A rapid high-yield isolation method for nuclear envelope ghosts

A method is presented for the isolation of nuclear envelopes from isolated Tetrahymena macronuclei. In principle, nuclei are treated with DNase and RNase at low Ca²⁺/Mg²⁺ concentration followed by an extraction with 1 NaCl. The major advantages of this method are: (1) Unfragmented nuclear envelopes are obtained in the form of ghosts consisting of two juxtaposing nuclear membranes interrupted by pores as revealed by thin-section and freeze-etch electron microscopy. (2) The ghosts are obtained in high yield (60%) within a short period (1 h). (3) The nuclear envelopes largely retain their lipid composition. An average ghost contains about 96% of total phospholipids of an average nucleus. Nuclei and ghosts reveal an almost identical pattern of phospholipids and fatty acids as shown by thin-layer and gas-chromatography. (4) The lipids in the ghosts largely remain arranged in bilayers as probed by electron spin resonance using 5-doxylstearic acid as a spin label.     

A rapid method for separating small vesicles from suspension.

Techniques for rapidly aggregating suspensions of small vesicles made either of natural biological membrane or of phospholipid or phospholipid-protein mixtures by addition of one of the basic polypeptides, polylysine or protamine, have been investigated. Either filtration or centrifugation may be used to rapidly (15 to 30 s) and completely (over 98%) separate the vesicular aggregate from the suspending medium. At low values of the polylysine-to-vesicle weight ratio, aggregation is observed to increase with increasing polylysine concentration. At high values of this same ratio, aggregation decreases with increasing polylysine concentration. With protamine, like polylysine, aggregation increases with protamine concentration at low values of the weight ratio. At high values of the ratio, aggregation induced by protamine does not rapidly decrease with increasing protamine concentration as it does with polylysine. Explanations are given for these observations. While leakage induced by polylysine may be troublesome under some conditions with phospholipid vesicles or reconstituted systems, protamine aggregation has been found to induce much less leakage than polylysine aggregation. The leakage rate induced by protamine was not found to be significantly different from the control leakage rate for the first few minutes after addition of protamine under any of the conditions tested. Since this provides ample time for filtration, the protamine aggregation-filtration technique seems to be the method of choice for separation of many types of small vesicles from the suspending medium. It combines the advantages of rapid separation, complete separation, low rate of vesicle leakage, and versatility in being able to separate the vesicles from uncharged molecular components such as sugars where ion-exchange techniques will not work (1), as well as from ionic components. Low cost and simplicity are further advantages of the technique.     

A rapid method for the analysis of N tau-methylhistidine in human urine

A novel, automated method is described for the determination of N^τ-methylhistidine in human urine. The method uses a modification (H. Nakamura and J. J. Pisano (1976) Arch. Biochem. Biophys.177, 334–335) of the reaction of fluorescamine with amines, which renders it specific for certain imidazoles. Interference due to histidine and histamine is selectively removed by prior reaction with aldehydes. The fluorescence yield for N^τ-methylhistamine is 280, 440, 50, and 1.7, respectively. The concentrations of N^τ-methylhistidine in human urine as determined by this technique correlate well (r = 0.99) with those determined by ion-exchange chromatography. Furthermore, the technique is rapid (6–7 samples/h net throughout), is reproducible (coefficient of variation 1.8%), requires no prior treatment of the sample, and is implemented with widely available equipment.     

A new method for the isolation of renal basement membranes

A method is described for the isolation of basement membranes from rabbit renal cortex in which the detergent $\text{N-}\text{lauroyl}$ sarcosine is used as the disruptive agent. The isolated membranes have been compared with membranes prepared using ultrasonication and they were comparable both in terms of purity and gross chemical composition. Glomerular and tubular basement membranes were isolated by first separating glomeruli from tubules by density gradient centrifugation followed by detergent treatment of the separated tissues.The detergent method has the advantage that the basement membranes retained their native structure to a large degree, whereas sonicated membranes were severely fragmented. Collagen fibres were a significant contaminant in both preparations and were revealed more clearly by negative staining than by examination of thin sections. Studies with the detergent-treated membrane revealed that a few proteins, which seemed to be membrane components, were extracted with 1 M NaCl and that these proteins were lost from the basement membranes during sonication used in the conventional isolation procedure.     

