No abstract available

[en] The synthesis of N-maleoylmethionine sulphone (MMS), a membrane-impermeant protein-labelling reagent, is described. Radioactively labelled MMS can be readily prepared at high specific radioactivity from [³⁵S]methionine. The permeability of the erythrocyte membrane to the reagent was assessed by determining the extent of inactivation of glyceraldehyde 3-phosphate dehydrogenase after treatment of erythrocytes with MMS. Some inactivation of this enzyme was found when high concentrations (20mM) of the compound were used, but this could be prevented by pretreatment of the erythrocytes with 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid, suggesting that MMS slowly enters the cells via the anion-transport system. Treatment of erythrocytes with [³⁵S]MMS resulted in the labelling of six major components. Labelling of erythrocyte membranes resulted in the intense labelling of many additional components. MMS inhibited erythrocyte glucose transport. Cytochalasin b protected glucose transport against inactivation by MMS. Labelling experiments on erythrocytes in the presence and in the absence of cytochalasin b showed that the cytochalasin b-protected material was a broad band in the band-4.5 region. (author)

[en] Previous studies on protein turnover in ³H-labelled L-cell cultures have shown recovery of total ³H at the end of a three-day experiment to be always significantly in excess of the ³H recovered at the beginning of the experiment. A number of possible sources for this error in measuring radioactivity in cell proteins has been reviewed. ³H-labelled proteins, when dissolved in NaOH and counted for radioactivity in a liquid-scintillation spectrometer, showed losses of 30-40% of the radioactivity; neither external or internal standardization compensated for this loss. Hydrolysis of these proteins with either Pronase or concentrated HCl significantly increased the measured radioactivity. In addition, 5-10% of the cell protein is left on the plastic culture dish when cells are recovered in phosphate-buffered saline. Furthermore, this surface-adherent protein, after pulse labelling, contains proteins of high radioactivity that turn over rapidly and make a major contribution to the accumulating radioactivity in the medium. These combined errors can account for up to 60% of the total radioactivity in the cell culture. Similar analytical errors have been found in studies of other cell cultures. The effect of these analytical errors on estimates of protein turnover in cell cultures is discussed. (author)

[en] The radiation-inactivation method allows the determination of the Msub(r) of enzymes and receptors by monitoring the decay of biological activity as a function of absorbed dose. The presence of regulatory or effector proteins (inhibitors or activators) associated with an enzyme or receptor, or released in the preparation after tissue homogenization, may affect the decay of biological activity. How the activity is affected, however, will depend on the type of inhibition (competitive or non-competitive), the inhibitor or activator concentration, the dissociation constant of the enzyme-effector system, and the effector Msub(r) relative to that of the enzyme. Since little is known on how effector proteins influence radiation inactivation of enzymes and receptors, we have considered a theoretical model in an effort to provide a framework for the interpretation of experimentally obtained data. Our model predicts that competitive and non-competitive inhibitors of enzymes could be distinguished by analysing irradiated samples with various substrate concentrations. Inhibitors will decrease whereas activators will increase the apparent target size of enzymes or receptors. (author)

[en] A sensitive radioimmunoassay was developed for the energy-transducing adenosine triphosphatase (F₁-ATPase, EC 3.6.1.3) of Micrococcus lysodeikticus and the assay was extended to the α-, β- and γ-subunits of the enzyme. These subunits were isolated and cross-reactions studied. (author)

[en] A rapid and simple enzymic method is described for the synthesis of [³²P]phosphoenolpyruvate from [³²P]Psub(i), with a reproducible yield of 74%. The final product was shown to be a good substrate for pyruvate kinase (EC 2.7.1.40). (author)

[en] A convenient, inexpensive assay was developed for measuring relative changes in cyclic GMP in whole mouse neuroblastoma cells (clone NIE 115) based on labelling the cellular GTP pool with [8-³H]guanine. The time course of cell labelling and the distribution of radioactivity among possible products were studied; GTP is the only major labelled species. Radioactive cyclic GMP produced from the radioactive GTP on cell stimulation is isolated by column chromatography and its identity has been rigorously established by paper chromatography and ion-exchange chromatography. The assay was used to study the time course of the cyclic GMP changes that occur after stimulation of neuroblastoma cells with carbamoylcholine and the dependence of the cyclic GMP changes on the carbamoylcholine concentration. The assay gives results comparable with those obtained by using a radioimmunoassay for cyclic GMP and should be applicable to other whole-cell and tissue-slice systems. (author)

[en] A method is described for radiolabelling proteins with O-(4-diazo-3,5-di[¹²⁵I]iodobenzoyl)sucrose (DD¹²⁵IBS). When proteins so labelled were degraded within lysosomes, the radioactive fragments were largely retained within the organelle. High specific radioactivities were obtained without changing the properties of the protein. The validity of the method was demonstrated in vivo in rats using the short-lived protein lactate dehydrogenase, isoenzyme M₄, and the long-lived protein bovine serum albumin. Derivatization with DD¹²⁵IBS did not alter the clearance of either protein. Uptake of DD¹²⁵IBS-labelled lactate dehydrogenase, isoenzyme M₄, by liver and spleen of rats was determined. Radioactivity in these tissues increased up to about 2 h after injection (at this time the protein has been almost completely cleared from the blood) and subsequently declined with a half-life of approx. 20h. After differential fractionation of liver, radioactivity was largely found in the mitochondrial and lysosomal fraction. The results of these studies establish that DD¹²⁵IBS covalently coupled to plasma proteins should be a useful radioactive tracer for identifying the tissue and cellular sites of catabolism of relatively long-lived circulating proteins. (author)

[en] A radioiodinated, intracellularly trapped adduct of cellobiose and tyramine was used to determine the sites of plasma protein degradation in vivo. Proteins derivatized with the radioiodinated ligand were recognized as underivatized proteins both in vitro and vivo. On degradation of derivatized low-density lipoprotein, the rate of leakage from cultured fibroblasts was only 5% during 24 h. Similarly, on injection of labelled proteins into rats and rabbits, urinary excretion of the label was less than 10% of total labelled catabolic products recovered 24 h after injection. Examination of the tissue contents of label at two times after injection of labelled asialofetuin or apolipoprotein A1 in rats, and asialotransferrin in rabbits showed that the label did not detectably redistribute between tissues after initial uptake and catabolism; a significant leakage from liver was quantitatively accounted for by label appearing in gut contents and faeces. A simple double-label method was devised to provide a correction for intact protein in trapped plasma, the extravascular spaces, and within cells. By using this method it becomes unnecessary to fractionate tissue samples. (author)

[en] A simple method has been developed for labelling human plasma lipoproteins to high specific radioactivity with radioactive cholesteryl esters in vitro. After isolation by preparative ultracentrifugation, the selected lipoprotein was incubated for 30 min at 4⁰C in human serum (d > 1.215) that had been prelabelled with [4-¹⁴C]cholesteryl oleate or [1,2-³H]cholesteryl linoleate, and was then re-isolated by ultracentrifugation. All major lipoprotein classes were labelled by the procedure. Specific radioactivities of up to 18 d.p.m. .pmol^-1 (46d.p.m. .ng^-1) were achieved. When radiolabelled high-density lipoprotein was infused intravenously, the radioactive cholesteryl ester behaved in vivo indistinguishably from endogenous cholesteryl esters produced by the lecithin (phosphatidylcholine): cholesterol acyltransferase reaction. (author)