Isolation and characterization of three positional isomers of diglucosylcyclomaltoheptaose

Three positional isomers of diglucosylcyclomaltoheptaose [(G)2-beta-cyclodextrin], 6(1),6(4)-di-O-(a-D-glucopyranosyl)-cyclomaltoheptaose (1), 6(1),6(3)-di-O-(a-D-glucopyranosyl)-cyclomaltoheptaose (2), and 6(1),6(2)-di-O-(a-D-glucopyranosyl)-cyclomaltoheptaose (3) were isolated by h.p.l.c. on a reversed-phase column from the mother liquors of a large-scale preparation of beta CD with Bacillus ohbensis cyclomaltodextrin glucanotransferase (EC 2.4.1.19) and were characterized by h.p.l.c. analysis of partial hydrolyzates and by 13C-n.m.r. spectroscopy. Their molecular weights were confirmed by f.a.b.-m.s. Their characteristic chromatographic behavior on four h.p.l.c. columns of different separation modes was found to be very useful for their identification. It is particularly noteworthy that the first application of a graphitized carbon column to CDs enabled a fine separation of all three positional isomers.     

Isolation and characterization of hamster brain polyribosome-cytomatrix complexes.

We have developed a method for the isolation of a brain subcellular fraction enriched in both highly aggregated polyribosomes and cytoskeletal proteins. This method is based on gentle dispersion of brain tissue and low speed centrifugation. This fraction is enriched in typical cytoskeletal proteins as glial fibrillary protein, neurofilament proteins and actin. Messenger RNA did not seem to be involved in the polyribosome association to the cytomatrix as shown by the effect of exposure to micrococcal nuclease. On the other hand, in vivo disruption of protein synthesis by acute experimental phenylketonuria, hypothermia or heat-shock did not cause the release of ribosomes from the cytomatrix.     

Isolation and characterization of galanin from sheep brain.

Galanin is a well-known, naturally occurring peptide, which has been characterized from both cDNA sequences and direct peptide analysis. Previous structural studies have been made using intestinally derived material. This report concerns galanin isolation from sheep brain and its sequence determination. Sheep galanin shows great similarity to pig galanin, differing by one amino acid substitution, that being a histidine residue, as in cow and rat galanin, instead of tyrosine at position 26.     

Studies on brain cytosol neuraminadase. I. Isolation and partial characterization of two forms of the enzyme from pig brain.

1. Two forms of cytosol neuraminidase (EC 3.2.1.18) (neuraminidase A and neuraminidase B) were isolated and purified from pig brain homogenate, by proceeding through the following steps: centrifugation of brain homogenate at 105 000 X g (1h); ammonium sulphate fractionation (35-55% saturated fraction); column chromatography on Biogel A 5 m; column chromatography on hydroxy apatite/cellulose gel; affinity chromatography on Affinose-tyrosyl-p-nitrophenyloxamic acid. The separation of the two forms of neuraminidase was provided by chromatography on hydroxylapatite/cellulose gel. Neuraminidase A was purified about 500-fold; neuraminidase B about 400-fold. 2. The pH optima and the maximum activities in various buffers were different for neuraminidase A and B (for instance the pH optimum was in sodium acetate/acetic acid buffer, 4.7 for neuraminidase A and 4.9 for neuraminidase B). Ions affected in a different way the two enzymes: K+ activated neuraminidase A but not neuraminidase B; Na+ and Li+ inhibited neuraminidase A at a higher degree than neuraminidase B. Neuraminidase B seemed to be moderately activated by some bivalent cations (Ca2+; Mg2+; Zn2+); neuraminidase A did not. The Km values for sialyllactose were different: 2.2-10(-3) M for neuramindase A; 0.46-10(-3) M for neuraminidase B.     

