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The yield and purity of synaptic plasma membranes (SPM) and synaptic junctions (SJ) from rat brain has been examined as a function of the concentration of rho-iodonitrotetrazolium (INT)--succinate used during their preparation. An INT concentration of 1 mg/g brain tissue (wet weight) was sufficient to obtain SPM and SJ of purity comparable to that obtained using 4--6 times that concentration of dye (1--3). At this lower level of INT the yield of SPM increased by about 100%, whereas mitochondrial contamination remained at 10--13% of the total SPM protein. At concentrations of INT below 0.5 mg/g brain tissue (wet weight) the contamination of SPM by mitochondria increased rapidly. At very low concentrations of INT (0.13 mg/g tissue) the contaminating protein of mitochondrial origin was 40--50% of the total protein in the SPM fraction. Examination by gel electrophoresis of SPM, SJ, and mitochondrial fractions with different degrees of cross-contamination allowed the assignment of marker polypeptides for mitochondrial, junctional, and nonjunctional plasma membranes. Under the conditions used to prepare SJ, a variable amount of particulate material floated over 1.0 M sucrose. It consisted of SJ and many membrane vesicles and had a protein composition similar to that of SJ contaminated by extrajunctional membrane proteins. An analogous fraction arose during in the preparation of postsynaptic densities.

1. 1. Gill tissue from eels was mechanically disrupted and subjected to density gradient centrifugation to yield an enriched population of chloride cells and an enriched population of respiratory epithelial cells. 2. 2. Chloride cells isolated in this way appeared morphologically intact in the light microscope but electron microscopic examination showed that the plasma membrane was damaged. 3. 3. The specific activity of Na + /K + -ATPase in sea water fish was more than thirty times greater in the chloride cell population than in the respiratory epithelial cell population. 4. 4. Cells released from gill tissue (prior to density gradient centrifugation) catalysed the reduction of iodonitrotetrazolium violet in the presence of succinate; the specific activity of succinate dehydrogenase measured in this way was 2·5 times greater in cells from sea water fish than in those from fresh water fish. 5. 5. Density gradient separation of cells reacted with succinate and iodonitrotetrazolium showed that the enhanced succinic dehydrogenase activity of sea water gills was localized in the chloride cells.

The relative efficiencies of phenazine methosulfate (PMS), 1-methoxy-phenazine methosulfate (MPMS) and Meldola Blue (MB) as electron carriers were determined biochemically (non-enzymic NADH-tetrazolium salt-test) and by quantitative histochemistry (heart and kidney slices; succinate dehydrogenase, SDH; lactate dehydrogenase, LDH). MPMS developed the highest electron transfer velocity in biochemical assays. The reaction was independent of the pH value between 7.0-8.5. PMS and MB always showed a lower transfer ability in biochemical tests which was higher with iodonitrotetrazolium chloride (INT) than with nitro blue tetrazolium chloride (NBT). A distinct pH dependence was demonstrable with MB in this respect, preferentially using INT as tetrazolium salt. Quantitative histochemical results with electron carriers are often at variance with biochemical ones. MPMS leads to somewhat higher demonstrable activities only in the determination of the NAD-dependent LDH, whereas MB results in somewhat higher LDH activity than PMS (reaction medium with agarose). MB and PMS yielded almost equally high activities in the demonstration of the flavoprotein-dependent SDH using a reaction medium with agarose. With an aqueous reaction medium, PMS resulted in higher SDH activities than MB. MPMS always had the lowest efficiency in electron transfer ability using an aqueous or agarose containing reaction medium (SDH). With PVA in the reaction medium (SDH determination) PMS was clearly superior to MPMS. MB showed only a small transfer activity under these conditions because PVA seems to bind MB almost completely. It is concluded that in histochemistry an appropriate electron carrier and electron carrier concentration must be determined for different incubation conditions, tissues, tissue preparations and dehydrogenases studied. General statements about the efficiency or inefficiency of an electron carrier as a result of only one incubation condition does not seem to be justified.

Synaptosomal plasma membrane (SPM) and other subcellular fractions were isolated from the forebrain of 1-day-old chickens by a procedure based on that of Davis and Bloom (16) and Cotman and Taylor (13). The procedure involves the centrifugation through a discontinuous sucrose gradient of a crude synaptosomal-mitochondrial fraction which has been lysed and weighed with iodonitrotetrazolium. SPM isolated by this method contains only small amounts of lysosomal or mitochondrial membranes and is practically devoid of contaminating microsomal membranes, as estimated by enzyme marker assays. The purity of chick-brain SPM prepared by this method is compared to the purity of chick-brain fractions obtained by two other laboratories, using different methods (4, 59). The SPM were extracted with Triton X-100 and all fractions solubilized in sodium dodecyl sulfate (sds). The delipidated proteins of all fractions were subjected to SDS-polyacrylamide electrophoresis on slab gels and stained for protein. A distinct difference was observed between the patterns given by the Triton-soluble and -insoluble fractions. Electron microscopy of the synaptic junction fraction showed numerous junctional complexes.

