The in vivo stimulation of mannose incorporation into mannosylretinylphosphate, dolichylmannosylphosphate, and specific glycopeptides of rat liver by high doses of retinylpalmitate

Excess vitamin A stimulated the incorporation of mannose into rat liver mannosylretinylphosphate (MRP), dolichylmannosylphosphate (DMP), and into glycoproteins by over 200% during a 20-min labeling period. The glycoproteins were digested with pronase and separated into three components by molecular sieve chromatography. The stimulation of mannose incorporation was greatest in the Peak II glycopeptide (Mr = 6500). In contrast, the incorporation of galactose into glycolipids or glycopeptides was not altered by vitamin A treatment. Analysis of the glycopeptide stimulated by vitamin A treatment showed it to contain mannose, glucose, galactose, and glucosamine in the respective molar ratios of 7:10:17:1 and to be rich in glutamic acid, serine, glycine, aspartic acid, and threonine. The results suggest that excess vitamin A stimulates the incorporation of mannose into glycoproteins by enhancing the synthesis of lipid intermediates involved in specific mannosyl transfer reactions.     

Separation of DNA restriction fragments by ion-exchange chromatography on FPLC columns Mono P and Mono Q

Separation of DNA restriction fragments by FPLC ion-exchange chromatography on Mono Q and Mono P columns was investigated. The columns were found to be particularly suitable for the separation of fragments up to 500–600 bp long. Larger fragments can also be separated although less effectively. We found the following practical working ranges for the parameters investigated: pH, 4 to 11; flow rate, 0.05 to 0.6 ml/min corresponding to separation times between 2 and 20 h. (better resolution is achieved at lower flow rates); gradient slope; between 0.5 mm eluting salt/ml buffer and over 5 mm/ml (better resolution is achieved at lower gradient slopes; eluting ionic strength was found to be independent of gradient slope); gradient composition, chloride salts of smaller monovalent cations eluted the DNA at lower ionic strengths but separations obtained were similar; additives, substances such as urea, formamide, and EDTA can be added without chromatographic effects; sample amount: amounts from 2.5 to 200 μg were applied, corresponding to single peak content of from 42 ng to 74μg DNA. Yields were generally over 90% and the chromatographed DNA was fully accessible to restriction enzyme cleavage. Separations occurred predominantly according to DNA size, but AT-rich fragments were retarded in a predictable way.     

Separation of paraproteins from human plasma by membrane chromatography

Membrane chromatography using a commercially available blotting membrane was performed in a dead-end filtration mode to separate paraproteins from plasma of patients suffering from paraproteinemia. The affinity membrane was found to display distinct specificity to monoclonal IgG1. A dissociation constant (K_d) of 3.2 μM and a maximum binding capacity of 1.43 mg/cm² IgG1 paraprotein were obtained from the adsorption isotherm of the affinity membrane. The membrane was found to absorb immunoglobulins species-dependently because no binding of immunoglobulins from mouse, rat and rabbit could be observed.     

Separation of bacterial luciferase from oxidoreductases by affinity chromatography

The NADH-dependent and NADPH-dependent oxidoreductase activities associated with bacterial luciferase in Vibrio (Beneckea) harveyi can simultaneously be removed from purified luciferase through a Blue Sepharose CL-6B column. The result achieved with this one-step affinity chromatography is similar to that obtained with two sequential "reverse-affinity" chromatographies.     

Analysis of thiohydantoins from amino acids by gas-liquid chromatography on short glass capillary columns

A method for separation of amino acid methyl or phenyl thiohydantoins by GLC on a short glass capillary column is described. Calculations of required parameters of the capillary column are presented. By the described methods, nineteen of twenty silylated methylthiohydantoins were separated in one run. The last one (histidine) can be identified on the same column by starting the analysis at a higher temperature. Cysteine and arginine were analysed as S-methylcysteine and ornithine, respectively.     

Separation of urinary oestrogen sulphates from oestrogen glucosiduronates on diethylaminoethyl-Sephadex columns

1. Gradient elution (sodium chloride concentration gradient) on DEAE-Sephadex columns is used to separate urinary oestrogen conjugates of pregnancy urine. Changes in the shape of the gradient alter the chromatograms in a predictable manner so that the dispersion of peaks can be modified at will. 2. Suitable choice of the gradient results in the separation from each other of oestrogen sulphates, oestrogen 16(or 17)-glucosiduronates, oestrogen 3-glucosiduronates and free oestrogens. 3. Evidence for the presence of oestriol 3-sulphate, oestrone 3-sulphate, 17β-oestradiol 3-sulphate, 16-oxo-17β-oestradiol 3-sulphate and oestriol 16(or 17)-sulphate in the sulphate fraction of DEAE-Sephadex chromatograms of pregnancy urine is provided.     

Heterogeneity of tropocollagen as investigated by chromatography on hydroxyapatite columns

The chromatography of native acid-soluble tropocollagen from calf skin on hydroxyapatite columns has been investigated. Resolution of a number of chromatographic peaks has been obtained by using shallow slopes of the eluting phosphate gradient. The results obtained suggest a heterogeneity of tropocollagen molecules which might be due to different distributions of absorbing sites on the molecules. The results obtained have also been used to study the mechanism of chromatography of proteins on hydroxyapatite and the resolving power of the columns.     

Affinity chromatography of nonhistone chromosomal proteins from lymphocytes on DNA-agarose columns

A two step procedure is presented consisting of hydroxyapatite and DNA-agarose chromatography which allows the isolation of nonhistone chromosomal proteins with different affinities towards single stranded DNA. The application of this fractionation scheme to nonhistone chromosomal proteins from bovine lymphocytes is described.