonucleolysis.

Use of Adsorption Chromatography on Sephacryl S-500 for Improved Separation of Isoforms of Soluble Photosynthetic Catalysts from Cyanobacteria

Abstract Sephacryl S-500, a gel-filtration matrix, adsorbs water-soluble proteins at high concentrations of ammonium sulfate. It has been used here as a matrix for adsorption chromatography, separating isoforms of cytochrome C553 and ferredoxin from Oscillatoria sp. and Microcystis aeruginosa, respectively. The isoforms of these proteins were separated at 35% and 40% saturated ammonium sulfate, respectively. Using this adsorption chromatography, we have purified high levels of individual isoforms with improved resolution over conventional procedures such as polyacrylamide gel electrophoresis and ion-exchange chromatography.

Lixiviación de fertilizantes de magnesio en Ultisoles de Puerto Rico

Se hicieron seis experimentos en columnas de suelo para determinar la solubilidad y el movimiento vertical de aplicaciones superficiales de sales de Mg en tres Ultisoles típicos de la zona montañosa de Puerto Rico. La lixiviación del Mg proveniente de la aplicación del fue óxido de magnesio mucho más lenta que la de las sales de sulfato de magnesio y sulfato de potasio-magnesio. La adición de sulfato de amonio a la banda de óxido de magnesio aumentó la solubilidad y el movimiento vertical del Mg en la columna de suelo de 15 a 85%. La urea tuvo un efecto mínimo en la solubilidad del óxido de magnesio mientras que el cloruro de potasio y el superfosfato triple fueron inefectivos.

Protein Adsorption of Sephacryl S-Type Gels Using High Concentrations of Ammonium Sulfate

Abstract A variety of proteins adsorbed to Sephacryl gels, S-200, S-500 and S-1000 and Fractogel TSK-HW65(F) in the presence of high concentrations of ammonium sulfate. The adsorption of these proteins to individual gel matrices showed some variations. The strength of adscrption. of cytochrome c, myoglobin and chymotrypsinogen A to Sephacryl S-200 increased with temperature and pH, suggesting that hydrophobic interaction is involved in the adsorption. The chromatographic behaviour of these three proteins on Sephacryl S-type gels and Fractogel varied with the ammonium sulfate concentration of the eluant buffer. At 30% saturation, cytochrome c and myoglobin eluted together and followed by chymotrypsinogen A. At 45% saturation, however cytochrome c eluted before myoglobin and chymotrypsinogen A remained tightly bound to the column. Temperature, pH, flow-rate and glycerol were some of the factors affecting the separation of these three proteins on Sephacryl S-200.

Peptide synthesis by stabilized trypsin: Industrial kinetic studies under extreme experimental conditions

Studies of the kinetics of peptide synthesis catalysed by trypsin derivatives have been carried out. We have studied equilibrium (ECS) and kinetically (KCS) controlled synthesis using benzoyl arginine or benzoyl arginine ethyl ester as acyl donors and various amino acid derivatives as nucleophiles. In both cases, kinetics studies were carried out under conditions of industrial interest: (a) by using very active and very stabilized enzyme derivatives as catalysts, (b) under the extreme experimental conditions necessary for improving the performance of these synthetic processes (high concentrations of apolar organic co-solvents in ECS or high concentrations of ammonium sulfate in KCS), and (c) by using high concentrations of substrates. The hydrophobic adsorption of nucleophile substrates on the active center of the enzyme seems to be the key kinetic step in both of these synthetic processes, as synthetic activity in both ECS and KCS greatly increases with pH and with nucleophile concentration. In ECS, the presence of high concentrations of apolar organic solvents (necessary for achieving important synthetic yields) greatly inhibits the adsorption of nucleophiles, and hence the best performance of these processes would be obtained using a moderate excess of the nucleophile substrate in order to quantitatively convert the acyl donor substrate into synthetic product at the highest reaction rates. In KCS, we found that the use of a ‘salting out’ agent ( e.g., ammonium sulfate) greatly improves the hydrophobic adsorption of nucleophiles. In addition, the presence of ammonium sulfate, perhaps as a ‘water-ordering agent’, promotes a dramatic reduction in the rate of hydrolysis of the acyl-enzyme complex when the active center of each trypsin molecule is partially blocked by the adsorbed nucleophile at saturating concentrations. Thus synthetic/hydrolytic ratios are dramatically improved by the presence of this salt, and consequently synthetic yields approach 100%. By using these two different synthetic strategies we have been able to quantitatively convert arginyl derivatives into dipeptides under extreme conditions in which our stabilized trypsin derivatives are very active and very stable. We have calculated catalyst productivities of 2 and 300 tons of dipeptide per year per liter of our trypsin catalysts by using ECS or KCS synthetic strategies, respectively.

