Perparation and comparative analytical study of cat liver microsomes]

Cat liver homogenates have been fractionated by differential centrifugation. Four particulate fractions (1 000 X g, 10 000 X g, and 145 000 X g) and a supernatant have been obtained. The biochemical composition of these fractions has been established from the assay and distribution pattern of 22 enzymatic and chemical constituents including marker enzymes for mitochondria, lysosomes, peroxisomes, plasma membranes, endoplasmic reticulum, Golgi apparatus and cell sap. The microsomal fraction was characterized by a moderate contamination with large cytoplasmic granules and by a low yield in protein and cholesterol. It contained 50 per cent of Golgi complex and about 40 per cent of plasma membranes. Morphological analysis of subcellular fractions was performed and confirmed biochemical results.     

Centrifugal,biochemical, and electron microscopic analysis of cytoplasmic particulates in liver homogenates

A combined centrifugal, biochemical, and electron microscopic study of the cytoplasmic particulates present in 0.88 M sucrose homogenates of rat liver has been carried out. Size distribution analyses of particles containing pentose nucleic acid (PNA) and exhibiting several types of enzymatic activity revealed three major size groups within the range of particle radius between 10 and 500 mmicro. A different array of biochemical properties was associated with each size group. The largest particles, with an average radius (assuming spherical shape) in the region of 220 to 260 mmicro, contained all of the succinic dehydrogenase activity of the cytoplasmic extract, 29 per cent of the diphosphopyridine nucleotide (DPN)-cytochrome c reductase activity, and minor amounts of PNA and acid phosphatase activity. Cytologically, this group of particles was identified with the mitochondria. All of the uricase activity, 58 per cent of the acid phosphatase activity, and 26 per cent of the PNA was apparently associated with a second size group of particles (average radius 120 mmicro) which were tentatively identified by electron microscopy with vesicular structures derived from the ergastoplasm of the intact cell. The third particle group demonstrated by centrifugation exhibited a major size distribution peak at 25 mmicro and a second smaller peak at 55 mmicro. Over 50 per cent of the total cytoplasmic PNA and DPN-cytochrome c reductase activity was associated with particles in this size group. Electron microscopy revealed a morphologically heterogeneous population of particles within this size range.     

The Chemical Fractionation of Rabbit and Swine Thymus

A chemical fractionation procedure, previously found applicable to bovine thymus and bovine and ovine palatine tonsils, was used to fractionate rabbit and hog thymus. With respect to the chemical fractionation steps, yields of fractions, and optical and electrophoretic properties, extracts from hog and rabbit thymus were indistinguishable from similar extracts prepared from calf thymus. The study provides composition and yield data applicable to the thymus of a small mammal readily available in most laboratories.     

The chemical fractionation of lymphatic organs

A method for chemically fractionating lymphatic organs has been described. The method has been shown to be applicable to bovine palatine tonsils, sheep palatine tonsils, and bovine thymus. Approximately 50 per cent of the dry weight of tonsils and about 30 per cent of thymus has been found to be soluble in the 0.15 M NaCl extract. Four components have been isolated which together account for 65 per cent by weight of the material in the extracts. Four other components have been identified and partially defined by means of electrophoretic mobility, solubility, or some other chemical or physical property.     

Natural polyphenols-iron interaction

The iron-binding capacity of different fractions of natural polyphenols extracts was determined by chromatographic and electrophoretic methods. Their effects on iron-induced calcium homeostasis changes in liver tissue suspension showed that mate tea and green tea extracts provoke a very significant inhibition of the iron effects, whereas it is much less significant with red wine extract. The biological importance of this phenomenon is discussed.     

