scholarly journals The major proteoglycan of adult rabbit skeletal muscle. Relationship to small proteoglycans of other tissues

1991 ◽  
Vol 274 (1) ◽  
pp. 219-223 ◽  
Author(s):  
N Parthasarathy ◽  
L Chandrasekaran ◽  
M L Tanzer
Keyword(s):  
Skeletal Muscle ◽  
Ion Exchange ◽  
Gel Electrophoresis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Rabbit Skeletal Muscle ◽  
Adult Rabbit ◽  
Bovine Bone ◽  
Adult Skeletal Muscle ◽  
N Terminus

We have been interested in examining the putative biological role(s) of the major proteoglycan of adult skeletal muscle. The small proteoglycans of adult rabbit skeletal muscle and tendon were extracted and purified by sequential density-gradient ultracentrifugation, ion-exchange chromatography and gel filtration. They appeared to be homogeneous by the criterion of gel electrophoresis in SDS and to yield one major product, the core protein, after digestion with chondroitin ABC lyase, also observed after gel electrophoresis. Two major products were obtained when the intact proteoglycans were cleaved by CNBr, and those peptides were separated by SDS/PAGE and by ion-exchange chromatography. Sequencing of the N-terminal amino acids of either the intact proteoglycans or the CNBr-cleaved products allowed for comparison of the muscle and tendon proteoglycan with derived amino acid sequences previously reported for bovine bone proteoglycan. The bone and tendon proteoglycan sequences were remarkably similar, whereas those of the muscle proteoglycan differed from the other two molecules. The major site of glycosaminoglycan substitution was on a peptide fragment distant from the N-terminus, and a presumptive serine residue at position 4 from the N-terminus also appeared to be substituted, perhaps with a small glycosaminoglycan chain. These results provide some insight into the diversity of small proteoglycans of the PG-II class and provide a basis for exploring their mode of genetic expression.

Rapid separation of bovine caseins by mass ion exchange chromatography

1989 ◽  
Vol 56 (3) ◽  
pp. 391-397 ◽  
Author(s):  
K. F. Ng-Kwai-Hang ◽  
J. P. Pélissier
Keyword(s):  
Liquid Chromatography ◽  
Ion Exchange ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Rapid Separation ◽  
Rapid Isolation ◽  
Time Required

SummaryThe rapid isolation of major bovine caseins in gram quantities was investigated. Whole casein was precipitated from individual cow's milk by adjusting the pH to 4·6 and the precipitated casein was suspended in 4·5 M urea (pH 8·0) containing 0·02 M imidazole and 0·03 M β-mercaptoethanol, and bound on a QAE Zeta Prep 250 cartridge. Stepwise elution with the urea/imidazole β-mercaptoethanol buffer and varying amounts of NaCl gave five well resolved peaks, which were identified by polyacrylamide gel electrophoresis and fast protein liquid chromatography to be pure γ-casein, κ-casein. β-casein, β-casein and αs-casein, respectively. The ion exchange cartridge was regenerated by flushing with buffer containing 0·50 Μ-NaCl followed by equilibration with starting buffer before separation of next sample. The time required to run each sample including cartridge regeneration and equilibration was 4 hours.

Plasma clearance, metabolism, and tissue accumulation of 3H-labeled catecholamines in trout

1986 ◽  
Vol 250 (3) ◽  
pp. R519-R525 ◽  
Author(s):  
N. P. Nekvasil ◽  
K. R. Olson
Keyword(s):  
Skeletal Muscle ◽  
Ion Exchange ◽  
Plasma Clearance ◽  
Large Mass ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Salmo Gairdneri ◽  
Tissue Accumulation ◽  
Two Component ◽  
Kidney Liver

Plasma clearance, metabolism, and tissue accumulation of [3H]norepinephrine (NE) and [3H]epinephrine (E) were measured after injection into the dorsal aorta of chronically catheterized trout, Salmo gairdneri. Sucrose, an inert volume marker, was injected with the catecholamines (CAs). Ion-exchange chromatography was used to separate unmetabolized CAs from deaminated and O-methylated metabolites in plasma. Both CAs are cleared from plasma at an exponential two-component rate. By 10 min postinjection, CA-specific extraction lowered plasma [3H]NE by 65% and [3H]E by 50%. Over 80% of the 3H remaining in plasma 10 min after injection was metabolized to O-methylated and deaminated products. Thus trout are able to quickly and efficiently lower circulating CA levels through tissue accumulation and metabolism. Kidney, liver, spleen, and atrium accumulate more CA than other tissues, although most tissues bind CA to some extent. Gills preferentially accumulate CAs over sucrose. Skeletal muscle has a low affinity for CAs but by virtue of its large mass may be an important organ in CA metabolism. NE is removed from the circulation faster, and more NE is bound to tissues than E. A blood-brain barrier for E but not NE was observed.

scholarly journals Properties and N-terminal sequence of allophycocyanin from the unicellular rhodophyte Cyanidium caldarium

1977 ◽  
Vol 163 (3) ◽  
pp. 571-581 ◽  
Author(s):  
A S Brown ◽  
R F Troxler
Keyword(s):  
Molecular Weight ◽  
Ion Exchange ◽  
Phosphate Buffer ◽  
Alpha Helix ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Polyacrylamide Gels ◽  
Cyanidium Caldarium ◽  
N Terminus ◽  
Circular Dichroic

