scholarly journals Assimilation of glucose carbon in subcellular rat brain particles in vivo and the problems of axoplasmic flow

1967 ◽  
Vol 105 (3) ◽  
pp. 927-936 ◽  
Author(s):  
R. Vrba
Keyword(s):  
Particulate Matter ◽  
High Speed ◽  
Specific Activity ◽  
Sucrose Density Gradient ◽  
Mitochondrial Fraction ◽  
Axoplasmic Flow ◽  
Brain Lipids ◽  
Mitochondrial Fractions ◽  
High Speed Supernatant

1. Rats were injected with [U−14C]glucose and the content of 14C in proteins and lipids of the cerebral P1 (‘nuclear’), P2 (‘mitochondrial’), P3 (‘microsomal’) and high-speed supernatant fractions was measured 7, 22 and 93hr. after injection of labelled glucose. 2. The crude brain mitochondrial fractions (P2) were subfractionated on continuous sucrose gradients (0·32–1·8m-sucrose) and the 14C content of the proteins and lipids of about 20 subfractions was measured. 3. About 40–50% of the 14C assimilated by brain proteins was found in the P2 (‘mitochondrial’) fraction. About 68–70% of the 14C assimilated by brain lipids was also recovered from the lipids of the P2 fraction. 4. Between 22 and 93hr. after injection of [U−14C]glucose both the amount of 14C in the protein of the P2 (‘mitochondrial’) fraction and the specific activity of this protein increased. The specific activity of the protein of all other particulate fractions (P1, P2 and P3) and subfractions (obtained from sucrose-density-gradient subfractionation of fraction P2) when related to the specific activity of the high-speed supernatant protein also increased during 93hr. after injection of [U−14C]glucose. The amount of 14C in the protein of the high-speed supernatant and the specific activity of this protein decreased during the same period. 5. The distribution of 14C in the lipids of all subcellular particulate fractions remained unchanged during the period 22–93hr. after injection of [U−14C]glucose. 6. It was concluded that a diffusion occurs of some supernatant proteins into subcellular particulate matter of the cerebrum and no significant preference for any subcellular particulate matter was observed. The lipids occur in the cerebrum mainly in a non-diffusible state, which is consistent with the view that they form almost entirely a part of the structure of the cerebrum. 7. The data obtained do not lend further support to the concept of axoplasmic flow within the cerebrum or the concept of a one-directional flow of mitochondria or other subcellular particles within the cerebrum.

scholarly journals Mitochondrial Electron Transport Enzymes in Normal and Leukemic Human Leukocytes

Blood ◽  
1968 ◽  
Vol 31 (6) ◽  
pp. 710-718 ◽  
Author(s):  
AUDREY E. EVANS ◽  
GODFREY S. GETZ
Keyword(s):  
Electron Transport ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Polymorphonuclear Leukocytes ◽  
Gradient Analysis ◽  
Sucrose Density Gradient ◽  
Mitochondrial Fraction ◽  
Human Leukocytes ◽  
Mitochondrial Fractions ◽  
Succinate Cytochrome C Reductase

Abstract Assays of TD transhydrogenase and succinate cytochrome C reductase carried out on the homogenate of normal and leukemic human leukocytes and on the 1.5 M. fraction (fraction 3) of a continuous sucrose density gradient analysis of a crude mitochondrial fraction, provided strong evidence for the mitochondrial location of TD transhydrogenase. A slightly higher activity of these enzymes was found in the homogenate of cells from chronic lymphocytic and acute leukemia compared to those of normal polymorphonuclear leukocytes, normal lymphocytes, and cells from chronic myeloid leukemia. These differences were very much more marked when purified mitochondrial fractions from these cells were examined. The significance of these findings is discussed.

scholarly journals DISTRIBUTION OF NEWLY SYNTHESIZED AMYLASE IN MICROSOMAL SUBFRACTIONS OF GUINEA PIG PANCREAS

1966 ◽  
Vol 30 (3) ◽  
pp. 519-530 ◽  
Author(s):  
P. Siekevitz ◽  
G. E. Palade
Keyword(s):  
Guinea Pig ◽  
High Speed ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Enzymic Activity ◽  
Pellet Fraction ◽  
Light Region ◽  
Centrifuge Tube ◽  
In Vivo Labeling