Chromatography of .BETA.-Glucuronidase from Bovine Liver. A Study of the Enzyme Binding Sites of Prepared Adsorbents.

beta-Glucuronidase from bovine liver was adsorbed to the adsorbents prepared with CH-Sepharose 4B and either the competitive inhibitor or its analogs such as p-aminophenyl 1-thio-beta-D-glucuronic acid, -glucoside, -galactoside, and N-acetyl glucosaminide. The adsorbed enzyme was eluted at 0.1 or 0.5 M NaCl by a stepwise gradient. Chromatography of the enzyme was also performed by using the adsorbents prepared with Epoxy-activated Sepharose 6B and amine compounds or other compounds. In order to see whether the hydroxyl groups of the sugar parts in the ligand are necessary for the adsorption of the enzyme, chromatography was performed by using the adsorbents prepared with sugar derivatives as the ligand. As a result, it was found that beta-glucuronidase had an affinity for adsorbents prepared with either acetyl derivatives or methoxy derivatives of glycosides and CH-Sepharose 4B. From the results of elution of the enzyme with NaCl from adsorbents having amide bonding, it was clarified that the affinity of the enzyme for adsorbents without glycosides in the ligands correlated with acidity of the amide in the adsorbents. Hydrogen bond chromatography was performed with the prepared adsorbents. The enzyme was adsorbed under a high concentration of ammonium sulfate, and the elution of the adsorbed enzyme from adsorbents was examined by the degradation of salt. The enzyme was most easily eluted from aminoethyl 1-thio-beta-D-glucuronic acid-CH Sepharose 4B at 0.9 M ammonium sulfate and at 0.5 M concentration of the salt with p-aminophenyl 1-thio-beta-D-glucuronic acid-CH Sepharose 4B.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and function studies of the cGMP-stimulated phosphodiesterase.

Studies of cGMP binding to both the native cyclic GMP-stimulated phosphodiesterase and to two unique isolated chymotryptic fragments lacking the catalytic domain suggest that the enzyme contains two noncatalytic cGMP-binding sites/homodimer. In the presence of high concentrations of ammonium sulfate, 2 mol of cGMP are bound/mol of cGMP-stimulated phosphodiesterase homodimer. Under these conditions, linear Scatchard plots of binding are obtained that give an apparent Kd of approximately 2 microM. The inclusion of 3-isobutyl-1-methylxanthine produces a curvilinear plot. In the absence of ammonium sulfate, the dissociation of cGMP from the holoenzyme is rapid, having a t1/2 of less than 10 s, and addition of ammonium sulfate to the incubation greatly decreases this rate of dissociation. The native enzyme is resistant to degradation by chymotrypsin in the absence of cGMP; however, in its presence, chymotrypsin treatment produces several discrete fragments. Similarly, in the presence but not in the absence of cGMP, dicyclohexylcarbodiimide causes an irreversible activation of the enzyme without cross-linking the nucleotide to the phosphodiesterase. Both observations provide evidence that a different conformation in the enzyme results from cGMP binding. Only the conformation formed upon cGMP binding is easily attacked by chymotrypsin or permanently activated by treatment with dicyclohexylcarbodiimide. One major chymotryptic cleavage site exposed by cGMP binding is at tyrosine 553, implying that this region takes part in the conformational change. Limited proteolysis experiments indicate that these noncatalytic binding sites are located within a region of internal sequence homology previously proposed to include the cGMP-binding site(s) and that they retain a high affinity and specificity for cGMP independent of the catalytic domain of the enzyme. The products formed by partial proteolysis can be separated into individual catalytically active and cGMP-binding fractions by anion exchange chromatography. Gel filtration and electrophoresis analysis of the isolated fractions suggest that the cGMP-binding peak has a dimeric structure. Moreover, it can be further resolved by polyethyleneimine high performance liquid chromatography into two peaks (Peaks IIIA and IIIB). Peak IIIA binds 2 mol of cGMP/mol of dimer with an apparent Kd of 0.2 microM. Peak IIIB, however, has greatly reduced cGMP binding. Further digestion of these fragments with cyanogen bromide show that the differences between Peaks IIIA and IIIB are due to one or more additional proteolytic nicks in IIIB that remove a few residues near its C terminus, most probably residues 523-550 or 534-550. This in turn suggests that this region is essential for cGMP-binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Thiophilic adsorption chromatography: The separation of serum proteins

Human serum proteins were separated on a matrix obtained by reaction of β-mercaptoethanol with divinyl sulfone-activated agarose (the so-called T-gel). Binding of almost all serum proteins was observed at high concentrations of ammonium sulfate. Elution was achieved by gradually lowering the concentration of salt in the washing buffer. Fractions obtained during elution were analyzed by fused rocket immunoelectrophoresis. Proteins were recovered in high yields and with an excellent separation in this one-step procedure (“Thiophilic adsorption chromatography”). A rapid and straightforward procedure giving essentially pure immunoglobulins from crude rabbit serum with at least 80% yield by the T-gel is also presented.

