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 new method for the isolation of plasma membranes from amphibian embryos

Summary A method for the isolation of plasma membranes is described. N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP), a heterobifunctional reagent, is covalently linked to protein amino groups in plasma membranes of intact cells. After homogenization of the cells the plasma membranes can be separated from other cell components by selective coupling to reduced Thiopropyl-Sepharose 6B and then recovered after treatment with 2-mercaptoethanol.     

A simple, versatile, nondisruptive method for the isolation of morphologically and chemicaly pure basement membranes from several tissues.

A simple procedure has been developed for the isolation of ultrastructurally pure, intact basement membranes from bovine retinal and brain blood vessels, rabbit renal tubules and rat renal glomeruli. By this procedure, cell membranes and intracellular materials are selectively solubilized with 4% sodium deoxycholate to yield morphologically and chemically intact basement membrane preparations. Therefore, this method appears to be a versatile, nondisruptive procedure for the isolation and characterization of basement membranes from a variety of tissues. Its applicability has been demonstrated by the preparation for the first time of isolated basement membranes from non-renal mammalian blood vessels.     

A rapid method for the isolation of kidney brush border membranes.

A simple rapid method for the preparation of purified brush border membranes from rabbit kidney proximal tubules is described. The method is based on hypotonic lysis, Ca2+ aggregation of contaminants and differential centrifugation. In contrast to most other published methods, the brush border membranes are free of contamination by basolateral membranes.     

Studies on the filtration properties of isolated renal basement membranes.

1. The filtration properties of films of renal basement membrane were studied in vitro using pressure filtration chambers. 2. Retention of cytochrome c by the films was found to be dependent upon the filtration pressure indicating that it was transferred across the films by convective as well as diffusive flow. In contrast, serum albumin was transferred by diffusive movement only. 3. When solutions containing both cytochrome c and IgG were filtered it was found that increasing the filtration pressure reduced the flux of cytochrome c across the films. A similar phenomenon occurred when serum was filtered, less protein passed through the films at high filtration pressures. These phenomena are explained by concentration-polarisation effects. 4. The flux of cytochrome c through the films was found to decrease in a non-linear manner as the films thickness was increased. With thin films, the flux of cytochrome c increased in a non-linear manner as the concentration of the protein in the overstanding solution was increased. With thicker films the flux was linearly dependent on concentration. These findings are interpreted as supporting the view that movement of cytochrome c occurs, at least in part, by convective flow.     

An improved method for the isolation of adipocyte plasma membranes.

An improved procedure is outlined for the isolation of an adipocyte plasma membrane fraction containing much less endoplasmic reticulum contamination than plasma membranes prepared by the procedures that are currently in commen use. It is also shown that ¹²⁵I-labeled diazotized diiodosulfanilic acid can be used as a nonpermeable reagent which selectively labels two protein components in plasma membranes of intact adipocytes.     

A rapid method for the isolation of kidney brush border membranes

A simple rapid method for the preparation of purified brush border membranes from rabbit kidney proximal tubules is described. The method is based on hypotonic lysis, Ca²⁺ aggregation of contaminants and differential centrifugation. In contrast to most other published methods, the brush border membranes are free of contamination by basolateral membranes.     

A new method for the selective isolation of phosphoserine-containing peptides

Meyer et al. [(1986) FEBS Lett 204, 61-66] have shown that phosphoserine can be converted to S-ethylcysteine by beta-elimination and addition of ethanethiol. I have utilised this modification to develop a rapid method for the selective purification of phosphoserine-containing peptides from complex mixtures. Changing phosphoserine to S-ethylcysteine increases the hydrophobicity of a peptide, altering its mobility during reverse-phase chromatography. The number of S-ethylcysteine residues in a peptide can be quantified at the picomolar level, following acid hydrolysis and conversion to the phenylthiocarbamyl derivative. The procedure may be particularly powerful for the analysis of peptides that are phosphorylated at multiple sites in vivo.     

Composition of renal basement membranes isolated under various conditions

GBM isolated from a surgical biopsy directly or after a 22 hr incubation period--to imitate the usual interval between death and isolation--appeared to be nearly identical in amino acid composition. Sonication and detergent procedures for isolation of GBM and TBM lead to preparations of different chemical composition. Phosphorus analysis and electron micrographs indicate the presence of material of supposedly cellular origin in sonicated but not in detergent-treated bovine and human GBM. Detergent-treated bovine and human GBM preparations are more enriched in the collagen-typical amino acids than sonicated samples. SDS-PAGE analyses show a nearly identical polypeptide pattern. Sonicated and detergent-treated bovine TBM preparations are free of cellular material. They show in SDS-PAGE a similar heterogeneous polypeptide pattern, but with lower intensities of three components with molecular weights between 30 and 60 kdalton. Sulfated GAG's are present in higher concentration in sonicated than in detergent-treated GBM and TBM. Collagen is not extracted from glomeruli and tubules by detergent treatment.     