Amylase distribution was studied in guinea pig pancreas microsomes fractionated by centrifuging, for 2 hr at 57,000 g in a linear 10 to 30% sucrose gradient, a resuspended high speed pellet obtained after treating microsomes with 0.04% deoxycholate (DOC).1 Amylase appeared in the following positions in the gradient: (a) a light region which contained ∼35% of total enzymic activity and which coincided with a monomeric ribosome peak; (b) a heavy region which contained ∼10% of enzymic activity in a sharp peak but which had very little accompanying OD260 absorption; (c) a pellet at the bottom of the centrifuge tube which contained ∼20% of the enzymic activity. After 5 to 20 min' in vivo labeling with leucine-1-C14, radioactive amylase was solubilized from these three fractions by a combined DOC-spermine treatment and purified by precipitation with glycogen, according to Loyter and Schramm. In all cases, the amylase found in the pellet had five to ten times the specific activity (CPM/enzymic activity) of the amylase found in the light or heavy regions of the gradient. The specific radioactivity (CPM/mg protein) of the proteins or peptides not extracted by DOC-spermine was similar for all three fractions. Hypotonic treatment of the fractions solubilized ∼80% of the total amylase in the fraction from the heavy region of the gradient, but only ∼20% of the amylase in the monomer or pellet fraction. Electron microscope observation indicates that the monomer region of the gradient contained only ribosomes, that the heavy region of the gradient contained small vesicles with relatively few attached ribosomes, and that the pellet was composed mostly of intact or ruptured microsomes with ribosomes still attached to their membranes. It is concluded from the above, and from other evidence, that most of the amylase activity in the monomer region is due to old, adsorbed enzyme; in the heavy region mostly to enzyme already inside microsomal vesicles; and in the pellet to a mixture of newly synthesized and old amylase still attached to ribosomes. Furthermore, the ribosomes with nascent, finished protein still bound to them are more firmly attached to the membranes than are ribosomes devoid of nascent protein.

scholarly journals Studies on bone enzymes. Distribution of acid hydrolases, alkaline phenylphosphatase, cytochrome oxidase and catalase in subcellular fraction of bone tissue homogenates

1965 ◽  
Vol 97 (2) ◽  
pp. 389-392 ◽  
Author(s):  
G Vaes ◽  
P Jacques
Keyword(s):  
Cytochrome Oxidase ◽  
Subcellular Fraction ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Distribution Patterns ◽  
Mitochondrial Fraction ◽  
Acid Hydrolases ◽  
Mitochondrial Fractions ◽  
Tissue Homogenates ◽  
Isopycnic Centrifugation

1. When bone homogenates were fractionated according to the scheme developed for liver by de Duve, Pressman, Gianetto, Wattiaux & Appelmans (1955), all the enzymes assayed except cytochrome oxidase were found to occur partly in soluble and partly in particulate fractions. Among the particle-bound enzymes, the highest specific activity was found in the heavy-mitochondrial fraction for cytochrome oxidase, in the microsomal fraction for alkaline phenylphosphatase and in the light-mitochondrial fraction for eight acid hydrolases and for catalase. 2. Combined heavy-mitochondrial and light-mitochondrial fractions were subfractionated by isopycnic centrifugation in density gradients of sucrose or glycogen. In the various systems tried, cytochrome oxidase showed a relatively narrow distribution range with a sharp peak; the acid hydrolases and catalase showed flat and irregular distribution patterns, differing slightly in shape from one enzyme to the other. However, it was not possible to achieve a marked separation between the various enzymes under study. 3. It is concluded from these results that the acid hydrolases belong to special cytoplasmic particles, probably lysosomes, and that these particles are physically and enzymically heterogeneous. Catalase appears to be non-mitochondrial and could also belong to the lysosomes; but the possibility of an association with another type of particle must be kept in mind in view of what is known of liver catalase. Alkaline phenylphosphatase is largely attached to microsomal elements.