Allophycocyanin from the unicellular rhodophyte Cyanidium caldarium was purified by (NH4)2SO4 fractionation and ion-exchange chromatography on brushite (calcium phosphate) columns and on DEAE-Sephadex A-25 columns. The specific absorption coefficient (A0.1%1cm) at 650nm of purified allophycocyanin was 6.35 in 0.05M-potassium phosphate buffer, pH7.0. Absorption maxima of allophycocyanin occurred at 650, 618 (shoulder), 350 and 275 nm. Circular-dichroic spectra displayed positive-ellipticity bands at 658 and 630 nm and a major negative-ellipticity band at 340nm. Computer analysis of the circular-dichroic spectrum of allophycocyanin from 207 to 243 nm indicated 42% alpha-helix and 58% beta-form. The estimated molecular weight of purified allophycocyanin on calibrated Sephadex G-200 columns at pH7.0. was 196000. Electrophoretic examination of allophycocyanin on sodium dodecyl sulphate/polyacrylamide gels revealed a single band with apparent mol.wt. 16000. The presence of two polypeptide subunits, with nearly the same molecular weight, was revealed on polyacrylamide gels by using a modified electrophoresis buffer. Spectral analysis of the allophycocyanin subunits resolved by ion-exchange chromatography on Bio-Rex 70 columns indicated that a single phycocyanobilin chromophore was present on each polypeptide chain. Treatment of allophycocyanin with 8M-urea (pH3.0) and subsequent removal of urea by dialysis against water yielded a derivative phycobiliprotein with spectroscopic characteristics similar to those of phycocyanin. The original allophycocyanin spectrum was regenerated after incubation in phosphate buffer, pH7.0. Automated sequences analysis of the N-terminus of allophycocyanin showed that (a) the sequences of the two subunits were different from one another and were different from the subunits of phycocyanin from the same alga, (b) the subunits occurred in a molar ratio of 1:1 and (c) the sequences homology at the N-terminus among alpha- and beta-subunits of allophycocyanin from blue-green and red algae approached 90%.

scholarly journals Purification and Partial Characterization of a Murein Hydrolase, Millericin B, Produced by Streptococcus milleri NMSCC 061

2000 ◽  
Vol 66 (1) ◽  
pp. 23-28 ◽  
Author(s):  
M. Beukes ◽  
G. Bierbaum ◽  
H.-G. Sahl ◽  
J. W. Hastings
Keyword(s):  
Ion Exchange ◽  
Specific Activity ◽  
Gel Filtration ◽  
Fold Increase ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Antimicrobial Substances ◽  
Streptococcus Milleri ◽  
N Terminus

ABSTRACT Streptococcus milleri NMSCC 061 was screened for antimicrobial substances and shown to produce a bacteriolytic cell wall hydrolase, termed millericin B. The enzyme was purified to homogeneity by a four-step purification procedure that consisted of ammonium sulfate precipitation followed by gel filtration, ultrafiltration, and ion-exchange chromatography. The yield following ion-exchange chromatography was 6.4%, with a greater-than-2,000-fold increase in specific activity. The molecular weight of the enzyme was 28,924 as determined by electrospray mass spectrometry. The amino acid sequences of both the N terminus of the enzyme (NH2 SENDFSLAMVSN) and an internal fragment which was generated by cyanogen bromide cleavage (NH2 SIQTNAPWGL) were determined by automated Edman degradation. Millericin B displayed a broad spectrum of activity against gram-positive bacteria but was not active againstBacillus subtilis W23 or Escherichia coli ATCC 486 or against the producer strain itself. N-Dinitrophenyl derivatization and hydrazine hydrolysis of free amino and free carboxyl groups liberated from peptidoglycan digested with millericin B followed by thin-layer chromatography showed millericin B to be an endopeptidase with multiple activities. It cleaves the stem peptide at the N terminus of glutamic acid as well as the N terminus of the last residue in the interpeptide cross-link of susceptible strains.

scholarly journals Purified Haemoglobin Preparations in the Evaluation of HbA1c Determination by Ion Exchange Chromatography

1993 ◽  
Vol 30 (3) ◽  
pp. 265-271 ◽  
Author(s):  
Margo P Cohen ◽  
Janice Witt ◽  
Van-Yu Wu
Keyword(s):  
Ion Exchange ◽  
Gel Electrophoresis ◽  
High Performance ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Clinical Samples ◽  
Ion Exchange Resins ◽  
Substantial Portion ◽  
Blood Samples ◽  
Exchange Resins

The use of ion exchange resins for the estimation of HbA1c in clinical samples rests on the assumption that HbA1c is effectively and efficiently separated from other N-terminally modified haemoglobins and from HbAo. To test this assumption, we applied highly purified preparations of HbA1a+1b, HbA1c and HbAo to ion exchange minicolumns, using conditions of application simulating actual blood samples and the first and second elution buffers provided by the manufacturer. The authenticity and purity of the applied haemoglobin preparations were documented by high performance liquid chromatography, gel electrophoresis and carbohydrate content. About 40% of the applied HbA1a+1b eluted in the first fraction; 45% eluted in the second fraction, and 10% to 15% required 1 mol/L NaCl to elute from the column. Of the applied HbA1c, 65–80% eluted where expected in the second fraction, about 20% required 1 mol/L NaCl to elute from the column, and the remainder eluted with HbA1a+1b. Some 3–6% of pure HbAo applied to minicolumns emerged in the second fraction, with the remainder eluting as expected after making the buffer 1 mol/L in NaCl. The results indicate that the fraction eluting from ion exchange minicolumn chromatography that is designated ‘HbA1c’ contains HbA1a+1b, and that a substantial portion of the HbA1c in an applied sample does not elute in this fraction.

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