Chick brain actin and myosin. Isolation and characterization

Brain actin extracted from an acetone powder of chick brains was purified by a cycle of polymerization-depolymerization followed by molecular sieve chromatography. The brain actin had a subunit molecular weight of 42,000 daltons as determined by co-electrophoresis with muscle actin. It underwent salt-dependent g to f transformation to form double helical actin filaments which could be "decorated" by muscle myosin subfragment 1. A critical concentration for polymerization of 1.3 microM was determined by measuring either the change in viscosity or absorbance at 232 nm. Brain actin was also capable of stimulating the ATPase activity of muscle myosin. Brain myosin was isolated from whole chick brain by a procedure involving high salt extraction, ammonium sulfate fractionation and molecular sieve chromatography. The purified myosin was composed of a 200,000-dalton heavy chain and three lower molecular weight light chains. In 0.6 M KCl the brain myosin had ATPase activity which was inhibited by Mg++, stimulated by Ca++, and maximally activated by EDTA. When dialyzed against 0.1 M KCl, the brain myosin self-assembled into short bipolar filaments. The bipolar filaments associated with each other to form long concatamers, and this association was enhanced by high concentrations of Mg++ ion. The brain myosin did not interact with chicken skeletal muscle myosin to form hybrid filaments. Furthermore, antibody recognition studies demonstrated that myosins from chicken brain, skeletal muscle, and smooth muscle were unique.     

Adult rat brain synaptic vesicles I. Isolation and characterization

Synaptic vesicles were isolated from adult rat brain in a form which seemed to be 90–95% pure by chemical and enzymatic assay. The only significant contaminant was the synaptosomal plasma membrane. Contamination with Golgi apparatus and lysosomes appeared limited although some uncertainty remains on this point. The vesicles are sufficiently pure for valid analytical studies to be performed, but the possibility of internal heterogeneity of the preparations must be taken into account.     

Isolation and partial characterization of chick brain synaptic plasma membranes.

An isolation procedure for synaptic plasma membranes from whole chick brain is reported that uses the combined flotation-sedimentation density gradient centrifugation procedure described by Jones and Matus (Jones, D. H. and Matus, A. I. (1974) Biochim. Biophys. Acta 356, 276-287) for rat brain. The particulate of the osmotically shocked and sonicated crude mitochondrial fraction was used for a flotation-sedimentation gradient step. Four fractions were recovered from the gradient after 30 min centrifugation. The fractions were identified and characterized by electron microscopy and by several markers for plasma membrane and other subcellular organelles. Fraction 2 was recovered from the 28.5-34% (w/v) sucrose interphase and contained the major part of the activities of the neuronal plasma membrane marker enzymes. The specific activities of the (Na+ +K+)-activated ATPase (EC 3.6.1.3), acetylcholinesterase (EC 3.1.1.7) and 5'-nucleotidase (EC 3.1.3.5) were, respectively, 4.5, 2.0 and 1.2 times higher than in the homogenate. However, Fraction 2 also contained considerable amounts of activities of putative lysosomal and microsomal markers in addition to lower amounts of mitochondrial and myelin markers. Although no prepurification of synaptosomes from the crude mitochondrial fraction was performed, the synaptic plasma membranes obtained showed many properties analogous to similar preparations from rat brain described in recent years.     

Isolation and partial characterization of a sulfogalactoglycerolipid from rat brain.

The brain of adult rats were analyzed for the presence of 35-SO4-containing glycolipids following intraventricular injection of Na2-35SO4. Radiochromatographic analyses revealed the presence of two minor 35-SO4-containing glycolipids, in addition to sulfogalactosylceramide. One of these two minor sulfolipids was isolated and tentatively identified as a 1-O--alkyl-2-0-acyl-3-(3'-sulfogalactosyl)-glycerol, a compound recently demonstrated to be the major glycolipid of mammalian testis. The alkyl and acyl compositions of the compound from rat brain are more heterogeneous than those from rat testis. The non-sulfated form of the galactoglycerolipid was also detected in rat brain. The amount of the sulfogalactoglycerolipid in rat brain is 0.19 mumol per gram wet weight, approximately one-third of the amount in rat testis (per gram wet weight), and is approximately one-fifteenth that of sulfogalactosylceramide in rat brain. The possible significance of the common occurrence in brain and testis of sulfated and non-sulfated galactolipids is discussed.     

Isolation and characterization of two types of sarcoplasmic reticulum vesicles.

