A gel filtration method has been developed for the complete removal of sodium dodecyl sulfate (SDS) from proteins and peptides. The protein or peptide (20 μg–10 mg) containing SDS (up to 30–60 mg) is dissolved in a mixture of propionic acid, formic acid, and water (2:1:2, ). Under these conditions, protein-SDS (or peptide-SDS) complexes, as well as SDS micelles, are dissociated. Subsequently, protein and SDS can be separated on a small Sephadex G-25 superfine column. The recovery of protein is typically 90% or more. 相似文献
We have investigated the induction of competence (IL-2 responsiveness) and progression in human T lymphocyte proliferation triggered by phorbol ester and calcium ionophore. The degree of proliferation induced with the phorbol ester, phorbol 12,13-dibutyrate (PDB) and the calcium ionophore ionomycin was dependent on the duration of exposure to these agents, with more than 6 h required for obtaining maximum proliferation. Following brief exposure to both agents for 30 min, which did not cause significant proliferation, T cells became competent to proliferate in response to exogenous interleukin 2 (IL-2). These competent T cells also progressed to DNA synthesis following incubation with PDB in the absence of ionomycin. Induction of competence to proliferate in response to either PDB or IL-2 was blocked by EGTA, suggesting that transmembrane Ca2+ flux was obligatory at this stage. Since other phorbol esters and synthetic diacylglycerols also stimulated DNA synthesis in competent cells, it is likely that progression was triggered by activation of protein kinase C. Following a brief exposure to PDB and ionomycin, subsequent incubation with PDB induced gene expression and secretion of IL-2 and augmented the expression of IL-2 receptors in the competent cells. Thus, we have demonstrated that Ca2+ mobilization is required for rendering T cells competent to express functional IL-2 receptors, to produce IL-2 in response to subsequent incubation with PDB, and that sustained activation of protein kinase C seems necessary for IL-2 production and subsequent progression of competent T cells to DNA synthesis. 相似文献
Summary A reproducible method is described for the separation and quantification of ascorbic acid and dehydroascorbic acid by ion-pairing
reverse-phase high performance liquid chromatography and detection by absorbance at 232 nm. Lowest detectable concentrations
with a linear response of detection were 5 nmol for ascorbic acid and 50 nmol for dehydroascorbic acid. This method was applied
to the analysis of C3H/10T1/2 cells and culture medium after influx or efflux experiments and single or multiple treatments
with ascorbic acid. Subsequent measurement of the radioactivity in the eluted fractions increased the detectability of both
ascorbic acid and dehydroascorbic acid to 10 to 20 pmol.
This research was supported by grant CA 09320 and CA 31574 from the National Cancer Institute, Bethesda, MD, and grant BC441
from The American Cancer Society. 相似文献
A simpler method for determining aldosterone secretion rate (ASR) has several applications. High performance liquid chromatography (HPLC) has several advantages over traditional chromatographic methods for purification to constant specific activity of aldosterone liberated from its 18-glucuronide by acid hydrolysis. We found it necessary to introduce several modifications to remove urochromes before HPLC. Two methods for determining ASR were developed. With Method A a more traditional initial procedure was followed, and Sephadex LH-20 chromatography allowed removal of considerable urochromes before HPLC. However, aldosterone recovery was improved with Method B, which employed several bonded phase silica derivatives (Sepralytes) and a PBE 94 column to remove urochromes before HPLC. With this procedure the Sephadex LH-20 chromatography was not required. Aldosterone purification to constant specific activity was achieved by HPLC on a diol column with a normal phase system, and quantification was performed by RIA. ASR determinations were equivalent with both methods. This methodology should be applicable to other steroid secretory rate determinations and to applications involving purification of steroid conjugates. 相似文献
Ribulose-1,5-bisphosphate carboxylase activity was found in endosperm of germinating castor bean seed Ricinus communis and was localized in proplastids. The endosperm carboxylase has been extensively purified and is composed of two different subunits. The molecular weights of the native carboxylase and its subunits were 560,000, 55,000, and 15,000 daltons, respectively. The Michaelis-Menten constants, Km, for the endosperm carboxylase with respect to ribulose 1,5-bisphosphate, bicarbonate, CO2, and magnesium in millimolar are 0.54, 13.60, 0.92, and 0.57, respectively. The endosperm carboxylase was activated by Mg2+ and HCO3−. The preincubation of the carboxylase with 1 millimolar HCO3− and 5 millimolar MgCl2 resulted in activation by low and inhibition by high concentrations of 6-phosphogluconate.
In studies of dark 14CO2 fixation by endosperm slices, [14C]malate and [14C]citrate were the predominantly labeled products after 30 seconds of exposure of the tissue to H14CO3−. In pulse-chase experiments, 87% of the label is malate, and citrate was transferred to sugars after a 60-minute chase with a small amount of the label appearing in the incubation medium as 14CO2. The minimal incorporation of the label from 14CO2 into phosphoglyceric acid indicated a lack of the endosperm ribulose-1,5-bisphosphate carboxylase participation in the endosperm's CO2 fixation system. The activities of key Calvin cycle enzymes were examined in the endosperms and cotyledons of dark-grown castor bean seedlings. Many of these autotrophic enzymes develop in the dark in these tissues. The synthesis of ribulose-1,5-bisphosphate carboxylase in the nonphotosynthetic endosperms is not repressed in the dark, and high levels of enzymic activity appear with germination. All of the Calvin cycle enzymes are present in the castor bean endosperm except NADP-linked glyceraldehyde 3-P dehydrogenase, and the absence of this dehydrogenase probably prevents the functioning of these series of reactions in dark CO2 fixation.
A partially purified M protein, extracted from a mouse-virulent strain of type 3, group A streptococcus, was administered subcutaneously in gradually increasing amounts at weekly intervals to 21 children in a Family Program. Seven children with type 3 bactericidal antibody in prevaccination sera showed a secondary response. Of 14 children with no detectable type 3 bactericidal antibody prior to vaccination, 13 developed definite type 3 antibody during or soon after vaccination. This response appeared to be of the primary variety in at least some of the 13 children because (i) the total incidence of antibody response (20 of 21) was greater than can be accounted for by the documented incidence of clinical and subclinical type 3 infections among children of our Family Program during a period of 14 years, (ii) the response in the 13 children with no detectable antibody prior to vaccination was more delayed than in those showing a definite secondary response, and (iii) on the average, the amount of vaccine needed for a response in these 13 children was 15 to 28 times greater than that required for the secondary response. Local skin reactions were variable among the vaccinated children. Systemic reactions were infrequent and not severe. The giving of multiple injections of partially purified M protein did not seem to cause cutaneous hypersensitivity. 相似文献
This paper describes some new characteristics of the phosphoenolpyruvate carboxykinase CO(2)-oxaloacetate exchange reaction in purified preparations of Rhodospirillum rubrum. The enzymatic activity has been purified 169-fold. Nucleotide diphosphates substitute for nucleotide triphosphates in the exchange reaction. Nucleotide diphosphates will not support the synthesis of phosphoenolpyruvate from oxaloacetate. This reaction differs significantly from the CO(2)-oxaloacetate exchange reaction in higher plants and animals. 相似文献