The osmotic behavior of corn mitochondria

The volume changes undergone by corn (Zea mays L.) mitochondria suspended in solutions of constant or varying osmolarity were studied. Within the range of osmotic pressure from 1.8 to 8.4 atmospheres, corn mitochondria behave as osmometers, if allowance is made for an osmotic “dead space” of about 6.9 μl/mg protein. The final equilibrium volume of mitochondria swollen in solutions containing both ribose and sucrose were shown to depend upon the concentration of impermeable solute (sucrose) present and not upon the concentration of ribose present. Osmotic reversibility was found for mitochondria swollen in isotonic solutions of KCI or ribose. The passive swelling of corn mitochondria may be due to the osmotic flow of water coupled to the diffusion of a permeable solute.     

PERMEABILITY OF MICROSOMAL MEMBRANES ISOLATED FROM RAT LIVER

Water compartments, permeability, and the possible active translocation of various substances in rat liver microsomes were studied by using radioactive compounds and ultracentrifugation. The total water of the microsomal pellet, 3.4 µl/mg dry weight, is the sum of water in the extramicrosomal and intramicrosomal spaces, or 56 and 44%, respectively. Sucrose space accounts for 77% of the intramicrosomal water and the hydration water ~ 14%, leaving almost no sucrose-impermeable space when using the ultracentrifugation approach. With increasing sucrose concentration, microsomes do not show an osmotic response. The intramicrosomal water decreases greatly in the presence of Cs⁺ and Mg⁺⁺ in rough but not in smooth microsomes. Uncharged substances of molecular weight of up to at least 600 freely penetrate microsomal membranes, which already become impermeable to charged substances at a molecular weight of 90. These substances also induce an osmotic response. The vesicles can be made permeable to charged substances after water treatment and cooling, which, however, does not increase glucose-6-phosphatase and inosine diphosphatase (IDPase) activities, and these enzymes can still be activated by deoxycholate. IDPase, reduced nicotinamide adenine dinucleotide-cytochrome c reductase, and reduced nicotinamide adenine dinucleotide phosphate-dependent hydroxylation reactions, performed in vitro, also disproved the hypothesis of an accumulation of charged substances inside of vesicles of being a major pathway. The products of the enzymic reactions as well as the glucuronidated form of a hydroxylated product can be recovered on the cytoplasmic side of membranes, and little accumulation occurs in the intravesicular compartment.     

Precocious Germination during In Vitro Growth of Soybean Seeds

Immature Glycine max (L.) Merrill seeds were grown and matured in liquid medium at 25°C under fluorescent light. In standard medium containing minerals, 146 millimolar sucrose and 62.5 millimolar glutamine (osmolality 0.24), precocious germination seldom occurred with a starting seed size of less than 300 milligrams fresh weight. Frequency of precocious germination increased with increased starting seed size. Sucrose concentration strongly affected precocious germination while glutamine concentration had no effect. Starting with 300 to 350 milligrams fresh weight seeds, treatments which reduced the sucrose concentration or lowered the osmolality of the culture medium stimulated precocious germination, and increased the fresh weight growth but not the dry weight growth of seeds. Increasing the osmolality to 0.38 with sucrose or mannitol prevented precocious germination without reducing dry weight accumulation in seeds. In medium with initially low osmolality, precocious germination was inhibited by addition of 1 to 100 micromolar abscisic acid to the medium without a reduction in seed growth. During growth and maturation of large soybean seeds in vitro, precocious germination and other abnormal tissue growth can be prevented by high sucrose or mannitol concentrations in the medium or by addition of abscisic acid.     

