Difference in hydrophobicity between mitochondria-bindable and non-bindable forms of hexokinase purified from rat brain

Hexokinase able to bind to mitochondria was purified to homogeneity from rat brain by two successive DEAE-cellulose chromatographic steps. The enzyme lost only the binding ability with almost undetectable change in molecular weight on mild chymotrypsin digestion. The bindable hexokinase was adsorbed to a Phenyl-Sepharose column and eluted with a Lubrol PX gradient, whereas non-bindable hexokinase and yeast hexokinase were not adsorbed under the similar conditions. These results suggest that mitochondria-bindable hexokinase has a hydrophobic region on its surface, which is responsible for the specific interaction with mitochondria.     

Peptide Mapping Studies of the Chromogranins and of Two Chromaffin Granule Proteoglycans

Abstract: The chromogranins, a family of related acidic glycoproteins, and two chondroitin sulfate/dermatan sulfate proteoglycans were isolated from the soluble contents of bovine adrenal chromaffin granules by chromatography on DEAE-cellulose. These chromaffin granule matrix glycoconjugates were treated with trypsin, and the resulting peptides were fractionated by HPLC. The two proteoglycans, which differ in their concentration of glycosaminogly-cans and glycoprotein oligosaccharides, yielded almost identical peptide patterns and would both appear to have the same protein moiety. The peptide profile of the proteoglycans differs, however, from that of the chromogranins, which they closely resemble in terms of amino acid composition. The various chromogranin fractions obtained by gel filtration were also found to have significant differences in the chromatographic patterns of their tryptic peptides.     

Nondenaturing procedure for rapid preparation of ferredoxin from Clostridium pasteurianum

A procedure is described for the rapid preparation of ferredoxin in high yield from crude extracts of C. pasteurianum. The method involves two successive chromatographic treatments on DEAE-cellulose in concentrated ammonium sulfate. Ferredoxin prepared by this method has an absorption spectrum and iron content similar to ferredoxin prepared by other methods.     

A novel procedure for the rapid purification of plastocyanin from pea (Pisum sativum) leaves.

Plastocyanin is soluble at high concentrations (greater than 3 M) of (NH4)2SO4 but under these conditions will adsorb tightly to unsubstituted Sepharose beads. This observation was utilized to purify plastocyanin from pea (Pisum sativum) in two chromatographic steps. Sepharose-bound plastocyanin was eluted with low-ionic-strength buffer and subsequently purified to homogeneity by DEAE-cellulose chromatography.     

Isolation and characterization of cartilage proteoglycans immunoreactive with an antibody to skin proteodermatan sulfate core protein

Low density proteoglycans showing cross-reaction with an antibody to skin proteodermatan sulfate (PDS) core protein were isolated from the bovine articular cartilage, by CsC1 density gradient centrifugation followed by repeated DEAE-cellulose chromatography. The size and amino acid composition of the core proteins of the immunoreactive proteoglycans, eluted at 0.25M and 0.5M NaC1 on DEAE-cellulose column, were quite similar to that of PDS. The glycosaminoglycan components of both proteoglycans were shown to be composed of a hybrid structure of chondroitin sulfate and dermatan sulfate, based on chondroitinase treatments followed by two-dimensional electrophoresis.     

Tyroslyl-tRNA synthetase from baker's yeast. Rapid isolation by affinity elution, molecular weight of the enzyme, and determination of essential sulfhydryl groups.

Tyrosyl-tRNA synthetase (EC 6.1.1.1) has been isolated from baker's yeast with an overall purification factor of more than 5000. After opening the cells, pH 4.8 precipitation, ammonium sulfate fractionation, removal of the nucleic acids with DEAE-cellulose and chromatography on CM-Sephadex, the critical purification step is the elution of the cation-exchanger-bound tyrosyl-tRNA synthetase with tRNATyr. The homogeneous enzyme exhibits a molecular weight of 40 000 as estimated by sedimentation equilibrium centrifugation and dodecylsulfate-gel electrophoresis under reducing and non-reducing conditions. Gel filtration experiments show a molecular weight of about 100 000 indicating the existence of an active dimeric form. The possibility of proteolytic cleavage of the enzyme is excluded. The reaction of tyrosyl-tRNA synthetase with p-chloromercuribenzoate and N-ethylmaleimide reveals two repidly reacting sulfhydryl groups per subunit of molecular weight 40 000, as demonstrated by the inhibition of aminoacylation and the isolation of enzyme-inhibitor complexes. In addition an efficient purification method is described for isolating tRNATyr from soluble ribonucleic acid from baker's yeast in three chromatographic steps in a yield of 28%.     

Purification of a Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart by specific interaction with its Ca2+-dependent modulator protein.

A Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart can be eluted from a DEAE-cellulose column either in the free form by buffers containing 0.1 mM ethylene glycol bis(beta-aminoethyl ether)N-N,N'N'-tetraacetic acid (EGTA) or as a complex of the enzyme with its protein modulator by buffers containing 0.01 mM CaCl2. A purification procedure based primarily on the significantly different affinity of the two forms of the enzyme for DEAE-cellulose was developed for the purification of the enzyme from bovine heart. The procedure involves ammonium sulfate fractionation, three chromatographic steps on DEAE-cellulose, and gel filtration on Sephadex G-200 with a 5000-fold purification over the crude extract. The purified enzyme has a specific activity of 120 mumol of cAMP/mg/min, can be activated 5-fold by Ca2+, but is only 80% pure as judged by analytical disc gel electrophoresis. The purified enzyme is unstable but can be stabilized by addition of Ca2+ and the protein modulator; this is in contrast to the less pure preparations of Ca2+-activatable phosphodiesterase which are destabilized by the protein modulator in the presence of Ca2+.     

Extraction and purification of proteoglycans from various types of connective tissue

A new procedure for the isolation of proteoglycans has been described. Tissues are extracted with 4 M guanidinium chloride. the extracting solven is then exchanged for 7 M urea and the extract is chromatographed on a DEAE-cellulose column previously equilibrated with 7 M urea. Non-proteoglycan proteins were eluted with urea in weak salt solutions. Subsequently proteoglycans were eluted with strong salt solutions. By the procedure proteoglycans from tissues containing only small amounts of proteoglycans can be obtained virtually free from collagen in a form suitable for further fractionation.     

Some properties of soluble and solubilized particle-bound hexokinase

Abstract— Particle-bound hexokinase of rat brain homogenates was solubilized by successive treatment with 0-9 M-NaCl or 003 M-ATP (pH 8.0) and 0-5% (w/v) Triton X-100. This solubilized hexokinase and the soluble hexokinase present in cytoplasm of rat brain homogenates were chromatographed on DEAE-cellulose and some kinetic properties of the isolated hexokinase peaks were studied. The chromatographic separation was greatly influenced by the EDTA-concentration of the buffer used. No significant differences were observed in the chromatographic pattern and in the apparent K_m-values for ATP and glucose and the apparent K_t for glucose-6-phosphate (versus ATP) between the soluble and particulate hexokinase solubilized by different reagents. On agarose-electrophoresis the solubilized particulate enzyme migrates as one single band, the soluble hexokinase separates into one major and two minor bands.     

A simple method for large-scale purification of plasma-derived apo-transferrin

We investigated and optimized a purification process, suitable for industrial scale, to obtain pharmaceutical grade apo-Tf (apo-transferrin), preserving its physiological properties and functions. Apo-Tf was obtained from fraction IV subfraction 1 and IV subfraction 4 (fraction IV-1,4), a waste product of the Cohn fractionation process, performing a single chromatographic run and two viral inactivation/removal steps. The structural integrity and the biological activity of the final product were extensively tested. The yield of apo-Tf produced was 80% on laboratory scale and 90% in scale-up lots, and the purity was higher than 95%. The purified protein preserves iron- and receptor-binding activities and shows a normal glycosylation pattern. The single chromatographic step process presented here provides an efficient means to prepare commercial quantities of the protein. The final product is sterile and two viral inactivation/removal steps were introduced into the process.     

Fractionation and isolation of the multiple forms of prorennin (prochymosin)

The heterogeneity of prorennin was studied by chromatography on DEAE-cellulose and microgranular DEAE-cellulose columns, as well as by polyacrylamide-gel electrophoresis. Prorennin prepared by alum treatment, salting-out and chromatography was resolved into three components by a compound gradient of sodium phosphate on microgranular DEAE-cellulose. Polyacrylamide-gel electrophoresis confirmed the chromatographic results, but crystalline rennin was shown to consist of four bands. When prorennin was isolated directly by chromatography, four zymogen components were resolved on microgranular DEAE-cellulose with a modified compound gradient of sodium phosphate. Polyacrylamide-gel electrophoresis confirmed the existence of four multiple forms of prorennin as well as homogeneity of the chromatographic fractions.     

Mitochondrial DNA polymerase, deoxyribonuclease and ribonuclease H activities from brain of chick embryo

R-DNA polymerase, D-DNA polymerase, DNase and RNase H activities in mitochondria from chick embryonic brain were studied by ion-exchange chromatography. Two main fractions were separated according to their chromatographic behaviour: a fraction M Ib which is eluted with the washing buffer from two successive DEAE-cellulose columns and a fraction M IV which is eluted at 400 mM KC1 from a phosphocellulose column. Although the two fractions contain both the DNA polymerase and the degrading activities, all the specific activities are higher in fraction M IV than in fraction M Ib. Heat inactivation experiments have shown that R-DNA polymerase is inactivated in both fractions, whereas RNase H and DNase are not affected. Thus, degrading activities and R-DNA polymerase activity seem to be catalyzed by different molecular entities. However the fact that in most cases these activities co-chromatograph suggests that the corresponding molecules form rather stable complexes.     

Immunochemical study of beta-glucuronidase inhibitor from porcine sublingual gland. Relationship between the antigenic determinant and the active site of the inhibitor.

Following removal of sialic acid by neuraminidase treatment the activity of the beta-glucuronidase inhibitor was remarkably decreased, but the antigenic determinant was not affected. A partial common antigen to the inhibitor was isolated from porcine small intestine, by successive fractionation of trypsin extraction of the latter on Sephadex G-150, DEAE-cellulose and Sepharose 4B column chromatography. The immunologic and characteristic properties of the common antigen were compared with those of the beta-glucuronidase inhibitor is not identical with its antigenic determinant.     

