Melvin Fried

Use of water-soluble polymers for the isolation and purification of human immunoglobulins

Abstract By the use of fractional precipitation with high molecular weight nonionic polymers, immune globulins of marked homogeneity were isolated in high yield from either whole serum or commercial samples of immunoglobulins. The isolated fractions were characterized by immunochemical and ultracentrifugal analyses. When the pH was varied from 4.9 to 8.6 and the ionic strength from 0.1 to 2.0, little effect on the precipitation of the immunoglobulins was noted. The immunochemical studies indicated that this fractionation technique is not effective in separating the γ G , γ M , and γ A activities present in the γ-globulins isolated from whole serum or in commercial immunoglobulin preparations. Preliminary experiments indicate that this technique may be used for the isolation of other serum proteins, especially albumin and the α-globulins. In these instances, however, it is important that ionic strength and pH be controlled. The mechanism for the precipitation of serum proteins by PEG has not been established, but it is suggested that the precipitation involves a local dehydration and consequent change in the dielectric constant of the medium immediately surrounding the protein molecules. It would appear that the high-polymer precipitation technique may be an effective and simple method for the isolation of serum proteins with preservation of much of their native properties.

The biosynthesis of plasma lipoproteins in higher animals

Abstract 1. 1. The distribution of lipoproteins was determined in the plasma or serum of five animal species. 2. 2. The incorporation, in vivo, of radioactive lysine into ultracentrifugally isolated plasma lipoproteins and other plasma of these animals was measured. 3. 3. The specific activities of the lipoproteins were highers than those of albumin or other plasma proteins and reached a maximum and declined earlier indicating more rapid synthesis and turnover. 4. 4. The lowest density lipoproteins appeared to be synthesized and metabolized most rapidly, consistent with their role in lipiod transport.

Metabolism of lipoprotein lipid in the isolated perfused rat heart

Abstract 1. 1. The disappearance from the perfusate, localization within heart lipids, and oxidation of 14C-labeled lipid from three ultracentrifugally separated lipoprotein fractions were studied in the isolated perfused rat heart. 2. 2. With d 3. 3. With d 1.006−1.070 lipoprotein, 14CO2 and 14C-label in the heart lipids accounted for all of the triglyceride fatty acid uptake. The final concentration of triglycerides in the perfusate was very low and no net increase in free fatty acid in the buffer was observed. The amount of triglyceride fatty acid recovered in CO2 was less than, and that in heart lipids equal to, that observed with d 4. 4. With d 1.070−1.210 lipoprotein, the lack of change in concentration or specific activity of phospholipid with perfusion suggested that phospholipids were not extracted, or metabolized by the perfused heart. The 14C-labeled lipid extracted was primarily free fatty acid; the major non-oxidative fate of this free fatty acid was incorporation into tissue phospholipids.

[22] Water-soluble nonionic polymers in protein purification

Publisher Summary Fractionation techniques employing nonionic water-soluble high polymers are of increasing significance in studying biologically important macromolecules and cell particulates. A two-phase system of dextran and polyethylene glycol (PEG) is employed in a countercurrent distribution procedure to separate two different strains of Escherichia coli. Several types of viruses, namely—bacteriophage, tobacco mosaic virus, vaccinia virus, and various polio and echo virus strains—are separated by distribution between two phases composed of buffers plus either dextran and PEG or dextran and methyl cellulose. PEG solutions are used to precipitate plant viruses and infectious bacteriophage particles are separated by sedimentation with PEG. An aqueous dextran and PEG two-phase system is employed to separate single-stranded DNA and double-stranded DNA, and the characteristics of the distribution of various RNA and DNA preparations in dextran and methyl cellulose systems are also measured.

The uptake of dissolved free fatty acids from seawater by a marine filter feeder, Crassostrea virginica

Abstract 1. 1. Crassostrea virginica can remove free fatty acids from seawater at naturally occurring concentrations of 0.25 μM. 2. 2. The uptake is saturable for both palmitate and stearate but not oleate; above 3 μM the uptake is greatly enhanced by micellar formation. The V max for palmitate is 1.67 nmole/g per hr and stearate is 1.22 nmole/g per hr. 3. 3. The most rapidly labeled lipid in the CHCl 3 extracts of the animal bries is phosphatidyl choline. The V max for the palmitate incorporation into phosphatidyl choline is 850 nmole/g per hr.