The localization of proteolytic activity in rat liver mitochondria and its relation to mitochondrial swelling and aging

1. On storage of rat liver mitochondria at 0°, water content, total amino acid content and leakage of protein all rose steadily over a 72hr. period. The initial ratio of intramitochondrial to extramitochondrial amino acid concentration lay between 18 and 24. Initially this rose, but it then fell to 1·9 at the end of storage. The concentration gradient between internal and external amino acids was relatively constant throughout the period. These processes were accentuated at 22° and 40°, the concentration gradient reaching 70μmoles/ml., water content rising to 8·3mg./mg. dry wt. and protein leakage reaching 42% of total mitochondrial protein. `Swelling agents' produced no correlated changes in amino acid production and swelling. 2. Added glutamate was not concentrated within the pellet of whole or disrupted mitochondria. Endogenous amino acids were distributed evenly between the pellet and the supernatant of disrupted mitochondria. It is concluded that amino acids are produced within mitochondria and that adsorption and uptake from the medium do not contribute significantly to amino acids in the pellet. 3. β-Glycerophosphate, a lysosome protectant, increased amino acid production by rat liver mitochondria. Treatment with Triton X-100 and disruption by freezing and thawing showed that 56% of proteolytic activity was `free' in whole mitochondria, whereas only 11% of acid phosphatase activity, a lysosomal enzyme, was `free'. 4. `Light' mitochondria contained 30% more neutral proteolytic activity but 300% more acid phosphatase activity than `heavy' mitochondria. 5. Electron micrographs of mitochondrial preparations showed less than one particle in 500 that could be identified as a lysosome. Treatment with Triton X-100 disrupted the structure of roughly 50% of the mitochondria; the rest appeared to retain their membrane, cristae and ground substance. Freezing and thawing caused gross swelling and loss of ground substance and rupture of external membranes. 6. Of the recovered proteolytic activity, 81% at pH7·4 and 70% at pH5·8 were found in the high-speed supernatant of broken mitochondria. A further fivefold increase in specific activity was found in the first protein fraction obtained by Sephadex G-50 gel filtration. 7. Between 60 and 80% of proteolytic activity was found in the 40–60%-saturated ammonium sulphate precipitate. Almost all of the soluble-fraction proteolytic activity could be recovered in a pH5·0 supernatant. 8. The results give no support to the view that mitochondrial neutral proteolytic activity reflects lysosomal content. 9. The possible role of intramitochondrial amino acid production and the proteolysis of internal barriers in passive swelling of mitochondria is discussed.     

Cat Heart Muscle in Vitro : IX. Cell ion and water contents in anisosmolal solutions

Cell contents of water, K, Na, and Cl have been determined in cat right ventricular papillary muscles immersed in solutions with and without NaCl when the external osmolality was varied with sucrose. The plot of cell water/kilogram dry weight (corrected for sucrose content) vs. (external osmolality)^-1 suggests that not less than 82% of water present in cells at physiological external osmolality is free to move across the cell membrane in response to an imposed osmotic gradient. Cells fail to increase their water content in very hypotonic solutions. For osmolalities greater than 5 times isosmolal, at which the mannitol space and the Cl³⁶ space are both equal to 100% of muscle water, rather large amounts of univalent cation appear to remain "bound" to the tissue.     

The intramitochondrial volume measured using sucrose as an extramitochondrial marker overestimates the true matrix volume determined with mannitol.

The matrix volume of isolated liver and heart mitochondria has been estimated at various osmolarities and in various osmotic supports using 36Cl- and [14C]sucrose, D-mannitol, D-3-methoxyglucose and choline as extramitochondrial markers. The use of 3-methoxyglucose was only possible at 0 degree C since it entered mitochondria at physiological temperatures. All extramitochondrial markers used gave linear plots of apparent matrix volume against the reciprocal of the osmolarity, but the slope of this plot was greater when sucrose was used than with the other extramitochondrial markers. When extrapolated to infinite osmolarity the mean matrix volume was zero when mannitol was used, but about 0.6 microliter/mg of protein for sucrose and Cl- and -0.4 microliter/mg of protein when choline was used. At physiological osmolarity (about 330 m-osmol) the mean matrix volume of de-energized liver mitochondria in KCl medium estimated using mannitol was 0.46 microliter/mg of protein, whereas that obtained using sucrose was 1.68 microliters/mg of protein. Values in mannitol, choline and sucrose media were similar when mannitol but not sucrose was used as extramitochondrial marker. It is argued that the 3H2O/[14C]mannitol space more accurately reflects the true mitochondrial matrix volume than does the 3H2O/[14C]sucrose space. The consequences of this for measurements of the protonmotive force and the intramitochondrial concentration of metabolites are discussed.     