Heterogeneity of proteoglycans in monkey corneal stroma

The proteoglycans of the cynomolgus monkey corneal stroma were isolated and characterized by using a combination of physiochemical and biochemical methods. Proteoglycans were biosynthetically radiolabeled by incubating whole corneas in medium containing [³⁵S]sulfate and either [³H]serine or [³H]mannose as precursors. Macromolecules were extracted from the corneal stromas with 4 m guanidine-HCl. After dialysis into 8 m urea, proteoglycans in the extracts were initially purified by DEAE-cellulose chromatography. A portion of the proteoglycan fraction was digested with chondroitinase ABC, and the keratan sulfate proteoglycans were then isolated by rechromatography of the digest on DEAE-cellulose. Another portion of the proteoglycan fraction was digested with endo-β-galactosidase and the dermatan sulfate-proteoglycans were then isolated by chromatography of the digest on Sepharose CL-4B. Each proteoglycan population was further fractionated by chromatography on concanavalin A-Sepharose and by CsCl density gradient centrifugation. Four subpopulations for both the keratan sulfate proteoglycans and the dermatan sulfate proteoglycans were isolated. Based on differences in binding to concanavalin A-Sepharose, buoyant densities, and glycosaminoglycan content, subpopulations of each proteoglycan differ by the number and properties of both the glycosaminoglycan chains and the mannose-containing oligosaccharides attached to their protein core.     

UPTAKE AND METABOLISM OF 5-METHYL-TETRAHYDROFOLIC ACID BY RAT BRAIN SLICES

DNA-dependent DNA polymerases were partially purified from nuclei of cells from the occipital lobe of human brain. The purification procedure included successive DEAE-cellulose and phosphocellulose column chromatography, gel filtration and sucrose density gradient centrifugation steps. Four enzymes corresponding to DNA polymerases-α, β, γ, and terminal deoxynucleotidyl transferase were found. Brain DNA polymerases could be differentiated from one another by size, template preferences and sensitivity to sulfhydryl blocking agents.     

Immunochemical study of β-glucuronidase inhibitor from porcine sublingual gland Relationship between the antigenic determinant and the active site of the inhibitor

Following removal of sialic acid by neuraminidase treatment the activity of the β-glucuronidase inhibitor was remarkably decreased, but the antigenic determinant was not affected.A partial common antigen to the inhibitor was isolated from porcine small intestine, by successive fractionation of trypsin extraction of the latter on Sephadex G-150, DEAE-cellulose and Sepharose 4B column chromatography. The immunologic and characteristic properties of the common antigen were compared with those of the inhibitor, and it was concluded that the active site of the β-glucuronidase inhibitor is not identical with its antigenic determinant.     

Purification and Properties of Cytokinin-Binding Proteins from Tobacco Leaves

A cytokinin-binding protein complex was purified 700-fold fromleaves of tobacco (Nicotiana sylvestris). The purification procedureconsisted of four chromatographic steps on columns of DEAE-cellulose,Mono Q, Phenyl Superose and Superose 12, respectively. The purifiedcytokinin-binding protein complex behaved as a 130-kDa globularprotein on gel filtration. This complex contains two proteinspecies whose molecular masses are estimated to be 57 kDa and36 kDa. Binding to benzyl[8-¹⁴C]adenine was inhibited by adenine,ATP, zeatin and cAMP but not by indoleacetic acid. Scatchardanalysis indicated the existence of at least two cytokinin-bindingsites in the purified complex. The dissociation constant forthe high-affinity site was 2.1 10^-5 M. (Received October 19, 1992; Accepted February 27, 1993)     

Purification and characterization of DNA-relaxing enzyme from Haemophilus gallinarium.

A DNA-relaxing enzyme capable of concerted nicking and closing of DNA backbone bonds has been purified from Haemophilus gallinarum by two chromatographic steps and gel filtration. The enzyme efficiently catalyzes the removal of superhelical turns from a negatively twisted DNA and requires Mg2+ for this activity. Slight removal of superhelical turns from a positively twisted DNA generated by binding of ethidium bromide is found, but only at high enzyme concentrations. The DNA-relaxing activity is inhibited markedly with heat-denatured DNA, whereas native DNA and RNA have almost no affect on this activity.     

α-galactosidase of Poterioochromonas malhamensis

In cell-free extracts of Poterioochromonas malhamensis, α-galactosidase (α-d-galactoside galactohydrolase, EC 3.2.1.22) was stable at pH 8. The pH optimum of the enzyme was 7. The enzyme was purified 10-fold through chromatographic steps involving DEAE-cellulose, hydroxylapatite and Sephadex G-200. The apparent MW was 360 000 by sucrose density-gradient centrifugation. All activity was lost on subjecting the enzyme to polyacrylamide-gel electrophoresis. The substrate specificity of the enzyme was examined and some kinetic values determined. The enzyme displayed an unusual activity curve with respect to isofloridoside.