CENTRIFUGAL,BIOCHEMICAL, AND ELECTRON MICROSCOPIC ANALYSIS OF CYTOPLASMIC PARTICULATES IN LIVER HOMOGENATES

A combined centrifugal, biochemical, and electron microscopic study of the cytoplasmic particulates present in 0.88 M sucrose homogenates of rat liver has been carried out. Size distribution analyses of particles containing pentose nucleic acid (PNA) and exhibiting several types of enzymatic activity revealed three major size groups within the range of particle radius between 10 and 500 mµ. A different array of biochemical properties was associated with each size group. The largest particles, with an average radius (assuming spherical shape) in the region of 220 to 260 mµ, contained all of the succinic dehydrogenase activity of the cytoplasmic extract, 29 per cent of the diphosphopyridine nucleotide (DPN)-cytochrome c reductase activity, and minor amounts of PNA and acid phosphatase activity. Cytologically, this group of particles was identified with the mitochondria. All of the uricase activity, 58 per cent of the acid phosphatase activity, and 26 per cent of the PNA was apparently associated with a second size group of particles (average radius 120 mµ) which were tentatively identified by electron microscopy with vesicular structures derived from the ergastoplasm of the intact cell. The third particle group demonstrated by centrifugation exhibited a major size distribution peak at 25 mµ and a second smaller peak at 55 mµ. Over 50 per cent of the total cytoplasmic PNA and DPN-cytochrome c reductase activity was associated with particles in this size group. Electron microscopy revealed a morphologically heterogeneous population of particles within this size range.     

Cell Fractionation of Anterior Pituitary Glands from Beef and Pig

Fresh anterior pituitary glands from beef and pig were separated by differential centrifugation into subcellular fractions. Nuclei and debris were obtained at 700 g for 15 minutes, secretory granules at 7000 g for 20 minutes, mitochondria at 34,000 g for 15 minutes, and microsomes at 78,000 g for 3 hours. Electron micrographs were taken of the individual fractions. Each fraction was analyzed for nitrogen, pentosenucleic acid (PNA), and phospholipide. Beef and pig anterior lobes were quite similar in their intracellular composition as seen in the subcellular fractions. Succinic dehydrogenase was localized in mitochondria, while alkaline phosphatase was concentrated in the microsomes. A proteinase with pH optimum at 8.2 was exclusively localized. in microsomal and supernatant fractions. Acid phosphatase, acid ribonuclease, and acid proteinase were distributed among the subcellular fractions in another pattern, indicating the presence of a particle type distinct from mitochondria and microsomes. The distribution of cytoplasmic PNA paralleled that of alkaline phosphatase.     

Proteins of the extracellular fluid of mouse brain: extraction and partial characterization

An extraction procedure for the isolation of proteins from the brain extracellular fluid (ECF) was developed and applied to studies of the ECF components of mouse brain. Perfused intact brains were incubated in an isotonic medium for periods of up to 2 h at 0 degrees C to allow the release of ECF into the medium without disruption of the integrity of the tissue. The validity of the extraction procedure was established by (a) the fact that the total yield of ECF proteins was constant per unit weight of brain tissue, (b) the absence of tyrosine hydroxylase, an enzyme marker of the cytoplasmic fraction, from the extracts, and (c) the distinctive features of the one- and two-dimensional gel electrophoretic patterns of ECF proteins as compared with those of the cytoplasmic and membrane fractions. The results indicate that the extracellular fluid of mouse brain contains a mixture of proteins with a wide distribution of molecular weights (10,000-100,000 daltons) at a concentration level of about 0.3%.     

Migration of putative progenitor T cells in response to thymus-derived chemotactic factors

Progenitor T cells reach the thymus through the circulation from hematopoietic organs and then migrate toward the site of differentiation in the thymus. The mechanism that regulates such intrathymic migration is not well understood. In order to clarify this mechanism, in vitro chemotactic activity for murine thymocytes was assayed in the extracts and culture supernatants of thymic tissue elements. A potent thymocyte chemotactic activity was found in the extract and culture supernatant from Ig-, Ia- thymic stromal cells. Peanut agglutinin-positive (PNA+1), Thy 1+, TL-, Lyt 1+2-, L3T4- thymocytes, Ig-, Thy 1- bone marrow cells, and mononuclear cells of spleen and peripheral blood, but neither B cells nor lymph node cells, were chemotactically attracted by the factor(s). The chemotactic activity was found in none of the following materials tested: the extract and culture supernatant of thymocytes, culture supernatant of lymph node stromal cells, normal mouse serum, and zymosan-activated serum. The chemotactic activity was found in three molecular fractions by gel chromatography. The activity in all three fractions was destroyed by trypsin digestion or by heating at 56 degrees C for 30 min. These results suggest that Ig-, Ia- thymic stromal cells but not thymocytes secrete a chemotactic factor(s) for progenitor T cells with three molecular species. The factor is considered to play an important role in the migration of intrathymic progenitor T cells into the site of differentiation.     

