2,4,6-trinitrobenzenesulfonic acid modification of the carboxyl-terminal region (C-domain) of calreticulin

The role of the primary amino groups of lysine sidechains in Ca²⁺ binding to calreticulin was evaluated by chemical modification of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS). TNBS binding to calreticulin could be described by two steps: (i) a fast reaction, with low affinity, and (ii) a slow reaction with a relatively high affinity. Inclusion of Ca²⁺ and/or Mg²⁺ decreased both the amount of TNBS bound to calreticulin and the apparent affinity constant of the slower reaction. In contrast, the properties of the faster reaction for TNBS binding were not sensitive to Ca²⁺ and/or Mg²⁺. Analysis of TNBS binding to the carboxyl-terminal (C-domain) and aminoterminal (N-domain) of calreticulin revealed that theC-domain andN-domain are responsible for the slow and fast component of the TNBS binding, respectively. In keeping with this, in the presence of Ca²⁺, TNBS binding to theC-domain was significantly reduced, whereas modification of theN-domain was unaffected. TNBS modification of calreticulin significantly decreased Ca²⁺ binding to the low affinity/high capacity Ca²⁺ binding site(s) which are localized to theC-domain but had no effect on the high affinity/low capacity Ca²⁺ binding localized to theN-domain.In theC-domain of calreticulin, which contains the low affinity/high capacity Ca²⁺ binding sites, acidic residues are interspersed at regular intervals with one or more positively charged lysine and arginine residues. Our results indicate that the aminogroups of the lysine sidechains in theC-domain of calreticulin have a role in the low affinity/high capacity Ca²⁺ binding that is characteristic of this region of the protein and which is proposed to contribute significantly to the capacity of the endoplasmic reticulum Ca²⁺ store. (Mol Cell Biochem130: 19–28, 1994)Abbreviations TNBS 2,4,6-Trinitrobenzenesulfonic Acid - GST Glutathione S-Transferase - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - EDTA Ethylenediaminetetraacetic Acid - EGTA Ethylene Glycol bis(-aminoethylether)-N,N,N,N-tetraacetic Acid - MOPS 4-Morpholinepropanesulfonic Acid     

An improved automated Biuret method for the determination of microgram protein concentrations.

The inherently simple biuret reaction is hampered by lack of sensitivity and reproducibility in the determination of low protein concentrations. A method utilizing a modified biuret formulation and colorimeter range expander permits protein determination in the 0.01–3.0 mg/ml range at a sampling rate of 60/hr without significant sample interaction. This inexpensive method is useful in investigations involving large numbers of samples with microgram protein content.     

An improved method for the fluorometric determination of tissue catecholamines

Catecholamines extracted from tissues are readily measured by fluorescence following trihydroxyindole derivatization. A complicating factor however, has been the variably high background fluorescence levels obtained which adversely affect the precision of the assay. Employing EDTA is shown to reduce the high background fluorescence to a consistently low level, and to improve the accuracy of the trihydroxyindole method. It is suggested that high background fluorescence readings obtained by previous workers were due to metal ions extracted from tissue and carried over into the derivatization procedure.Measurement of tissue catecholamines by fluorescent methods is improved by the addition of EDTA which gives a consistently low background with a concommitant increase in accuracy.     

An improved method for the determination of alpha-tocopherol in sheep liver

A method is described for the analysis of α-tocopherol in sheep liver, employing a direct attack on the tissue with alcoholic alkali in the presence of pyrogallol and hydroquinone. The alkali treatment is rapid and avoids drying, grinding, and solvent extraction. Combined with two-dimensional paper chromatography, it eliminates time-consuming freezing and column chromatographic techniques. Mean ± SE recoveries of α-tocopherol alone, or mixed with liver tissue, were 97.1 ± 0.05% and 87.1 ± 4.1%, respectively. The mean ± SE levels of α-tocopherol in the liver of sheep fed dry rations were 1.0 ± 0.1 μg/g, and in that of sheep fed green pasture, 18.5 ± 1.4 μg/g.     

An improved method for identifying 8-O-acetyl-N-acetylneuraminic acid

The periodic acid/thionin-Schiff/potassium hydroxide/periodic acid/fuchsin-Schiff sequence developed by Culling et al. frequently causes damage to sections and gives inconsistent results because of insufficient primary oxidation and difficulties in making the thionin-Schiff reagent. These disadvantages have been largely eliminated by more thorough primary oxidation and by replacing the original thionin-Schiff with a new cold thionin-Schiff. The effect of alkaline hydrolysis on thionin-aldehyde complexes was also studied and the reduction of color caused by this treatment was restored by a second thionin-Schiff reaction. The new sequence gives consistent results and imparts greater color to the thionin-Schiff reaction.     

An improved method for the determination of γ-carboxyglutamic acid in proteins,bone, and urine

A method of high-performance liquid chromatographic separation of the fluorescence derivative of γ-carboxyglutamic acid (Gla) is presented. Alkaline hydrolysates of protein samples were reacted with o-phthalaldehyde in the presence of ethanethiol for 2 min, and the fluorescence derivative of γ-carboxyglutamic acid was resolved from the other amino acids by a short column packed with silica-based anion exchanger under isocratic conditions. By this method, as low as 200 fmol of γ-carboxyglutamic acid can be quantitatively analyzed within 10 min. The method presented here shortened the analysis time for Gla and was at least 10 times more sensitive than the method we described previously (Anal. Biochem.117, 259–265, 1981). The application of this method to the formic acid-soluble or insoluble γ-carboxyglutamic acid-containing proteins in chicken bone and the concomitant increase of γ-carboxyglutamic acid content in chicken bone with age are reported.     

An improved bioassay for ACTH determination.

An improved method for the bioassay of ACTH has been developed using short-term culture of adrenal cell suspensions derived from intact rats. Six separate pools of adrenals were used and cells derived from the same pool were compared after acute dispersion and overnight culture. The ED50, defined as the concentration of ACTH required to produce half maximal steroidogenesis, for cultured cells was 35.4+/-2 pg/ml compared to 240+/-60 pg/ml for cells used immediately after dispersion. The minimum concentrations of ACTH necessary to produce a response significantly above basal levels were 0.98+/-.10 pg/ml and 12.1+/-3 pg/ml respectively. Response characteristics of the cultured cells were highly reproducible. The incubation volume employed in this study was 0.25 ml, so the ED50 expressed as dose of ACTH per tube was actually 8.8 pg for the cultured cells. Such sensitivity provides requisite methodology for the measurement of ACTH under varying physiological conditions.