A rapid method for assaying the metabolism of 7-ethoxyresorufin by microsomal subcellular fractions

Chemical activation of agarose with cyanogen bromide is a routine method when preparing gels for affinity chromatography and for immobilization of macromolecules. Two activation methods are in common use; the titration (1) and the buffer (2) methods.Manipulation of the gels during CNBr activation is complicated due to many steps, some of which have to be carried out as quickly as possible (1,2). In addition, handling the gel is harmful due to the poisonous vapors. In spite of these facts, little effort has been paid to facilitate the practical performance of the activation. We describe here a useful device to eliminate some of the practical troubles in the activation. The main advantages of the device are straight-forward working, speed, and the avoidance of CNBr vapors to a considerable extent. The device is also suitable for handling quantitative gel batches since the loss of gel is minimal.     

A new sensitive radial diffusion method for microdetermination of alpha-amylase

A rapid, convenient, and efficient hybridization method for the determination of virus-specific RNAs in Py virus-infected cells is described. The method involves carrying out the hybridization of viral RNAs present in the RNA isolated from infected cells directly on nitrocellulose filters carrying denatured, immobilized Py-DNA (15 μl RNA solution/25-mm² filter). Under optimal conditions quantitative hybridization of viral RNA sequences is obtained within 24 h. The efficiency of hybridization is increased significantly when RNA fragmented by alkali under carefully controlled conditions is used.     

A new method for the detection and assay of α-1,3-glucanases

A colorimetric method that is specific for the assay of α-1,3-glucanases is presented. The enzyme substrate consists of Cibacron Blue F3GA complexed with a dextranase-treated streptococcal glucan. The method is especially convenient for tests involving large numbers of samples, and can be adapted to quantitative as well as qualitative applications. The assay is sufficiently sensitive for screening bacterial samples as potential sources of α-1,3-glucanase.     

A photochemical method for rapid and precise determination of carbon monoxide levels in blood.

A simple and inexpensive flash photolysis apparatus for determination of the level of carbon monoxide saturation of blood samples is described. Saturation with CO is determined by observing the change in light transmission at 432 nm produced on photolysis of bound CO with a light flash. The procedure is highly specific for carbon monoxide, requires less than 5 μl of blood (obtainable from a finger prick), and has a resolution better than 0.1% in saturation. In addition the apparatus does not require frequent calibration.     

Immunocapillarymigration--a new method for immunochemical quantitation.

A new simple and rapid method for immunochemical quantitation called immunocapillarymigration is described. It is based upon the attachment of antibodies to a porous insoluble support and the subsequent capillarymigration of the antigen-containing solution in the porous support. The migration of the antigen solute is specifically delayed in comparison to the migration of the solvent and other solutes in the process and the relative delay decreases with increasing antigen concentration. When applied to the quantitation of transferrin in human plasma, immunocapillarymigration gave results which agreed with those obtained by single radial immunodiffusion.     

Transport of glycine,serine, and proline into spinach leaf mitochondria

The transport of radioactive glycine, serine, and proline into the matrix of spinach leaf mitochondria was studied using the silicone oil centrifugation technique. The uptake of all three amino acids showed a biphasic characteristic. At concentrations higher than 0.5 mm, an apparent diffusion process dominated. The uptake was not saturable at increasing amino acid concentrations, and there was no accumulation of amino acid in the matrix (i.e., concentration was similar to that in the medium). At concentrations lower than 0.5 mm, in addition to the diffusion process, an active uptake system that accumulated amino acid in the matrix became apparent. This system was partially inhibited by rotenone, antimycin A, and carbonylcyanide-m-chlorophenyl hydrazone. Also, uptake of glycine and serine was mutually inhibitory. These two amino acids exhibited comparatively less inhibitory effect on proline uptake, and proline also did not inhibit glycine or serine uptake. The results suggest that the active uptake system consists of at least two components with different degrees of amino acid specificity. The diffusion process dominates at amino acid concentrations of 0.5 mm or higher, whereas the active uptake system becomes more prominent as the amino acid concentration decreases.     

A new method for the determination of N-sulfate in heparin and its analogs.