Preparation,purification, and properties of E. coli virus T2

1. A method for the preparation of 8 to 10 liter quantities of T₂ virus lysates, titering 2 to 5 x 10¹¹ infectious units per ml. has been described. 2. Procedures have been developed for the concentration and purification of virus to a high specific infectivity. No fractionation procedure of the several used succeeded in further raising the specific infectivity of these purified preparations. 3. Some of the general properties of the better preparations have been determined. They exhibited titers of 2 x 10¹⁵ infective units per gm. of material or 1.2 x 10¹⁶ per gm. of nitrogen. 4. A study of the distribution of nitrogen among the various fractions of the virus showed that about 6 per cent of the total nitrogen is soluble in 4 per cent trichloracetic acid; that the protein nitrogen is about 40 per cent of the total and the nucleic acid nitrogen is 53 per cent. At least 96 per cent of the total phosphorus is in the nucleic acid fraction. Less than 0.5 per cent quantities of lipid and PNA were found.     

Cytoplasmic Ribonucleoprotein Components of the Novikoff Hepatoma

Prominent nucleoprotein sedimentation boundaries were demonstrable in cytoplasmic extracts of Novikoff hepatoma. Fractionation of the homogenates by differential centrifugation or a density gradient method revealed that 65 to 75 per cent of the cytoplasmic ribonucleic acid was present in the form of free ribonucleoprotein particles. After purification by differential centrifugation in dilute buffer, the particles contained 37 per cent RNA, very little lipid, and no demonstrable membrane material. Ultracentrifugal boundaries corresponding to those seen in the original extracts were present, the main component having an s20, _w of 81 S. Upon exposure to chelating agents, the particles dissociated through an intermediate component with sedimentation rate of 56 S to a final stage in which 46 and 28 S subunits were present in a weight ratio of 2:1. ATP and pyrophosphate were equally effective in causing dissociation. ADP was considerably less effective. Treatment of the purified particles with deoxycholate removed one-third of the protein and significantly altered the ultracentrifugal pattern. The particles now dissociated directly to the 46 and 28 S subunit when exposed to chelating agents. Upon electron microscopy, the 81 S particle appeared as an oblate spheroid 24 mµ in diameter. The 46 and 28 S subunits also appeared spheroidal.     

SUBCELLULAR LOCALIZATION OF [14C]ADENINE DERIVATIVES NEWLY-FORMED IN CEREBRAL TISSUES AND THE EFFECTS OF ELECTRICAL EXCITATION

Abstract— Guinea pig neocortical tissues were incubated with [¹⁴C]adenine, dispersed in cold isotonic sucrose and subcellular fractions prepared by centrifugation. Some 98 per cent of the assimilated ¹⁴C was found as acid-soluble nucleotides in the incubated tissues. In primary fractions obtained by differential centrifugation, about 60 per cent of the [¹⁴C]-nucleotides were in supernatant fractions, in distinction to ATP of which the greatest molar quantity (61 per cent of that in the dispersion) was in the crude mitochondrial fraction. When the crude mitochondrial fraction was separated by density gradient centrifugation, most ¹⁴C was found in synaptosomal fractions and about 85 per cent of this ¹⁴C was adenine nucleotides.
Electrical stimulation of incubating tissues immediately prior to their dispersion and centrifugation greatly diminished the proportion of ¹⁴C subsequently found in nucleotides (collectively) in the supernatant fraction, and increased their inosine and hypoxanthine. Stimulation increased the tissue's cyclic AMP but a preferential localization for this was not established. Results are tentatively interpreted in terms of liberation of an adenine derivative on excitation, and its action or reuptake at a tissue component different from that from which it was liberated. Fractionation of tissues which had been incubated with both [¹⁴C]-adenine and [³H]adenosine suggested that of the two compounds, more adenosine was taken up by synaptic regions in preference to other cellular regions of the tissue.     