Osmotically-induced alterations in volume and ultrastructure of mitochondria isolated from rat liver and bovine heart

Detailed studies correlating changes in mitochondrial optical density, packed volume, and ultrastructure associated with osmotically-induced swelling were performed. Various swelling states were established by incubating mitochondria (isolated in 0.25 M sucrose) at 0°C for 5 min in series of KCl and sucrose solutions ranging in tonicity from 250 to 3 milliosmols. Reversibility of swelling was determined by examining mitochondria exposed to 250 milliosmols media after they had been induced to swell. Swelling induced by lowering the ambient tonicity to approximately 130 (liver mitochondria) and 90 (heart mitochondria) milliosmols involves primarily swelling of the inner compartment within the intact outer membrane. Decreasing the ambient tonicity beyond this level results in rupture of the outer membrane and expansion of the inner compartment through the break. The maximum extent of swelling, corresponding with complete unfolding of the cristae and an increase in over-all mitochondrial volume of approximately 6-fold (liver mitochondria) and 11-fold (heart mitochondria), is reached at approximately 15 (liver mitochondria) and 3 (heart mitochondria) milliosmols. Exposure of liver mitochondria to media of lower tonicity results in irreversibility of inner compartment swelling and escape of matrix material. These changes appear to result from increased inner membrane permeability, possibly due to stretching.     

Growth of Corynebacterium glutamicum in glucose-limited continuous cultures under high osmotic pressure. Influence of growth rate on the intracellular accumulation of proline,glutamate and trehalose

In order to determine the response of Corynebacterium glutamicum to osmotic stress under different growth conditions, the bacteria were grown in glucose-limited continuous cultures at osmotic pressures of 0.4–2.4 osmol kg^–1 by addition of NaCl to the culture medium. Steady-state continuous cultures were obtained for all investigated osmotic pressures. Increasing the medium osmolality resulted in a higher specific glucose-uptake rate, a lower glucose-to-biomass conversion yield, as well as important changes in the cellular content. A short-term response to the addition of NaCl to a continuous culture was the rapid but transient uptake of Na⁺ ions. At steady state a higher osmotic pressure resulted in a strong increase of the intracellular concentrations of proline, from 5 mg/g to 125 mg/g dry weight, and of trehalose from 20 mg/g to 60 mg/g dry weight. The level of glutamate, which was the dominant intracellular amino acid at low osmotic pressure at 55 mg/g dry weight, was not affected by the addition of NaCl. The influence of the specific growth rate, between 0.1 h^–1 and 0.4 h^–1, on the intracellular metabolite concentration was also determined. The level of proline was found to increase strongly with the growth rate, whereas the trehalose content decreased slightly and the glutamate content did not change. The observed net increase in accumulated metabolites may be related to a requirement of a higher turgor pressure for rapid cell growth.     

Cat Heart Muscle in Vitro : I. Cell volumes and intracellular concentrations in papillary muscle

Methods have been developed for the simultaneous determination of total water, inulin space, and K and Na content in muscles of 0.5 to 10 mg. wet weight. These methods have been used to define steady state conditions with respect to intracellular K concentration in papillary muscles from cat hearts perfused and contracting isometrically at 27–28°C. and at 37–38°C. Cell volumes and intracellular ionic concentrations have been followed as a function of the external K concentration and compared with values predicted on the basis of electroneutrality and osmotic equilibrium.     