The inhibition of pyruvate decarboxylation in rat brain by α-ketoisocaproic acid☆

Abstract The conditions for optimal measurement of pyruvate decarboxylation in rat brain homogenates were determined to be: 6 mg of homogenate; 15 minutes incubation; 1 × 10−3 m pyruvate; pH 7.4; 37°. The apparent Km value for pyruvate decarboxylation under the above conditions was 6.7 × 10−4 m . The decarboxylation of pyruvate was competitively inhibited by 5 × 10−4 m KIC with a Ki of 1.0 × 10−4 m . At higher concentrations, a mixed type of inhibition was observed. These results are consistent with the current interpretation of the molecular lesion of branched-chain ketoaciduria, which holds that in the genetically determined absence of α-ketoisocaproic: α-keto-β-methylvaleric acid dehydrogenase αKIC accumulates and can inhibit both specific and general reactions leading to the symptomatology of the disease.

α-ketoisocaproic acid inhibition of pyruvate and α-ketoglutarate oxidative decarboxylation in rat liver slices☆

Abstract 1. 1. A metabolic system composed of rat liver slices was found to oxidatively decarboxylate pyruvate and α-ketoglutarate in a linear fashion beyond 60 minutes when incubated at 37° in Krebs phosphate buffer. 2. 2. Both pyruvate and α-ketoglutarate decarboxylation follow steady-state kinetics, exhibiting apparent K M values of 9.9 × 10 −3 and 2.0 × 10 −3 , m , respectively. 3. 3. α-Ketoisocaproic acid is a potent inhibitor of the oxidative decarboxylation of both pyruvate and α-ketoglutarate, yielding apparent K i values, in the liver system, of 1.0 × 10 −3 and 1.5 × 10 −3 , m , respectively. 4. 4. The importance of these observations in conjunction with the etiology of the metabolic disorder, branched-chain ketoaciduria, is discussed.

Lipoprotein geometry. II. Apoprotein exchange in human plasma high density lipoprotein

Abstract The relationships between the apoproteins of intact human serum high density lipoprotein particles, HDL 2 and HDL 3 , have been studied by observing the exchange of radioactively labeled apoproteins between one subclass and the other. This exchange process can be inhibited by chemically crosslinking the apoproteins of either the labeled or unlabeled subclass. These results are consistent with a dynamic relationship between HDL 2 and HDL 3 which appears dependent upon the association and perhaps the conformation of the apoprotein components of the lipoprotein particles.

Lipoprotein geometry. I. spatial relationships of human HDL apoproteins studied with a bifunctional reagent

Summary The spatial relationships of the apoprotein components of intact human serum high density lipoprotein particles have been studied using the bifunctional reagent 1,5-difluoro-2,4-dinitrobenzene. The results of crosslinking experiments indicate that the two major HDL apoproteins, apoA-I and apoA-II, lie close to one another in the intact lipoprotein particle. The reproducibility of these results, and the absence of other products signifies that they remain in a relatively fixed position.

Polymorphism in fowl serum albumin: III. KCl concentration effects on the yield and activity of chicken liver ribosomes

Abstract The effect of ionic strength during isolation on the activity of chicken liver ribosomes in a poly(U)-dependent phenylalanine-incorporating system was studied. Changing KCl concentration during isolation caused ribosome yield and activity to vary significantly. KCl concentrations of 150–250 mM in the isolation medium resulted in higher and more consistent ribosome yields and activity than 25–50 mM, although KCl concentration was the same in all assay mixtures (150 mM). Thus, the reduced activity characteristic of chicken ribosomes isolated at low KCl concentrations is not readily reversible in the assay mixture. The reduced yield and activity may result from a greater tendency of ribosomes isolated at low KCl concentrations to aggregate. The effects of cell-sap concentration, MgCl 2 concentration, and temperature during incubation were also determined, as well as the results of freezing ribosomes and cell sap before use. The optimum MgCl 2 concentration for phenylalanine incorporation was about 14 mM, and the optimum temperature for a 60-min incubation was 33–37 °C.