DISTRIBUTION OF POLYSOMES IN MOUSE BRAIN TISSUE

Abstract— Polysomes were isolated from several different fractions of mouse brain tissue. After homogenization, the extract was centrifuged to yield a post-mitochondrial supernatant fraction and a pellet fraction. Sucrose gradient analysis of the material in the post-mitochondrial supernatant fraction indicated that 80 per cent of the ribosomes were present in polysomes and that little, if any, of the pellet fraction was present. Sucrose gradient analysis of the solution obtained after washing the pellet showed that very little polysomal material was present. The remaining pellet fraction was resuspended in a detergent mixture of deoxycholate-Tween 40. Sucrose gradient analysis of the resulting detergent-soluble solution indicated that large amounts of ribosomal material, in which 60–70 per cent of the ribosomes were associated in polysomes, were present.
In brain tissue from young animals, 20 per cent of the polysomes were found in the post-mitochondrial supernatant fraction whereas 80 per cent of the polysomes were released from the pellet fraction by detergent treatment. In contrast, in brain tissue from adult animals, 40 per cent of the polysomes were found in the post-mitochondrial supernatant fraction, whereas 60 per cent of the polysomes were released from the pellet fraction by detergent treatment.     

Multiple disc gel bands of mitochondrial creatine and adenylate kinases

Abstract— The activities and electrophoretic patterns of creatine and adenylate kinases in the mitochondrial and high speed supernatant fractions of adult mouse brain were determined. Approximately 22 per cent of the activities of both kinases is firmly bound to the mitochondria. On acrylamide gel electrophoresis of creatine kinase, in addition to the major band previously described, there were several other bands found. Although present in both the mitochondrial and supernatant fractions these additional protein bands with creatine kinase activity were significantly more intense in the mitochondrial fraction. There was only onesecondary band of adenylate kinase activity in the mitochondrial fraction but additional bands were found in the soluble fraction.     

MICROSOMAL NUCLEOPROTEIN PARTICLES FROM PEA SEEDLINGS

Ultracentrifugal analysis of an extract of pea epicotyls, previously freed of debris and larger particles by centrifugation at 40,000 g for 10 minutes, has revealed the presence of a major component which possesses a sedimentation coefficient of 74 S. This component constitutes about 25 per cent of the TCA-precipitable material in the clarified epicotyl extract and is estimated to make up 1 to 2 per cent of the dry weight of the original tissue. In size, chemical composition, and morphology, the 74 S component resembles the nucleoproteins of the microsomes from animal tissues. The 74 S component of pea epicotyl extracts has been purified by repeated cycles of differential centrifugation to yield a preparation which is 80 per cent homogeneous in the analytical ultracentrifuge. It has been found to contain 30 to 37 per cent RNA as judged by a variety of analytical techniques. Approximately 55 per cent of the weight of the material is protein and a further 4.5 per cent phospholipide. Electron micrographs of air-dried specimens of the purified preparation show the 74 S constituent to be flattened spheres with an average height of 180 A and an average diameter of approximately 280 A. The molecular weight of the 74 S particles is computed from sedimentation, viscosity, and partial specific volume data to be 4.5 million ± 10 per cent in agreement with the value estimated from electron micrographs. The 74 S or microsomal component of pea epicotyls is rapidly aggregated in the presence of low concentrations of Mg ions or by somewhat higher concentrations of Ca or K salts. ATP on the contrary causes resolution of electrolyte-induced microsomal aggregates with simultaneous degradation of the particles to an ultracentrifugally inhomogeneous mixture of lower molecular weight materials.     

The Distribution of Nickel, Copper, Zinc, and Iron in Tree Leaves

The distributions of nickel, copper, zinc, and iron in freshleaves from four tree species were studied using differentialoentrifugation of cell homogenates to fractionate the cell organellesand atomic absorption spectrophotometry to measure the concentrationsin the various fractions. Results from each species were closelysimilar. Approximately 90 per cent of the nickel was presentin the supernatant fraction while approximately 80 per centof the iron was present in the chloroplast fraction. Both copperand zinc had subcellular distributions similar to one anotherand contained appreciable amounts of the elements in both chloroplastand supernatant fractions. High-voltage paper electrophoretic separations of the solubleextracts of leaves showed that while copper, zinc, and ironexisted principally as anionic complexes, nickel existed asa cationio species. It was considered that this cationic formof nickel was unlikely to be an unchelated aquated cation.     