Sodium and proline accumulation in Corynebacterium glutamicum as a response to an osmotic saline upshock

In order to understand the role of the medium osmolality on the metabolism of glumate-producing Corynebacterium glutamicum, effects of saline osmotic upshocks from 0.4 osnol. kg^–1 to 2 osmol. kg^–1 have been investigated on the growth kinetics and the intracellular content of the bacteria. Addition of a high concentration of NaCl after a few hours of batch culture results in a temporary interruption of the cellular growth. Cell growth resumes after about 1 h but at a specific rate that decreases with increasing medium osmolality. Investigation of the intracellular content showed, during the first 30 min following the shock, a rapid but transient influx of sodium ions. This was followed by a strong accumulation of proline, which rose from 5 to 110 mg/g dry weight at the end of the growth phase. A slight accumulation of intracellular glutamate from 60 to 75 mg/g dry weight was also observed. Accordingly, for Corynebacterium glutamicum an increased osmolality in the glutamate and proline synthesis during the growth phase.     

Temperature Dependence of Vasopressin Action on the Toad Bladder

Toad bladders were challenged with vasopressin at one temperature, fixed on the mucosa with 1% glutaraldehyde, and then subjected to an osmotic gradient at another temperature. Thus, the temperature dependence of vasopressin action on membrane permeability was distinguished from the temperature dependence of osmotic water flux. As the temperature was raised from 20° to 38°C, there was a substantial increase in the velocity of vasopressin action, but osmotic flux was hardly affected. In this range of temperature the apparent energy of activation for net water movement across the bladder amounted to only 1.2 kcal/mole, a value well below the activation energy for bulk water viscosity. It is suggested that osmotic water flux takes place through narrow, nonpolar channels in the membrane. When the temperature was raised from 4° to 20°C, both vasopressin action as well as osmotic water flux were markedly enhanced. Activation energies for net water movement were now 8.5 kcal/mole (4°–9°C) and 4.1 kcal/mole (9°–20°C), indicating that the components of the aqueous channel undergo conformational changes as the temperature is lowered from 20°C. At 43°C bladder reactivity to vasopressin was lost, and irreversible changes in selective permeability were observed. The apparent energy of activation for net water movement across the denatured membrane was 6.6 kcal/mole. Approximately 1 µosmol of NaCl was exchanged for 1 µl of H₂O across the denatured membrane.     

Water movement from intracristal spaces in isolated liver mitochondria

When analyzing mitochondria isolated in a sucrose medium that had been embedded for thin sectioning according to one low denaturation embedding technique, large intracristal spaces were present in close to 90% of the mitochondria. The two crista membranes were closely apposed in only 40% of all cristae. When the mitochondria were transferred to an incubation medium, the percentage of mitochondria with intracristal spaces was reduced to 40%. About 90% of all cristae were lacking any space separating the two crista membranes. The presence of inorganic phosphate in the medium was required for the closing of the intracristal spaces. The percentage of cristae lacking an intracristal space remained the same after addition of substrate for respiration (state 4) and of ADP (state 3). Inhibition or uncoupling of respiration led to an increase in the percentage of intracristal spaces, showing that oxidative phosphorylation is required to maintain the crista membranes closely apposed. The appearance and disappearance of the intracristal spaces was an indication of water movements across the crista membranes. The mean volume of the mitochondria increased 33% when they were transferred from the sucrose medium to the incubation medium, showing that the removal of water from the cristae was not caused by a passive osmotic effect. Addition of substrate made the volume decrease by 28%. After further addition of ADP, the volume decreased another 23%. No change in volume was associated with inhibition or uncoupling of respiration. The observations revealed that water can move into or out of the cristae independently of water movement out from the entire mitochondrion. Therefore, the water moving out from or into the cristae is translocated across the cristae membrane. The observations are interpreted to reveal the presence of a mechanism that actively prevents water from accumulating in the crista membrane. This mechanism allows for a low water activity to be maintained within the membrane. The variations in the frequency of intracristal spaces occurred without any simultaneous changes in the width of the space appearing between the two surface membranes after isolation of the mitochondria. The observations, therefore, do not agree with the concept that there is an outer compartment that communicates freely with intracristal spaces.     