A new method for the determination of N-sulfate in heparin and its analogs is described. The method is based on the determination of inorganic sulfate liberated by deamination with nitrous acid. The accuracy, simplicity, and validity of this method are evaluated by comparing it with previous methods.     

A rapid radioenzymatic assay for dopamine and N-acetyldopamine.

Dopamine (DA) was measured in various tissue extracts as [³H]methoxy-N-acetyldopamine after incubation with two partially purified enzymes, catechol-O-methyl transferase (EC 2.1.1.1) and N-acetyltransferase (EC 2.3.1.5), in the presence of [³H]adenosylmethionine and acetyl-CoA. This product can be separated quantitatively from labeled products of norepinephrine and epinephrine by solvent extraction. N-Acetyl-DA can be assayed by omitting the acetylating system from the incubation mixture. The procedure is rapid, convenient for processing large numbers of samples, and has a sensitivity of approximately 0.1 pmol. It has been used to measure DA in ganglia and in individual neurons from gastropod mollusks.     

The reactivity of uronic and aldonic acid derivatives towards trifluoroacetolysis. A new method for specific elimination of reducing 4-O-substituted hexose residues

Trifluoroacetolysis of d-glucuronic acid and methyl α-d-glucopyranosiduronic acid resulted in an initial phase of degradation followed by stabilisation of the compounds as their 6,3-lactones. The methyl ester of methyl 4-O-methyl-α-d-glucopyranosiduronic acid was largely stable towards trifluoroacetolysis. Aldonic acids substituted at O-3 or O-6 were stable towards trifluoroacetolysis because of the formation of γ-lactones. Aldonic acids substituted at O-4, and incapable of forming γ-lactones, were converted into the trifluoroacetylated enol of 3-deoxy-2-hexulosonic acid. Treatment of the 3-deoxy-2-hexulosonic acid with mild base eliminated the substituent at O-4.     

A fluorometric-HPLC assay for quantitating the binding of benzo[a]pyrene metabolites to DNA

A nonradiometric method is presented for quantitating low levels of benzo[a]pyrene (BP) derivatives that are covalently bound to the DNA of BP-treated mice. This method consists of hydrolyzing the DNA with acid to liberate the BP-adducts in the form of the isomeric tetrols of BP. These tetrols have fluorescence quantum yields of ～0.7 in deoxygenated solution at 298 K. Hence they are easily quantitated, following HPLC separation, by means of fluorescence detection. The sensitivity of the method is such that one bound BP residue per 10⁷ bases can be detected in 100 μg of DNA.     

A new,fast, and sensitive assay for NADH-ferredoxin oxidoreductase detection in clostridia

A new, simple and very sensitive assay for NADH-ferredoxin or flavodoxin reductase activity is described. The assay is based on the nonenzymatic reduction of the metronidazole by ferredoxin or flavodoxin. In the presence of NADH, ferredoxin or flavodoxin and cell-free extract of clostridia, no metronidazole reduction is observed; the reaction occurs only if acetyl-CoA is added to the reaction mixture. Metronidazole reduction is quantitated by the spectrophotometric measurement at 320 nm. In this assay the change in absorbance is linearly related to the amount of clostridial extract for concentration of 0.1 to 0.8 mg/ml and to the flavodoxin or ferredoxin for concentrations of 0.5 to 8 nmol/ml.     

A rapid,sensitive method for accurate determination of the lecithin/sphingomyelin ratio by thin-layer chromatography and reflectance spectrofluorometry

A highly reproducible thin-layer chromatographic procedure has been developed for accurate determination of the lecithin/sphingomyelin ratio. Two interfering compounds, phosphatidyl inositol and phosphatidyl serine, have been investigated and eliminated by adsorption onto DEAE-cellulose. A uniform fluorescence staining procedure employing 2′,7′-dichlorofluorescein has been developed. Accurate quantitation was performed by direct measurement of the reflected fluorescence intensity of the lecithin and sphingomyelin fluorophore spots with a spectrofluorometer equipped with a thin-layer scanning attachment. Stability and reproducibility studies are reported.     

The cluster-tray method for rapid measurement of solute fluxes in adherent cultured cells

The use of an inexpensive and simple modification of Costar 24-well cluster trays is described in a rapid and reproducible method for measuring substrate fluxes in adherent cultured eukaryotic cells.