Degradation of somatomedin A by various organ homogenates from rats

Degradative activities of somatomedin A (SMA) have been examined in various tissue homogenates of rat using trichloracetic acid precipitable radioactivity of 125I-SMA. Kidney and testis showed higher specific activities and liver and brain lower activities. They were dependent on SH reagents; 0.5 mM HgCl2 inhibited the degradative activity of liver completely and 1 mM dithiothreitol (DTT) augmented the activity slightly. The activities in liver were separated by differential centrifugation; about 90 per cent of the total activity in the whole homogenate was recovered in the supernatant fraction at 100,00 x g for 60 min, and 10 per cent in the precipitate. The pH profile of each fraction was different; that of the supernatant showed a single peak at pH 7.4 and that of the pellet revealed two peaks at pH 5.9 and 7.4. However, both fractions showed similar SH-dependency.     

A contribution to the standardization of methods for the preparation of seed proteins ofAllium cepa L.

The study of certain conditions for the extraction of seed proteins ofAllium cepa revealed that the best extractibility of proteins is obtained by the use of a buffered physiological solution at 20 °C in comparison with TRIS-glycine buffer at 5 °C. Using potassium phosphate buffer with 0.01 M mercaptoethanol and 0.4 M NaCl, an amount of proteins by up to 25 per cent higher passes into solution as compared with the physiological solution, but these extracts are unsuitable for the electrophoretic separation in polyacrylamide gels. The defatting of the seed meal under low temperature did not affect the qualitative composition of the protein complex studied, the addition of a 1 per cent soluble starch to the polyacrylamide gel. improved its resolution.     

Immunochemical characteristics of a factor causing antigen-specific lysis of thymus-dependent lymphocytes]

A factor capable of lysing in vitro, in the presence of a specific antigen, the cells of the lymph nodes and the thymus of intact mice was revealed in the supernatant obtained after the centrifugation of a suspension of viable cells of the lymph nodes and the thymus of the immunized mice. It was found by immunochemical methods that this factor had a mol. wt. of about 30000 dalton and an electrophoretic mobility in polyacrylamide gel exceeding that of mouse blood serum albumin. Besides, it was revealed by the precipitation reaction in agar that it was not an immunoglobulin or its chains.     

SOME PROPERTIES OF ISOLATED NEURONAL CELL FRACTIONS

Abstract— 1. Histochemical evidence was presented illustrative of the composition of neuronal and neuropil ('glial') fractions isolated according to a previously published procedure. The neuropil refers to all cortical tissue other than neuronal perikarya.
2. On the basis of cell counts and of DNA content, an average cell mass of 100-110 pg was calculated for cells in the neuronal fraction. Eight per cent of the total DNA was recovered in the neuronal fraction.
3. Both fractions synthesized ATP in vitro. Concentrations after 60 min incubation with glucose were: neuropil, 7–36 μmoles/mg protein; neuronal, 12–31 μmoles/mg protein.
4. Osmotic shock or homogenization resulted in changes in turbidity of the cell fractions which were interpreted as indicative of loss of cell structure. The free pool amino acids glutamate, glutamine, GABA, aspartate and alanine were retained in the precipitable material through several washes with isotonic solutions. Homogenization released 72 per cent of the neuronal and 68 per cent of the neuropil amino acids into the supernatant, but only 37 per cent and 19 per cent respectively of the protein.
5. By contrast with earlier reports, K⁺ accumulation has now been demonstrated in both neuronal and neuropil fractions. After incubation with glucose, K⁺ level were calculated as being 80 per cent of slice in the neuronal, and 65 per cent in the neuropil fraction. These results, and those of the osmotic shock experiments, were taken as indicative of the retention of some cell structure.
6. By comparison, cell fractions prepared by other procedures, using acetone-glycerol-water or tetraphenylboron for tissue disaggregation, produced preparations with limited metabolic capabilities; oxygen uptake, CO₂ and lactate production were all lowered substantially.