Effect of Sodium-Glucose Cotransport Inhibition on Polycystic Kidney Disease Progression in PCK Rats

The sodium-glucose-cotransporter-2 (SGLT2) inhibitor dapagliflozin (DAPA) induces glucosuria and osmotic diuresis via inhibition of renal glucose reabsorption. Since increased diuresis retards the progression of polycystic kidney disease (PKD), we investigated the effect of DAPA in the PCK rat model of PKD. DAPA (10 mg/kg/d) or vehicle was administered by gavage to 6 week old male PCK rats (n=9 per group). Renal function, albuminuria, kidney weight and cyst volume were assessed after 6 weeks of treatment. Treatment with DAPA markedly increased glucose excretion (23.6 ± 4.3 vs 0.3 ± 0.1 mmol/d) and urine output (57.3 ± 6.8 vs 19.3 ± 0.8 ml/d). DAPA-treated PCK rats had higher clearances for creatinine (3.1 ± 0.1 vs 2.6 ± 0.2 ml/min) and BUN (1.7 ± 0.1 vs 1.2 ± 0.1 ml/min) after 3 weeks, and developed a 4-fold increase in albuminuria. Ultrasound imaging and histological analysis revealed a higher cyst volume and a 23% higher total kidney weight after 6 weeks of DAPA treatment. At week 6 the renal cAMP content was similar between DAPA and vehicle, and staining for Ki67 did not reveal an increase in cell proliferation. In conclusion, the inhibition of glucose reabsorption with the SGLT2-specific inhibitor DAPA caused osmotic diuresis, hyperfiltration, albuminuria and an increase in cyst volume in PCK rats. The mechanisms which link glucosuria to hyperfiltration, albuminuria and enhanced cyst volume in PCK rats remain to be elucidated.     

Metabolism of propionate by sheep liver. Stimulation of the mitochondrial rate by factors from the cell sap

1. The rate of metabolism of propionate by aged sheep-liver mitochondria in the presence of oxygen + carbon dioxide (95:5) was 5·0 (± s.e.m. 0·8) μmoles/mg. of mitochondrial N/hr. 2. When aged in the presence of the mitochondrial supernatant the rate was increased. Mitochondria from 0·33g. of liver, when combined with the corresponding mitochondrial supernatant from 0·08g. of liver, metabolized propionate at a rate of 11·4 (± s.e.m. 1·2) μmoles/mg. of mitochondrial N/hr. This rate is comparable with rates previously obtained with aged nuclear-free homogenates. 3. Two factors in the mitochondrial supernatant were detected, which when combined reproduced the effect of the fresh supernatant and prevented loss of activity on aging. One of these was non-diffusible and was recovered by fractionation of the dialysed mitochondrial supernatant with ammonium sulphate. The second factor was present in an ultrafiltrate of fresh mitochondrial supernatant and in boiled mitochondrial supernatant; it was isolated and identified as l(+)-glutamate. 4. The effect of the non-diffusible factor was due to protection of the mitochondria from the aging process, whereas glutamate served both in this capacity and as a direct stimulant of propionate metabolism at low concentration.     

FORMATION OF MITOCHONDRIA IN NEUROSPORA CRASSA : A Study Based on Mitochondrial Density Changes

The choline concentration used in the growth medium influences the density of mitochondria produced by the chol-1 mutant of Neurospora. Isopycnic centrifugation in sucrose gradients can be used to determine the density of mitochondria, and can resolve into two populations, mitochondria derived from a mixture of cells grown at low (1 µg/ml choline chloride) and high (10 µg/ml choline chloride) choline levels. In an experiment in which cells are shifted from low to high choline growth conditions, mitochondria obtained after varying time periods show a gradual decrease in density tending toward the level typical of high choline mitochondria. Over a 90-minute period of observation, during which time there is an increase of mitochondrial protein mass of ~ 50 per cent over that initially present, the mitochondria change density as a single population. These results are consistent with the view that mitochondria grow by random accretion of new lecithin into existing mitochondrial structures, and also that the mitochondrial population increases by division.     