Fractionation of whale cartilage chondroitin sulfate on sepharose CL-4B in the presence of high concentration of ammonium sulfate. Relationship between molecular size and unit-disaccharide composition of the polysaccharide

Fractionation of whale cartilage chondroitin sulfate on sepharose CL-4B in the presence of high concentration of ammonium sulfate. Relationship between molecular size and unit-disaccharide composition of the polysaccharide

Vitamin K-dependent carboxylase: effect of ammonium sulfate on substrate carboxylation and on inhibition by stereospecific substrate analogs

(1) High concentrations of ammonium sulfate may stimulate the carboxylase activity of bovine liver microsomes about 10-fold. This effect results from an increase of the V max, whereas neither the apparent K m for a number of substrates nor the K i for substrate analogs is affected. (2) The effect of ammonium sulfate was only found in substrates lacking the pro-sequence. No effect was measurable on the carboxylation of pro-PT28 and endogenous precursor proteins. (3) If the pro-fragment was added as a peptide not covalently bound to a carboxylatable substrate, the carboxylation thereof was only slightly affected and ammonium sulfate remained active as a stimulator of carboxylase activity. (4) S-MeTPT is a much stronger inhibitor of carboxylase activity than is R-MeTPT. (5) The inhibition of carboxylase by the methylated tripeptides is competitive and independent of the type of substrate. Also pro-PT28, which contains the full pro-sequence, could be inhibited completely. (6) On the other hand the carboxylation of endogenous protein precursors could only be partly inhibited by the substrate analogs: even at high concentrations of S-MeTPT a residual endogenous substrate carboxylation of about 30% was left.

Stimulatory effect of ammonium sulfate at high concentrations on the aminoacylation of tRNA and tRNA-like molecules

The influence of various salts on the aminoacylation of tRNA Val and the tRNA-like structure from turnip yellow mosaic virus RNA by yeast valyl-tRNA synthetase has been studied. As expected, increasing the concentration of salts inhibits the enzymatic reaction. However, in the presence of high concentration of ammonium sulfate, and only this salt, the inhibitory effect is suppressed. Under such conditions, the aminoacylation becomes comparable to that measured in the absence of salt. It was shown that ammonium sulfate affects both the catalytic rate of the reaction and the affinity between valyl-tRNA synthetase and the RNAs. Because the affinity between the partners in the complex is increased when the concentration of the salt is high, it is suggested that hydrophobic effects are involved in tRNA/synthetase interactions.

Effect of aggregation of histone octamers in high-salt solutions on circular dichroism spectra

The circular dichroism (CD) of freshly prepared chicken erythrocyte core histones has been reexamined in high concentrations of ammonium sulfate and sodium chloride, conditions which cause drastic changes in the solubility and aggregative properties of these proteins. After sample clarification by ultracentrifugation, no significant net changes are detected in the secondary structure of the core histones in the range of 2.0-2.5 M ammonium sulfate. There is also no significant difference between the CD spectra of histone solutions in 2 M sodium chloride and clarified solutions of histones in high concentrations of ammonium sulfate. It was observed that sample clarification by ultracentrifugation immediately prior to taking CD spectra was necessary for signal stabilization, especially under conditions which begin to favor crystallization of the histones.