scholarly journals BRAIN MITOCHONDRIA

1963 ◽  
Vol 19 (2) ◽  
pp. 309-316 ◽  
Author(s):  
Diana S. Beattie ◽  
Howard R. Sloan ◽  
R. E. Basford
Keyword(s):  
High Speed ◽  
Brain Cortex ◽  
Lactate Production ◽  
Mitochondrial Fraction ◽  
Brain Mitochondria ◽  
Glycolytic Enzymes ◽  
Supernatant Fraction ◽  
Glycolytic Activity ◽  
High Speed Supernatant ◽  
Stimulation Of

A mitochondrial fraction prepared from calf brain cortex possessed negligible glycolytic activity in the absence of the enzymes of the high speed supernatant fraction. When mitochondria were added to a supernatant system supplemented with optimal amounts of crystalline hexokinase, a 20 per cent stimulation of glycolysis was observed. The supernatant fraction produced minimal amounts of lactate in the absence of exogenous hexokinase; the addition of mitochondria doubled the lactate production. The substitution of glycolytic intermediates for glucose as substrates as well as the addition of exogenous glycolytic enzymes to the supernatant fraction or supernatant fraction plus mitochondria indicated that the mitochondria contributed mainly hexokinase and phosphofructokinase. By direct assay of all of the enzymes of the glycolytic pathway, only hexokinase and phosphofructokinase were shown to be concentrated in the mitochondrial fraction. All other glycolytic enzymes were found to exhibit higher total and specific activities in the supernatant fraction.

scholarly journals Canine submandibular-gland hyaluronidase. Identification and subcellular distribution

1968 ◽  
Vol 110 (1) ◽  
pp. 9-17 ◽  
Author(s):  
Y. H. Tan ◽  
J. M. Bowness
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Submandibular Gland ◽  
High Speed ◽  
Specific Activity ◽  
Mitochondrial Fraction ◽  
Product Formation ◽  
Triton X ◽  
Submandibular Saliva ◽  
Testicular Hyaluronidase

1. Submandibular glands from four species of mammal have been shown to contain a hyaluronidase active at acid pH; glands from dog and cat had a much higher content of this enzyme than has been found in other sources. 2. Product formation from hyaluronate after 24hr. incubation was almost the same as with testicular hyaluronidase, indicating that the enzyme is an endo-poly-β-hexosaminidase. 3. When submandibular-gland homogenates were fractionated by the scheme developed for liver by de Duve, Pressman, Gianetto, Wattiaux & Appelmans (1955), all the enzymes assayed, except cytochrome c oxidase, were found to occur partly in the soluble fraction and partly in the particulate fractions. Among the particular fractions, the highest specific activity was found in the heavy-mitochondrial fraction for cytochrome c oxidase, in the microsomal fraction for alkaline phosphatase and in the light-mitochondrial fraction for acid phosphatase, β-N-acetylhexosaminidase and acid-active hyaluronidase. 4. Release of the enzyme activity from the sedimentable fractions occurred in 0·1% Triton X-100 or after high-speed homogenization. 5. Stimulation of dogs by pilocarpine was found to decrease the hyaluronidase content of the submandibular gland by 5% and to cause the occurrence of a corresponding amount of acid-active hyaluronidase in the submandibular saliva. 6. The results are discussed in relation to the subcellular localization of hyaluronidase.

scholarly journals Heterogeneity of rat liver mitochondrial fractions and the effect of tri-iodothyronine on their protein turnover

1970 ◽  
Vol 118 (1) ◽  
pp. 111-121 ◽  
Author(s):  
S. S. Katyare ◽  
P. Fatterpaker ◽  
A. Sreenivasan
Keyword(s):  
Rat Liver ◽  
Mitochondrial Fraction ◽  
Liver Mitochondria ◽  
Heavy Fraction ◽  
Rat Liver Mitochondria ◽  
Turnover Rates ◽  
Loosely Coupled ◽  
Mitochondrial Fractions ◽  
Isoelectric Ph

