Subcellular Fractionation Methods Research Articles

Biosynthesis and degradation of plasma lipoproteins.

The origin of serum lipoproteins has been shown to be confined mostly to the liver and to a more limited extent to the intestine. During the past decade the pathways of lipid and protein synthesis, which had been previously characterized in vitro, were localized to the endoplasmic reticulum of the parenchymall iver cell. With the help of electron microscopy, radioautography and newer methods of sub-cellular fractionation it became possible to identify the intrahepatic form of serum VLDL and to follow its intrahepatic path from endoplasmic reticulum through the Golgi apparatus to the sinusoidal cell surface. More recently it was shown that the Golgi apparatus participates in the glycosidation of serum lipoproteins. Of the various serum lipoproteins so far only the VLDL has been identified in the liver and the question whether HDL and LDL are also secreted as distinct particles has not been answered. Far less is known about the site of degradation of lipoproteins, especially of the protein moiety. Recently we have used iodinated rat high density lipoprotein as a model system to study this problem. Following injection into rats the HDL was cleared from the circulation with a half life of about ½ hrs. During the first 6 hrs about 10% of the label was taken up by the liver and about 5% by the small intestine. Ninety percent of the radioactivity recovered in the liver was precipitable by TCA. With the help of radioautography the label was seen predominantly over parenchymal liver cells and concentrations of label were seen over peribiliary areas. Electron microscopic radioautography has shown that the concentration of label could be localized to lysosomes of parenchymal liver cells. Thus the liver, which has been shown to be the main source of plasma lipoproteins seems to participate also rather actively in their catabolism.

Acetylcholinesterase and acetylcholine proteolipid receptor: Two different components of electroplax membranes

Electroplax membranes from Electrophorus electricus were separated by two methods of subcellular fractionation, and in both cases a parallelism between the content of acetylcholinesterase and the binding of cholinergic drugs was observed. Treatment of the membranes with the -SH reagents, p- hydroxymercuribenzoate and p- chloromercuribenzoate , produced a 16–28% inhibition in the binding of [ 14C]acetylcholine. With the -S-S- reagent, 1,4-dithiothreitol, followed by N- ethylmaleimide the inhibition was 64–67%. Membranes depleted of acetylcholinesterase with 1 M NaCl showed no quantitative change in the binding of dimethyl (+)-[ 14C]tubocurarine and methyl[ 14C]hexamethonium as compared to the controls. Furthermore the same amount of receptor proteolipid, which binds [ 14C]acetylcholine, was found after the 1 M NaCl treatment. It is concluded that: (a) acetylcholinesterase and the acetylcholine proteolipid receptor are two different macromolecules present in the electroplax membranes; (b) the receptor proteolipid, previously isolated from the intact tissue, is contained in the electroplax membrane.

THE INCORPORATION OF14C-THREONINE AND14C-GLUCOSAMINE INTO SUBCELLULAR FRACTIONS AND INTO BOVINE SUBMAXILLARY MUCIN BY SLICES OF BOVINE SUBMAXILLARY GLAND

Bovine submaxillary gland slices were used to investigate the subcellular site of biosynthesis of bovine submaxillary mucin (BSM). Slices were incubated with14C-threonine and14C-glucosamine in Krebs medium III at pH 7.4 and 37 °C for varying periods of time up to 180 minutes. Methods of subcellular fractionation based on those developed for liver proved unsatisfactory for use with submaxillary gland, probably because of the presence of large amounts of highly viscous mucin. A method of subcellular fractionation was developed which yielded five fractions: P (total soluble protein) and C (soluble BSM, purified by Cetavlon precipitation) were prepared from the 16,000 × g supernatant; and R (ribosomes), S (deoxycholate-soluble membrane-bound protein), and a residual fraction D were prepared from the 16,000 × g pellet. The incorporation of both tracers into these fractions was time-dependent and was inhibited by preincubation of the slices for 15 minutes with puromycin. The highest specific activities were recorded in the R and S fractions, implicating them as possible sites of glycoprotein biosynthesis. The time course of14C-threonine incorporation suggested that protein is made on ribosomes, is transferred to the endoplasmic reticulum, and is then ready for storage or secretion. The time course of14C-glucosamine incorporation suggested that carbohydrate is linked to the protein both while the protein is still associated with the ribosomes and after it has been released from them. The involvement of ribosomes in carbohydrate attachment to polypeptide received additional support from zone centrifugation analysis on sucrose-density gradients of ribosomes labeled with14C-glucosamine.

Site of fluoride accumulation in navel orange leaves.

Fluoride-polluted navel orange leaves, Citrus sinensis (Linn.) Osbeck, were fractionated into the subcellular components in hexane/carbon tetrachloride mixtures having various densities. Fluoride was determined at each fraction. Analyses were also made for the subcellular distribution of chlorophyll, nitrogen, and DNA to assess the extent of cross-contamination of each component. The fraction containing cell wall, nuclei, and partly broken cells apparently contained a major amount of fluoride. However, if allowance was made for the cross-contamination of chloroplasts and chloroplast fragments, the fraction of chloroplasts was found to be the site of the highest fluoride accumulation. When each particulate component was washed with water after drying, the combined washings contained more than 50% of the total fluoride of the isolated fractions. The usual method of subcellular fractionation with aqueous solvent shifted the major site of fluoride accumulation from the fraction of chloroplasts to that of the supernatant.