A purified preparation of sarcoplasmic reticulum from rabbit skeletal muscle has been found to consist of a heterogeneous population of vesicles. Isopycnic centrifugation was used to obtain "light" and "heavy" vesicles from the upper and lower ends of a 25 to 45% (w/w) linear sucrose gradient. Each fraction accounted for about 10 to 15% of the total vesicles. The remainder of the vesicles were of intermediate density and banded between the light and heavy fraction. Light vesicles were composed of about equal amounts of phospholipid and Ca-2+ pump protein which contained approx. 90% of the protein. Heavy vesicles contained in addition to the Ca-2+ pump protein (55-65% of the protein) two other major protein components, the Ca-2+ binding and M55 proteins which accounted for 20-25 and 5-7% of the protein of these vesicles, respectively. The sarcoplasmic reticulum subfractions had 32-P-labelled phosphoenzyme levels proportional to their Ca-2+ pump protein content and contained similar Ca-2+-stimulated ATPase activities. They were capable of accumulating Ca-2+ in the presence of ATP and of releasing the accumulated Ca-2+ when placed into a medium with a low Ca-2+ concentration. The vesicles differed significantly in that heavy vesicles had a greater number of non-specific Ca-2+ binding sites than light vesicles (approx. 220 vs 75 nmol of bound Ca-2+ per mg protein), in accordance with their high content of Ca-2+ binding protein. Electron dense material could be seen within the compartment of heavy but not light vesicles. Removal of Ca-2+ binding and M55 proteins from heavy vesicles resulted in empty membranous structures consisting mainly of Ca-2+ pump protein and phospholipid. Electron micrographs of sections of muscle showed dense material in terminal cisternae but not in longitudinal sections of sarcoplasmic reticulum. These experiments are consistent with the interpretation that (1) the electron dense material inside heavy vesicles may be referable to Ca-2+ binding and/or M55 proteins, and that (2) light and heavy vesicles may be derived from the longitudinal sections and terminal cisternae of sarcoplasmic reticulum, respectively.     

Isolation and characterization of two different forms of inositol phospholipid-specific phospholipase C from rat brain

Two different forms of inositol phospholipid-specific phospholipase C (PLC) have been purified 2810- and 4010-fold, respectively, from a crude extract of rat brain. The purification procedures consisted of chromatography of both enzymes on Affi-Gel blue and cellulose phosphate, followed by three sequential high performance liquid chromatography steps, which were different for the two enzymes. The resultant preparations each contained homogeneous enzyme with a Mr of 85,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One of these enzymes (PLC-II) was found to hydrolyze phosphatidyl-inositol 4,5-bisphosphate (PIP2) at a rate of 15.3 mumol/min/mg of protein and also phosphatidylinositol 4-monophosphate and phosphatidylinositol (PI) at slower rates. For hydrolysis of PI, this enzyme was activated by an acidic pH and a high concentration of Ca2+ and showed a Vmax value of 19.2 mumol/min/mg of protein. The other enzyme (PLC-III) catalyzed hydrolysis of PIP2 preferentially at a Vmax rate of 12.9 mumol/min/mg of protein and catalyzed that of phosphatidylinositol 4-monophosphate slightly. The rate of PIP2 hydrolysis by this enzyme exceeded that of PI under all conditions tested. Neither of these enzymes had any activity on phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, or phosphatidic acid. These two enzymes showed not only biochemical but also structural differences. Western blotting showed that antibodies directed against PLC-II did not react with PLC-III. Furthermore, the two enzymes gave different peptide maps after digestion with alpha-chymotrypsin or Staphylococcus aureus V8 protease. These results suggest that these two forms of PLC belong to different families of PLC.     

Isolation and characterization of two chondroitin lyases from Bacteroides thetaiotaomicron.

Two chondroitin lyases were isolated from the colon anaerobe Bacteroides thetaiotaomicron. Both enzymes had similar molecular weights (104,000 and 108,000) and similar isoelectric points (8.0 and 7.9, respectively). Both enzymes were active against chondroitin sulfates A, B, and C and unsulfated polysaccharides, such as chondroitin and hyaluronic acid, although one of the enzymes was twice as active against chondroitin as the other enzyme. Both had similar Km values for chondroitin sulfates A and C (40 to 70 micrograms/ml) and for chondroitin (300 to 400 micrograms/ml). Neither enzyme could degrade the highly sulfated mucopolysaccharide heparin, but heparin was a potent inhibitor of the activity of both enzymes. Although enzymes I and II were similar in many respects, a comparison of peptides resulting from partial digestion with N-chlorosuccinimide or papain demonstrated that the two proteins are not related.     