1. Rat liver mitochondria were separated into heavy, light and fluffy fractions by differential centrifugation under standard conditions. 2. All mitochondrial fractions possessed soluble as well as membrane-bound enzymes typical of mitochondria. 3. The heavy fraction represented the stable mitochondrial structures and the fluffy particles appear to be loosely coupled. 4. The light mitochondrial fraction lacked the ability of coupled phosphorylation. 5. A study of mobility and isoelectric pH indicated a similarity in the basic membrane structure of all the mitochondrial fractions. 6. The turnover rates of proteins in the heavy and fluffy particles were almost identical; however, this rate was rapid for the light mitochondrial fraction. 7. On treatment with 3,3′,5-tri-iodo-l-thyronine, succinoxidase activity was maximally stimulated much earlier in the light mitochondrial fraction than in the heavy fraction. The activity of the fluffy particles, however, remained almost unaffected. 8. Malate dehydrogenase activity in all the mitochondrial fractions was stimulated only at 40h after tri-iodothyronine treatment. 9. The pattern of incorporation of dl-[1-14C]leucine in vivo in the tri-iodothyronine-treated animals indicated a rapid initial incorporation and high synthetic ability of the light mitochondrial fraction. 10. The turnover pattern of proteins of the mitochondrial fractions from animals receiving repeated doses of tri-iodothyronine was remarkably different from the normal pattern and suggested that preformed soluble protein units may be incorporated in the light mitochondrial fraction during maturation to form the stable heavy mitochondria. 11. The amount of light-mitochondrial proteins decreased by 40% on thyroidectomy and increased by 160% on treatment with tri-iodothyronine. 12. The possible significance of these results is discussed in relation to mitochondrial genesis.

scholarly journals The incorporation of radioactive fatty acids into the phospholipids of nerve-cell-body membranes in vivo. Evidence for highly labelled neuronal nuclei

1980 ◽  
Vol 188 (1) ◽  
pp. 153-161 ◽  
Author(s):  
R. Roy Baker ◽  
Huu-yi Chang
Keyword(s):  
Fatty Acid ◽  
Nerve Cell ◽  
Cell Body ◽  
High Speed ◽  
De Novo ◽  
Nerve Cell Body ◽  
Double Labelling ◽  
Nuclear Fraction ◽  
Mitochondrial Fractions

1. Nerve cell bodies were isolated in bulk from cerebral cortices of 15 day-old rabbits after intrathecal injections of [3H]plamitate, [3H]oleate or [3H]arachidonate and [14C]glycerol. 2. Nuclear, microsomal and two mitochondrial fractions were isolated from homogenates of the radioactively labelled nerve cell bodies by using differential and discontinuous-gradient centrifugation. 3. After 7.5min in vivo, a high percentage (>80%) of the total 3H-labelled fatty acid radioactivity was found in the membrane fractions of the nerve cell bodies, whereas after 60min in vivo 50% of the total [14C]glycerol radioactivity was found in the high-speed supernatant. 4. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, and the radioactivity in neutral lipid and non-esterified fatty acid fractions were determined in the four subfractions, as were the distributions of several marker enzymes and nucleates. 5. With respect of 3H-labelled fatty acid, the phospholipids of the nuclear fraction had the highest specific radioactivities of the four subfractions. However, for [14C]glycerol labelling, generally the 14C specific radioactivities for individual phospholipids were comparable in the four subfractions. This latter observation suggests transport of phospholipids synthesized de novo between membranes of the nerve cell body. 6. Double-labelling experiments demonstrated that individual phospholipids and the combined neutral lipids of the nuclear fraction had higher labelling ratios of 3H-labelled fatty acid/[14C]glycerol than did the corresponding lipids of the microsomal or mitochondrial fractions. 7. On the basis of the labelling results and the marker studies, it is proposed that it is indeed the nuclei of the nuclear fraction that have these lipids highly labelled with 3H-labelled fatty acid, and the existence of nuclear acyl transferases that are responsible for this fatty acid incorporation is suggested.

