Tissue fractionation techniques

One further problem is the large overshoot in ABA production in wilted leaves. With applied ABA a doubling of the ABA content of the leaf is usually adequate for stomatal closure, while increases up to 40-fold have been reported in wilted leaves. However, extractions of whole leaves do not take into account the location of ABA within the leaf. Perhaps much of the hormone is sequestered in a compartment that has no access to the guard cells. Thus, it would be of much importance to determine the distribution of ABA at the tissue level as well as its intracellular location. Since ABA is a small water-soluble molecule, conventional fractionation techniques may not be suitable to determine its distribution in various organelles. A highly specific immunological method for detection of ABA has recently been developed (38, 39). It is conceivable that this technique could be further developed for determining the cellular localization of ABA as has already been done for the photoreceptor phytochrome (77, 78). 

From a strict biochemical point of view a clear-cut definition of the role of the liver in the biosynthesis of any particular plasma protein can be made only when the particular protein has been clearly and cleanly isolated, as in the case of fibrinogen. The practical difficulties of effecting such isolations on a small scale from isotopic labeling studies of the plasma proteins, such as we have described, seriously militate against such a detailed demonstration at present. The use of fractionation techniques with greater resolving power such as acrylamide gel electrophoresis already show some promise in our laboratory toward affording a more definitive picture of the biosynthetic role of the liver and the nonhepatic tissues in plasma protein production. 

Because of the favorable sorptive properties of the reversed-phase supports, batch adsorption and desorption can be a very effective way to desalt a chromatographed sample or to partially fractionate a peptide mixture during a purification procedure. For example, 1-2 gm of an oc-tadecyl silica packed into a silanized glass or plastic pipette can be used for the batch fractionation of small amounts of a crude peptide extract from tissues, such as the pancreas or pituitary, or from a synthetic experiment. A number of commercial products, such as the Waters Sep-Pak, have found use in this manner 10) as a purification or sample preparation aid. Protocols for batch extraction procedures on alkyl silicas have been discussed 17a,b) and applied to neuropeptides 10, 158, 166) and other hormonal peptides 88, 162, 167, 168). With these methods recoveries of peptides present in a tissue extract are generally higher than those found with classical fractionation techniques due in part to the fact that proteolytic degradation is minimized. 

Identification may be helped by changing the precipitating capacity of the antiserum. If the latter is absorbed with pure fractions of plasma proteins until no precipitation lines are formed in the Ouchterlony test, the pattern of ImEl will lack the corresponding lines. If the unknown antigen mixture contains fractions of tissues or tumors, the antiserum has to be absorbed first with pooled lyophilized human plasma. Another technique absorbs the antiserum specifically with a considerable excess of the unknown antigen a control ImEl shows the effect of absorption, thereby revealing the active antigen. Heremans (HI) has used this... 

In 1%7, Miflin reported that a particulate fraction from pea or barley roots was able to reduce nitrite using reduced benzyl viologen as the electron donor. Miflin (1970) and Bourne and Miflin (1970) isolated a barley root particle that reduced nitrate to ammonia when provided with pyruvate and ATP. The fractionation techniques used separated the nitrosome from the mitochondrial and peroxisomal fractions. Dalling et al. (1972b) found that 15% of the total nitrite reductase activity of wheat roots was associated with an organelle that was tentatively identified as a proplastid. A proplastid location for nitrite reductase in tissue culture cells has been indicated by Washitani and Sato (1977a,b). 

The findings made with tissue fractionation techniques conflict with histochemical studies of Koenig, who claims that gangliosides are mainly in lysosomes. These discrepancies may result from a variety of causes (1) the staining method used to identify the glycolip-id may be nonspecific and stain sialic acid-containing proteins (2) brain lysosomes may not be separated from microsomes and (3) possibly most of the glycoprotein is in the microsomes, but only that in the lysosomes is detectable by histochemical techniques. 

Evidence to substantiate this concept was immediately forthcoming. Investigations by Claude and by Schneider and their collaborators indicated that the succinoxidase and cytochrome oxidase activities of liver cells are largely concentrated in the mitochondria, a finding which explained the results of earlier studies on these insoluble enzymes. These workers also developed a technique for the fractionation of tissue extracts in hypertonic sucrose solution, a technical advance of considerable value, since previous work with distilled water or buffered saline extracts did not preserve the morphological and enzymological properties of the fractions satisfactorily. With this method, it was possible to isolate mitochondria from liver tissue in a state approximating that found in the intact cell, with the full preservation of many labile enzyme activities which it had been impossible to study by previous methods. 

Male rats weighing about 135 g were given ordinary commercial rat food with (controls without) 0.3% clofibrate for 10-14 days. Liver subfractions were obtained by conventional tissue fractionation techniques, except that EDTA (5 mmol/1) and 1-tartrate (25 mmol/1) were added to avoid degradation of CoA (3) during the procedure. Marker enzymes for the subfractions were assayed as described previously (2,3,4). 

The validity of these unconfirmed results is subject to two reservations. Firet, the use of a blender rather than a tissue grinder (pestle and tube) may introduce artifacts in the form of partial (or complete) destruction of. some cellular structure. Second, the cellular fractionation technique is based on liver. It is a widely accepted fact that the technique may have to be modified in its application to other organs and that certain enzymes, known to be characteristic of specific cellular structure, must he assayed as criteria of adequate fractionation and of the absence of artifact formation. 

Filtering cells and cell fractions from fluid media. These particles, after concentration by filtration, may be examined through subsequent quantitative or qualitative analysis. The filtration techniques also have applications in fields related to immunology and implantation of tissues as well as in cytological evaluation of cerebrospinal, fluid. 

This procedure was compared with sequential extractive techniques employing alkaline hydrolysis of dried plant tissue followed by extraction of the acidified mixture with ethyl acetate. Fractions were individually evaluated for phytotoxic properties. Selected fractions from those showing a positive response were analyzed by gas-liquid chromatography. Structural identification and characterization of the individual components in these selected fractions were accomplished by gas chromatography-mass spectrometry. 

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