EXPANSION OF THE INNER MEMBRANE COMPARTMENT AND ITS RELATION TO MITOCHONDRIAL VOLUME AND ION TRANSPORT

Glutaraldehyde has been used to fix mitochondria undergoing rapid volume changes associated with energized ion transport under oscillatory state conditions and valinomycin-induced potassium uptake. Fixation was found to prevent structural changes which normally occur during ion accumulation or loss. By correlating packed volume measurements with electron microscopy, it is shown that changes in volume associated with ion movements reflect changes in the inner membrane compartment and that this compartment can be related to the sucrose inaccessible space. The method can therefore be used to accurately determine volume changes that arise from ion translocation.     

Effect of Low-Osmolality Nutrient Media on Growth and Culturability of Campylobacter Species

The growth and culturability of Campylobacter jejuni NCTC 11351 and other campylobacters were examined in media having different osmolalities at a range of temperatures (4, 25, and 42°C). The medium osmolalities used ranged from the osmolality of full-strength nutrient medium (modified campylobacter broth having an osmolality of around 254 mosmol) down to 96 mosmol. The following two methods were used to produce media having different osmolalities: dilution of the nutrient medium with distilled water and reformulation of the medium such that the concentrations of various osmolytes were altered while the nutrient content of the medium was unchanged. The results obtained with the two experimental methods were similar, indicating that there was an osmotic threshold effect, such that none of the campylobacters examined (C. jejuni NCTC 11351 and ATCC 33291, Campylobacter lari, and Campylobacter coli) grew in media having osmolalities around 130 mosmol and at temperatures below at 42°C. Conversely, growth occurred in media having osmolalities of around 175 mosmol and above. Osmolar concentrations can be expressed in terms of osmolarity or osmolality. Osmolality is easier to evaluate, is the more commonly used term, and was used in the current study. In nutrient media having low osmolalities (i.e., 130 mosmol and below), the number of CFUs per milliliter declined rapidly regardless of the temperature, and no cells were recovered after 24 h. However, at nongrowth temperatures (25 and 4°C) in higher-osmolality media (175 mosmol and above) a significant population was recovered throughout the experiment (up to 96 h). In low-osmolality nutrient media, the cellular morphology was principally coccoid, while in the early stages of growth in full-strength media the morphology was predominantly rodlike. We propose that the formation of coccoid cells in these experiments was the result of osmotic stress in low-osmolality media. This osmotic effect was apparent regardless of the osmolyte used to reformulate the medium (NaCl, KCl, Na₂SO₄, NH₄Cl, and glucose were used).     

ADP Protects Cardiac Mitochondria under Severe Oxidative Stress

ADP is not only a key substrate for ATP generation, but also a potent inhibitor of mitochondrial permeability transition pore (mPTP). In this study, we assessed how oxidative stress affects the potency of ADP as an mPTP inhibitor and whether its reduction of reactive oxygen species (ROS) production might be involved. We determined quantitatively the effects of ADP on mitochondrial Ca²⁺ retention capacity (CRC) until the induction of mPTP in normal and stressed isolated cardiac mitochondria. We used two models of chronic oxidative stress (old and diabetic mice) and two models of acute oxidative stress (ischemia reperfusion (IR) and tert-butyl hydroperoxide (t-BH)). In control mitochondria, the CRC was 344 ± 32 nmol/mg protein. 500 μmol/L ADP increased CRC to 774 ± 65 nmol/mg protein. This effect of ADP seemed to relate to its concentration as 50 μmol/L had a significantly smaller effect. Also, oligomycin, which inhibits the conversion of ADP to ATP by F₀F₁ATPase, significantly increased the effect of 50 μmol/L ADP. Chronic oxidative stress did not affect CRC or the effect of 500 μmol/L ADP. After IR or t-BH exposure, CRC was drastically reduced to 1 ± 0.2 and 32 ± 4 nmol/mg protein, respectively. Surprisingly, ADP increased the CRC to 447 ± 105 and 514 ± 103 nmol/mg protein in IR and t-BH, respectively. Thus, it increased CRC by the same amount as in control. In control mitochondria, ADP decreased both substrate and Ca²⁺-induced increase of ROS. However, in t-BH mitochondria the effect of ADP on ROS was relatively small. We conclude that ADP potently restores CRC capacity in severely stressed mitochondria. This effect is most likely not related to a reduction in ROS production. As the effect of ADP relates to its concentration, increased ADP as occurs in the pathophysiological situation may protect mitochondrial integrity and function.     