Phosphofructokinase and glycolysis in the livers of rats fed a thrombogenic diet

1970 ◽  
Vol 48 (2) ◽  
pp. 222-224
Author(s):  
N. Simard-Duquesne
Keyword(s):  
Lactic Acid ◽  
High Speed ◽  
Mitochondrial Fraction ◽  
Pasteur Effect ◽  
Phosphofructokinase Activity ◽  
Reconstituted System ◽  
High Speed Supernatant

Liver supernatant phosphofructokinase activity is increased in rats fed a thrombogenic diet at about the same time as the incidence of endotoxin-induced thrombosis is known to increase. In the livers of these rats, the Pasteur effect, as determined by the production of lactic acid in a reconstituted system containing a high-speed supernatant with and without the mitochondrial fraction, is significantly decreased.

scholarly journals The biosynthesis of brain gangliosides. Separation of membranes with different ratios of ganglioside sialylating activity to gangliosides

1977 ◽  
Vol 168 (3) ◽  
pp. 325-332 ◽  
Author(s):  
C A Landa ◽  
H J F Maccioni ◽  
A Arce ◽  
R Caputto
Keyword(s):  
Membrane Fraction ◽  
Time Course ◽  
Mitochondrial Fraction ◽  
Subcellular Fractions ◽  
Synthetase Activity ◽  
Acetylneuraminic Acid ◽  
Mitochondrial Fractions ◽  
Brain Gangliosides

Brain subcellular fractions were analysed for ganglioside-sialylating activity by measuring the incorporation of N-[3H]acetylneuraminic acid from CMP-N-[3H]acetylneuraminic acid into endogenous ganglioside acceptors (endogenous incorporation) and into exogenous lactosyceramide (haematoside synthetase activity). The ratios of endogenous incorporation to gangliosides and of haematoside synthetase to gangliosides for the synaptosomal and mitochondrial fractions from a washed crude mitochondrial fraction were lower than those obtained for other membrane fractions. The differences appear to reflect intrinsic characteristics of each membrane fraction. The results of labelling in vitro and the time course of labelling of gangliosides of the different subcellular fractions in vivo after injection of N-[3H]acetylmannosamine are consistent with the possibility of a subcellular site for synthesis of gangliosides different from that of ganglioside deposition.

scholarly journals A Cytochemical Study on the Pancreas of the Guinea Pig

1958 ◽  
Vol 4 (5) ◽  
pp. 557-566 ◽  
Author(s):  
Philip Siekevitz ◽  
George E. Palade
Keyword(s):  
Guinea Pigs ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Pancreatic Cell ◽  
Cytochemical Study ◽  
Mitochondrial Fractions ◽  
Liver And Pancreas ◽  
Intracardiac Injection ◽  
Cell Fractions

DL-leucine-1-C14 was administered by intracardiac injection to guinea pigs and its in vivo incorporation into the proteins of various pancreatic cell fractions followed over a period of 2 hours. The pancreas was homogenized in 0.88 M sucrose and fractionated by differential centrifugation to give nuclear, zymogen, mitochondrial, microsomal, postmicrosomal, and final supernatant fractions. The proteins of these fractions, obtained by precipitation with trichloroacetic acid followed by washing, were counted. The proteins of the microsomal fraction showed the highest early specific activity and were followed by those of the zymogen and mitochondrial fractions. The microsomal fraction was broken up into two subfractions: one consisting of detached RNP particles, the other representing mainly the microsomal content and membranes. The incorporation of labelled leucine into the proteins of microsomal subtractions and in those of postmicrosomal fractions was studied comparatively in the pancreas of fasted and fed guinea pigs as well as in the liver and pancreas of fasted animals. A tentative cytological picture of protein synthesis and transport based on these findings is presented.

Sign in / Sign up

Export Citation Format

Share Document