Isolation and characterization of two sequence-specific endonucleases from Anabaena variabilis.

Two endonucleases, AvaI and AvaII, were isolated from Anabaena variabilis on the basis of their ability to make a limited number of breaks at specific points in bacteriophage lambda DNA. Neither enzyme has cofactor requirements beyond Mg2+. Endonuclease AvaI makes eight breaks in the phage lambda chromosome at which the 5'-terminal sequence is pPy-C-G-N. AvaII endonuclease cuts phage lambda DNA more extensively, yielding fragments with the 5'-terminal sequence G-T-C-N or G-A-C-N. Neither enzyme generates cohesive ends.     

Isolation and characterization of two alkaline ribonucleases from calf serum.

Treatment of calf serum at 60 degrees C and pH 3.5 followed by chromatography on carboxymethyl (CM) cellulose resulted in the separation of two major peaks of alkaline RNAse activity. One was eluted from CM-cellulose at 0.075 M KCl with an overall purification of 5400-fold and the other was eluted at 0.25 M KCl with a 6700-fold purification. The RNAse eluted from CM-cellulose at 0.075 M KCl was almost completely inhibited by anti-RNAse A serum and by the endogenous RNAse inhibitor and a 33% inhibition was observed in the presence of 5 mM MgCl2. This enzyme seems to be similar or identical to RNAse A. The other RNAse, eluted from CM-cellulose at 0.25 M KCl was not inhibited by anti-RNAse A or 5 mM MgCl2 and was much less sensitive to the endogenous inhibitor. Both enzymes degraded RNA endonucleolytically and the nucleoside monophosphates obtained after partial hydrolysis of RNA by the two serum RNAases were primarily 2'- or 3' -CMP and 2'- or 3' -UMP. Poly(A), native DNA and denatured DNA were degraded slowly or not at all. The RNAase A-like enzyme degraded poly(C) at a significantly faster rate, and poly(U) at a slower rate, than RNA. However, the other serum RNAase was more active with poly(U) than with RNA and almost inactive with poly(C) as the substrate.     

Isolation and characterization of calcineurin from bovine brain

Calcineurin was isolated from bovine cerebrum extracts by sequential chromatography on Affi-Gel blue and calmodulin affinity columns. Calcineurin so isolated was approximately 90% pure and was composed of equimolar amounts of subunit A (Mr = 61 000-63 000) and subunit B (Mr = 15 000-17 000) when examined by sodium dodecyl sulfate gel electrophoresis. A polypeptide (less than 10%) with Mr = 71 000 whose function and role remains to be investigated, was routinely detected in the calcineurin preparation. Both inhibitory activity (towards calmodulin-dependent cAMP phosphodiesterase) and phosphatase activity (with 32P-labelled myelin basic protein as substrate) were associated with calcineurin as evidenced by (i) coelution from Affi-Gel blue, Affi-Gel calmodulin, diethythaminoethyl-Sepharose, and Sephacryl S-200 chromatography columns; (ii) association with the same protein band on nondenaturing gels; (iii) similar stability upon storage at 4 degrees C and with repeated freezing and thawing; and (iv) parallel heat inactivation. Phosphatase activity of calcineurin was maximal with 32P-labelled myelin basic protein as the substrate. Using this substrate, enzyme activity was generally stimulated 5- to 10-fold in the presence of Ca2+ and calmodulin; half-maximal activation (A0.5) was observed with 25 nM calmodulin. Calmodulin increased the Vmax of the reaction without affecting the Km for the substrate. Optimum temperature and pH for the reaction were 45 degrees C and 7, respectively, in both the absence and presence of Ca2+ and calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a novel peptide amide from porcine brain.