A new method for the selective isolation of actinomycetes from soil

Summary A coal-vitamin medium was developed to isolate actinomycetes from soil, which was superior to other currently used media. It increased the number of actinomycetes and inhibited the growth of other soil bacteria. The pretreatment of soil suspension with peptone (6%) and lauryl sulfate (0.05%) at 50°C for 10 min, also greatly increased the number of actinomycetes from soil prior to incubation with new medium.     

An improved method for the isolation of spinach chloroplast envelope membranes

A three-phase, discontinuous sucrose gradient yielded two distinct fractions of envelope membranes from spinach (Spinacia oleracea L.) chloroplasts. Their buoyant densities were 1.08 g cm⁻³ and 1.11 g cm⁻³. Electron micrographs showed the lighter and heavier fractions to consist primarily of single and double membranes, respectively. The milligrams of lipid-milligrams of protein ratio for the complete envelope membrane (double membrane fraction) was 1.74. Thin layer chromatograms showed that the lipids of the complete envelope membranes were similar to those found in earlier preparations which consisted of single and double membranes. This isolation procedure is superior to earlier methods in that the percentage of complete envelope membranes is greater and the yield is almost three times as great. Enzymatic and chemical analyses and microscopic examination showed the complete envelope membranes were free of bacterial, fungal, microsomal, mitochondrial, and lamellar membrane contamination as well as stromal contamination. The specific activities of nonlatent Mg²⁺ -dependent ATPase (80 μmoles of phosphate released hr⁻¹ mg protein⁻¹) were about 10-fold higher than those values found with earlier preparations consisting of single and double membranes, indicating that the ATPase is largely lost in preparations containing single membranes. These higher values show that the ATPase is located in the double membrane and probably functions in the transport processes of the envelope membrane.     

A rapid method of isolation of platelet membranes for radioligand and enzymatic assays

A simple and rapid procedure for isolation of the total platelet membrane fraction by chromatography on Sepharose CL 6B has been developed. This method allows a rapid (25-30 min) one-step separation of the membrane fraction on 26 x 150 mm columns with a 20-21 mg recovery. The high degree of purity of membrane preparations was confirmed by a radioligand assay using [3H]adenosine and L-[3H]glutamic acid. The purity of membrane preparations is comparable with that of membrane fractions obtained by standard ultracentrifugation methods. The homogeneity of the membrane fraction was demonstrated by using marker enzymes of plasma, microsomal and mitochondrial membranes. This finding is very important in that it allows the isolation of fractions differing in their protein content with no effect on the method reproducibility. The high utility of the membrane preparations in receptor studies was demonstrated for high affinity binding sites for [3H]adenosine and L-[3H]glutamic acid.     

A new method for isolation of polyamines from animal tissues

A new method for isolation of polyamines from tissues was developed and compared with the butanol extraction method which has been widely used for quantitative determination of polyamines. In the new method protein-free tissue extracts are applied to a small Dowex-50 column. The column is washed with appropriate buffers to remove ninhydrin-positive contaminants and the polyamines are eluted. With this method, the overall recovery of polyamines, after separation by paper electrophoresis and subsequent colorimetric determination with ninhydrin, is always over 90% (average 95%). This method is much better than the butanol method, which gives variable recoveries of 70–90%. The new method also has the advantages over the butanol method that the isolated polyamines are purer and the procedure is simpler.     

A quantitative assay for the hydrolysis of structurally intact basement membranes

A quantitative assay has been developed for the hydrolysis of native bovine anterior lens capsule basement membrane fragments. The rationale for using structurally intact membrane fragments as the substrate in enzyme assays is that the proteinase susceptibility of the various basement membrane components differs when examined individually compared to when they are present in their native state. The assay is based upon the solubilization of 3H-bound protein from a finely ground suspension of [3H]acetylated basement membranes. The acetylation reaction and the fragmentation procedure do not alter the morphology or proteinase susceptibility of the membranes. The initial rate of release of radiolabeled digestion fragments by six different proteinases is approximately linear over the first 15% of hydrolysis, and the initial rates obtained are proportional to the amount of enzyme over a wide range of enzyme concentrations. The labeling index of 3810 cpm/micrograms of basement membrane used in this study permits the solubilization of 50 ng of protein to be detected easily. Some information about the size of the protein fragments solubilized can be obtained by addition of trichloroacetic (5% w/v)-tannic acid (0.25% w/v) reagent to the supernatants from the assays, since this reagent appears to selectively precipitate larger fragments. An additional feature of this assay is that, since the substrate is radiolabeled, one can selectively visualize and analyze the size distribution of the digestion products by carrying out sodium dodecyl sulfate-gel electrophoresis with fluorographic detection. Based on the observation that these intact membranes are extensively digested by a number of common proteinases which have widely different substrate specificities, it appears that the hypothesis that a highly specific proteinase or class of proteinases is necessary for basement membrane catabolism is specious .