Spectropolarimetric analysis of the core histone octamer and its subunits

The secondary structure of the calf thymus core histone octamer, (H2A-H2B-H3-H4)2, and its two physiological subunits, the H2A-H2B dimer and (H3-H4)2 tetramer, was analyzed by ORD spectropolarimetry as a function of temperature and solvent ionic strength within the ranges of these experimental parameters where assembly of the core histone octamer exhibits pronounced sensitivity. While the secondary structure of the dimer is relatively stable from 0.1 to 2.0 M NaCl, the secondary structure of the tetramer exhibits complex changes over this range of NaCl concentrations. Both complexes exhibit only modest responses to temperature changes. ORD spectra of very high and very low concentrations of stoichiometric mixtures of the core histones revealed no evidence of changes in the ordered structure of the histones as a result of the octamer assembly process at NaCl concentrations above 0.67 M, nor were time-dependent changes detected in the secondary structure of tetramer dissolved in low ionic strength solvent. The secondary structure of the chicken erythrocyte octamer dissolved in high concentrations of ammonium sulfate, including those of our crystallization conditions, was found to be essentially unchanged from that in 2 M NaCl when examined by both ORD and CD spectropolarimetry. The two well-defined cleaved products of the H2A-H2B dimer, cH2A-H2B and cH2A-cH2B, exhibited reduced amounts of ordered structure; in the case of the doubly cleaved moiety cH2A-cH2B, the reductions were so pronounced as to suggest marked structural rearrangements.

Structure-function relationships of mitochondrial ATPase-ATPsynthase using Schizosaccharomyces pombe yeast mutants with altered F 1 subunits

Phenotypic revertants have been selected from mutants of the yeast Schizosaccharomyces pombe devoid of either α or β subunits of mitochondrial ATPase-ATPsynthase. In contrast to parental mutants, phenotypic revertants are able to grow on glycerol respiratory medium and show immunodetectable α and β subunits. However, growth and cellular respiration are only partially restored as compared to the wild strain, indicating that the recovered subunits are mutated. ATPase activity of revertant submitochondrial particles shows markedly different parameters: more acidic optimal pH, absence of bicarbonate activation and decreased sensitivity to azide inhibition in the α subunit-modified R3.51. Opposite differences are observed in the β subunit-modified R4.3: more alkaline optimal pH, much higher bicarbonate activation, and increased sensitivity to azide. The ITPase activity of R4.3 submitochondrial particles is also more sensitive to azide as compared to the wild strain. ATPase activity of purified F 1 also exhibits marked differences: loss of bicarbonate-sensitive negative cooperativity, decreased sensitivity to both ADP and azide inhibitions in the R3.51 revertant. On the contrary, increased negative cooperativity and increased sensitivity to both ADP and azide inhibitions are observed for the R4.3 revertant enzyme which in addition exhibits a much lower maximal rate. The β subunit-mutation of R4.3 also increases the sensitivity of ITPase activity to tripolyphosphate inhibition, whereas the α subunit-mutation of R3.51 is without any effect. Soluble F 1 with β subunit-mutation is very sensitive to high ammonium sulfate concentrations required for enzyme precipitation and concentration and known to partially deplete the enzyme from its endogenous nucleotides. On the contrary, poly(ethylene)glycol is very efficient for preparing from any strain a pure and very stable enzyme retain-ing high amounts of endogenous nucleotides. The R4.3 revertant F 1 retains even more nucleotides than the wildstrain F 1 and is much less sensitive to high iodide concentrations which favor enzyme dissociation and precipitation. The tryptophan intrinsic fluorescence of F 1 is modified by both mutations that increase the maximal emission intensity. The most important effect is produced by β subunit-mutation which decreases the quenchable fraction, one-third to one-half tryptophans being no longer accessible to iodide. The overall results suggest that both mutations modify enzyme-nucleotide interactions: the α subunit-mutation of R3.51 would favor ADP release by lowering interactions with the adenine moiety, whereas the β subunit-mutation of R4.3 would lower ADP release by strengthening interactions with the phosphate chain moiety.

Purification and Characterization of Cell-associated Glucosyltransferase Synthesizing Insoluble Glucan from Streptococcus mutans Serotype c

Streptococcus mutans Ingbritt (serotype c) was shown to have a significant amount of cell-associated glucosyltransferase activity which synthesizes water-insoluble glucan from sucrose. The enzyme was extracted from the washed cells with SDS, renatured with Triton X-100, adsorbed to 1,3-alpha-D-glucan gel, and then eluted with SDS. The enzyme preparation was electrophoretically homogeneous, and the specific activity was 7.3 i.u. (mg protein)-1. The enzyme had an Mr of 158,000 as determined by SDS-PAGE, and was a strongly hydrophilic protein, as judged by its amino acid composition. The enzyme gradually aggregated in the absence of SDS. The enzyme had an optimum pH of 6.5 and a Km value of 16.3 mm for sucrose. Activity was stimulated 1.7-fold by dextran T10, but was not stimulated by high concentrations of ammonium sulphate. Below a sodium phosphate buffer concentration of 50 mm, activity was reduced by 75%. This enzyme synthesized an insoluble D-glucan consisting of 76 mol% 1,3-alpha-linked glucose and 24 mol% 1,6-alpha-linked glucose.