Peptide with C-terminal tyrosine amide was isolated from porcine brain by acid extraction and sequential steps of reverse phase HPLC. Microsequence, amino acid and mass spectral analyses revealed the structure: Ac-Ala-Ser-Glu-Lys-Arg-Pro-Ser-Glu-Arg-His-Gly-Ser-Lys- Tyr-amide. Since this peptide had the identical sequence to N-terminus of porcine myelin basic protein (pMBP) 1-14, we have designated porcine myelin peptide amide 14 (pMPA14). The final HPLC step yielded 20 micrograms of homogeneous peptide preparation from 20 kg brain tissue. Unlike other amidated peptides, pMPA14 may be produced by non enzymatic mechanism or unknown amidating enzyme. This unique amidation seems to occur exclusively to MBP in the brain.     

Synthesis of gamma-monodentate and beta, gamma-bidentate CrITP. Isolation and characterization of the two chelation isomers and the individual diastereomers of beta, gamma-bidentate CrITP

The two chelation isomers of CrITP, gamma-monodentate and beta, gamma-bidentate CrITP, as well as the diastereomers of beta, gamma-bidentate CrITP were synthesized, isolated, and characterized. Synthesis of these complexes was done using pH titration methods similar to that described by Cleland [W.W. Cleland, Methods Enzymol. 87, 159 (1982)], and separation of the two chelation isomers was accomplished with DEAE-sephadex A-25 using 0-0.3 N linear HCl gradient. Diastereomer separation (analytical and preparative scales) of beta, gamma-bidentate CrITP using reverse-phase high-performance liquid chromatography, and then analysis of the diastereomers with circular dichroism spectroscopy, shows four diastereomers that exist as two pairs of mirror-image isomers, similar to the four diastereomers of beta, gamma-bidentate CrATP as presented by Dunaway-Mariano and Cleland [D. Dunaway-Mariano and W.W. Cleland, Biochemistry 19, 1496 (1980)]. Reverse-phase high-performance liquid chromatography analysis of gamma-monodentate CrITP shows the presence of two major peaks, both of which convert to beta, gamma-bidentate CrITP upon incubation at pH 6.0 for 1 hr.     

Membranes of chromaffin granules. Isolation and partial characterization of two proteins

Membranes of chromaffin granules were isolated from the adrenal glands of four different species. The solubilized membrane proteins could be resolved into several bands by polyacrylamide-gel electrophoresis (alkaline and acid gel systems). Two major protein components appeared to be common to the chromaffin granule membranes of ox, horse, pig and man. The various membrane proteins of bovine chromaffin granules were separated by filtration on Sephadex G-200 in the presence of sodium dodecyl sulphate. Two major membrane proteins (A and B) were obtained in purified form. Treatment of protein A with 2-mercaptoethanol before electrophoresis resulted in two more rapidly migrating subunits, whereas protein B was unaffected by mercaptoethanol treatment. The amino acid compositions of the two purified proteins were determined. They are very similar to that of the total membrane proteins but significantly different from that of the chromogranins, the soluble proteins of chromaffin granules.     

Isolation and characterization of two vibrio-shaped methane-oxidizing bacteria

An enrichment method for, and the isolation of two related vibrio-shaped methane-oxidizing bacteria are described. Their morphological and physiological characteristics are given. As a name for the genus of the organisms Methy lovibrio is proposed.We wish to thank Miss W. E. de Boer and J. van der Toorn of this laboratory for making the photographs. One of the authors (P. J. Steennis) is indebted to the Royal Netherlands Fermentation Industries Ltd., Delft for a grant.     

Isolation and characterization of two Korean mistletoe lectins

Two isolectins (KML-IIU and the KML-IIL) were individually isolated from the previously reported Korean mistletoe lectin, KML-C, by using an immunoaffinity column. Molecular weights of the KML-IIU and the KML-IIL were 64 kDa and 60 kDa respectively. Both of the lectins were composed of heterogeneous A and B subunits linked with a disulfide bond, and showed the same carbohydrate-binding specificities for Gal and GalNAc. However, they are different not only in biophysical properties (glycosylation and amino acid compositions) but also bioactivities (cell killing and cytokine induction). The KML-IIL showed 17-145 times stronger in cytotoxicities to various human and mouse cancer cell lines than the KML-IIU. The KML-IIL also induced TNF-alpha secretion from mouse peritoneal macrophages 4.5 times better than the KML-IIU. The results demonstrated isolectins in Korean mistletoe were varied in bioactivities and the KML-IIL may be developed as an anti-cancer agent.