The intracellular localization of 3β-hydroxysteroiddehydrogenase/Δ5-isomerase (Δ5-3β-OHD) in bovine adrenal cortex was determined by density perturbation with density gradient centrifugation and the use of marker enzymes. Mitochondrial rich fractions after incubation with succinate and iodonitrotetrazolium (INT) were loaded with insoluble reduced formazan and separated on a linear sucrose gradient. A shift in median density was observed in the INT-treated mitochondria for protein, succinic dehydrogenase activity, and for Δ5-3β-OHD activity, but not for 21-hydroxylase, a microsomal enzyme marker. Density perturbation and gradient fractionation reveals a dual location of Δ5-3β-OHD in both mitochondrial and microsomal fractions.

The activities of three enzymes--two mitochondrial and one microsomal--were measured in isolated islets of Langerhans from 2-month-old and 12-month-old rats. Mitochondrial glycerophosphate dehydrogenase activity (expressed as nanomoles of iodonitrotetrazolium reduced per minute per milligram of protein), decreased (P less than 0.01) from a mean (+/- SEM) of 73.2 +/- 11.2 (2-month-old) to 34.7 +/- 5.9 (12-month-old). In contrast, activities of neither mitochondrial monoamine oxidase nor microsomal NADH cytochrome-c reductase changed with age. These results demonstrate that the activity of the glycerophosphate shuttle decreases as rats grow older, and it raises the possibility that the consequent difficulty in regenerating cytosolic NAD+ may play a role in the insulin secretory defect associated with aging.

The effect of mercuric chloride (HgCl2) on electron transport system (ETS) activity in sediment was studied using the reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to water insoluble iodonitrotetrazolium formazan (INT-formazan) by respiring microorganisms and sediment enzymes. The effect of HgCl2 on ETS activity was more pronounced in aerobically incubated sediment than in anaerobically incubated sediment. The EC50 value for the inhibition of ETS activity in aerobically incubated sediment at 10 and 20 °C was 40.9 ± 16.2 and 48.8 ± 4.1 μg g−1 HgCl2, respectively. The EC50 value for anaerobically incubated sediment in the presence of HgCl, at 4, 10, and 20 °C was 153.0 ± 8.1, 173.1 ± 8.2, and 199.1 ± 15.2 μg g−1 HgCl2, respectively.

The native flavin, FAD, of spinach ferredoxin--NADP+ reductase, has been replaced by a number of FAD analogues with modifications of the isoalloxazine ring system. The apoenzyme binds 8-mercapto-FAD in its thiolate anion form and 6-hydroxy-FAD in its neutral form. These results are consistent with classification of this enzyme as a dehydrogenase/electron transferase, an ascription originally made on the basis of its physiological function and in common with other properties of this class, e.g. stabilization of the neutral flavin semiquinone. The chemical reactivity toward methylmethanethiolsulfonate of the 8-mercapto-FAD . enzyme clearly shows that the flavin 8-position is exposed to solvent. On the other hand, the lack of reactivity with the 2-thio-FAD . enzyme indicates that the pyrimidine subnucleus of the flavin is buried within the protein molecule. The seven modified flavins examined all support NADPH--ferricyanide reductase activity, the catalytic velocity being directly proportional to the redox potential of the flavin. No such linear free energy relationship was found between redox potential and activity with ferredoxin or iodonitrotetrazolium as acceptor.

Everyone has a mucous thread in the inferior fornix. Round and oval vacuole-like formations are present in this mucous thread. A compound that is capable of staining such vacuoles, making it possible to determinate the contents of the vacuoles, has recently been found. A vital staining is indicated by means of which a distinction can be made between an infected and a sterile conjunctiva.

An indirect test for the detection in man of immediate type hypersensitivity to penicillin G is described. It is based on an iodonitrotetrazolium stain for succinic dehydrogenase in the basophil leucocyte granules. Rabbit basophils, enriched by differential centrifugation, and passively sensitized by exposure to plasma from subjects with known penicillin sensitivity, showed in the presence of penicillin a marked reduction in this INT formazan staining, as compared with those exposed to control plasma.

Canadian scientists have developed a new procedure for rapidly measuring alcohol concentrations in body fluids that may be useful in emergency situations. Bhushan Kapur, CChem, director of laboratories, and Yedy Israel, PhD, head of biochemical research at the Clinical Institute of Ontario's Addiction Research Foundation, described the results of a two-year study of the procedure at the recent meeting of the American Association for Clinical Chemistry in New York City. The new tool, called the "Alcohol Dipstick," features a glucose-test-like plastic strip for on-the-spot measurement of ethanol concentrations. A quarter-inch cellulose pad at one end of the strip is impregnated with a buffered solution containing yeast alcohol dehydrogenase (ADH), nicotinamide-adenine dinucleotide, pyrazole, iodonitrotetrazolium chloride (INT), and diaphorase. When the strip is dipped into saliva, urine, or serum that contains ethanol, it turns various shades of pink almost instantly because of a reaction between, on the one hand, the NADH that