Purification of the migration stimulating factor produced by fetal and breast cancer patient fibroblasts.

We have previously shown that (i) human skin fibroblasts of fetal and adult origin display distinctive migratory phenotypes, (ii) this difference in cell behavior results from the production of a soluble "migration stimulating factor" (MSF) by fetal cells, and (iii) skin fibroblasts from breast cancer patients commonly resemble fetal fibroblasts both in migratory phenotype and in production of MSF. Data are now presented indicating that MSF present in the conditioned medium of fetal and cancer patient fibroblasts is precipitated at 10% saturation ammonium sulfate and binds to heparin and cation-exchange resins. Based on this information, we have devised a scheme for the purification of MSF involving the sequential application of ammonium sulfate precipitation, heparin affinity, gel filtration, and reverse-phase chromatography. Purified MSF has an estimated molecular mass of 70 kDa; amino acid analysis reveals a relatively high level of proline (13.34 residues per 100). Our results further suggest that skin fibroblasts from breast cancer patients produce an additional factor with migration stimulating activity; this factor is precipitated at higher concentrations of ammonium sulfate and binds to anion-exchange resins. We have previously discussed the possible direct involvement of fetal-like fibroblasts in cancer pathogenesis. The availability of MSF obtained from cancer patient fibroblasts provides a potential means with which to examine the complex cellular interactions contributing to this process as well as develop a screening regime for identifying individuals at elevated risk of developing cancer.

Protein aggregation in high-performance liquid chromatography: hydrophobic interaction chromatography of beta-lactoglobulin A.

Aggregation of beta-lactoglobulin A under acidic buffer conditions was studied in hydrophobic interaction chromatography. At high ammonium sulfate concentrations, pH 4.5 and 4 degrees C, UV chromatograms revealed a maximum of three peaks for beta-lactoglobulin A concentrations greater than 5 mg/mL, suggesting three distinct aggregate species. The size of the smallest aggregate (tetramer) and its stoichiometric relationship to the other two aggregates (octamer and dodecamer) were determined from the chromatographic data and a simple mass balance model. These stoichiometries agreed with those determined in a separate study by on-line low-angle laser light scattering. In addition, the association constants describing the formation of octamer from two tetramer molecules and the formation of dodecamer from the octameric and tetrameric species were found to be (2.4 +/- 0.5) X 10(4) M-1 and (3.3 +/- 0.8) X 10(3) M-1, respectively. Analysis of the beta-lactoglobulin A system is based on a model in which aggregates form in solution upon injection before adsorbing to the column matrix. The column retains those species formed in solution and induces little change in the relative amounts of each species. These results illustrate another example by which multiple peaks can arise in high-performance liquid chromatography, beyond the previously described studies of protein conformational changes during chromatography.

Purification of an acidic plastocyanin from MicroBiomis aeruginosa

Plastocyanin and cytochrome c-553 are two functionally equivalent electron carriers in the photosynthetic chain of cyanobacteria. Microcystis aeruginosa, a unicellular cyanobacterium which grows well at a high pH (8.6) and which was not known to possess plastocyanin, has been studied for its ability to synthesize plastocyanin in culture media with and without Cu. In the absence of Cu, an acidic cytochrome c-553 alone was isolated. With the inclusion of 2 microM Cu, cytochrome c-553 synthesis was partially suppressed and an acidic plastocyanin was isolated. A newly developed procedure, using high concentrations of ammonium sulfate to fractionate water-soluble proteins on Sephacryl S-200 was successfully used to isolate and concentrate the plastocyanin, thus allowing it to be further purified to homogeneity. This protein has an isoelectric point of 4.8 which is similar to the pI value reported for other acidic plastocyanins from higher plants and green algae. Its N-terminal sequence of the first 15 amino acids has been determined; 9 of these amino acids are identical to those in the sequence of the basic plastocyanin from Anabaena variabilis.

Purification of a novel 30,000 Da calcium-binding protein from bovine cerebellum.

A novel very acidic calcium-binding protein (CaBP) was purified from bovine cerebellum, using 45Ca autoradiography as a marker, through a preparative procedure involving salting out with a very high concentration of ammonium sulfate, DE52 column chromatography, RNAase treatment, and HPLC gel filtration. This protein showed a molecular weight of 30,0000 dalton (Da) on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and of 120,000 on in gel filtration chromatography analysis under physiological ionic strength. The calcium binding activity of this 30,000 Da CaBP was monitored on the basis of calcium-dependent changes in tyrosine fluorescence (Kd = 3.0 microM).