The isolation of neurophysin-I and -II from bovine pituitary neurosecretory granules separated on a large scale from other subcellular organelles. Demonstration of slow equilibration of neurosecretory granules during centrifugation in a sucrose density gradient

1. An improved procedure for the isolation of neurosecretory granules from the posterior lobe of the bovine pituitary gland is described. 2. Of the total oxytocic and pressor activities present in the original tissue 80% was sedimentable. 3. The granules were separated from mitochondria by prolonged centrifugation in a sucrose density gradient. During a sedimentation period of 5hr. the granules moved progressively into denser regions of the gradient and the mitochondria remained at the top. 4. The biological activities of the granules were measured: the oxytocic activity was 11·56±1·63 and the pressor activity was 15·60±3·91 units/mg. of protein. 5. A protein was isolated from a lysate of granules prepared from 40 pituitary glands. Amino acid analysis showed that it consisted of a mixture of neurophysin-I and neurophysin-II in equal proportions. It accounted for 60% of the soluble granule protein and for 50% of the total granule protein. 6. The neurophysins present in the granules are associated with 19·1 units of oxytocic and 21·1 units of pressor activity/mg. of protein. 7. Starch-gel electrophoresis revealed the presence of both neurophysins in extracts of 15 pituitary glands studied individually. 8. We conclude that the polypeptide hormones, oxytocin and [8-arginine]-vasopressin, are normally closely associated with the two neurophysins within neurosecretory granules of the pituitary gland.     

A centrifugation study of rat-liver mitochondria, lysosomes and peroxisomes during the perinatal period.

We have investigated the intracellular distribution of several enzymes on homogenates of late foetal, early postnatal and adult rat livers. Homogenates were subjected to differential centrifugations in 0.25 M sucrose and four fractions were isolated which corresponded to the N (nuclear) ML (total mitochondrial) P (microsomal) and S (soluble) fractions of de Duve et al. (1955). In general the age of the animal did not significantly affect the distribution pattern. Reference enzymes of mitochondria, lysosomes and peroxisomes were mainly recovered in the total mitochondrial fraction (ML). Glucose-6-phosphatase and esterase, both located in the endoplasmic reticulum, were chiefly associated with the microsomal fraction P together with galactosyltransferase (a reference enzyme of the Golgi apparatus). 5'-Nucleotidase, (a plasma membrane enzyme) exhibits a bimodal distribution and is mainly recovered in the N and the P fractions. Such results indicate that the membrane composition of the fractions isolated by the fractionation scheme was used, does not appreciably differ for the late foetal, early postnatal and adult rat livers. An analytical fractionation of the mitochondrial (ML) fraction of livers at different stages of development was performed by isopycnic centrifugation in sucrose gradients and in glycogen gradients using sucrose solutions of various concentrations as the solvents. The distribution of mitochondria, lysosomes and peroxisomes were assessed by establishing the distribution of their reference enzymes. Some physical characteristics of the particles were deduced from the manner in which the distributions were influenced by the sucrose concentration of the centrifugation medium. The distribution of liver mitochondrial enzymes one day prenatal differs strikingly from that of enzymes one day postnatal; foetal mitochondria seem characterized by a high osmotic space and a high hydrated matrix density; neonatal mitochondria seem devoid of an osmotic space and the density of their hydrated matrix is markedly lower than that of the foetal mitochondria. As ascertained by the distribution of mitochondrial enzymes in a sucrose 2H2O gradient, the high density of a foetal mitochondria matrix does not mainly originate from a lower amount of hydration water. The behavior of lysosomal enzymes in media with increasing concentrations of sucrose suggests that lysosomes originating from late foetal rat liver are endowed with a very small osmotic space. As for the peroxisomes, our results do not display significant behavior differences in centrifugations that would indicate physicochemical changes of these particles during the perinatal period.