Isolation and characterization of noradrenalin storage granules of bovine adrenal medulla

A method is described for the preparation of (1) the heavy population of bovine adrenal chromaffin granules ($\text{S}{}_{\mathrm{<mtext>H</mtext>}}\text{(}\text{average sedimentation coefficient}\text{) = 12 400}\text{S}$ in 0.25 M sucrose) essentially free from contamination with mitochondria and other organelles, and (2) a subpopulation of this heavy population which is highly enriched in noradrenalin ($\text{?95\%}$ of the total catecholamine is noradrenalin). The method is based on isopycnic gradient centrifugation using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.5 M sucrose medium.The isolated population of noradrenalin granules appeared highly electron dense in transmission electron microscopy and revealed a rather narrow size distribution. The specific content of amine and adenine nucleotides (with reference to total granule protein) was markedly higher than for the total population of heavy chromaffin granules. The molar ratio of amines to adenine nucleotides was, however, lower in the noradrenalin granules, i.e. 4.8 vs. 11.9.     

Altered DNA sedimentation by the presence of erythrocytes in alkaline sucrose gradient centrifugation

The presence of a relatively small number of red cells was found to affect DNA sedimentation profile of normal lymphocytes and acute leukemia cells, as observed by the alkaline sucrose gradient centrifugation technique coupled with the fluorometric measurement of DNA. Significant alteration was observed at a nucleated cell/erythrocyte ratio of $\text{20}\text{1}$ to $\text{0.2}\text{1}$, resulting in retardation of the $\text{S}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ value and the entire sedimentation profile. This effect seemed to be rather specific to erythrocyte lysate, since corresponding amounts of erythrocyte ghost, IgM, bovine serum albumin, and an increased number of nucleated cells did not influence the profile to an appreciable degree.     

Isolation of a mitochondrial DNA topoisomerase from human leukemia cells

Mitochondria from human acute lymphoblastic leukemia cells contain an ATP-independent DNA topoisomerase which can relax negative and positive supercoils. This enzyme has been purified 200-fold by carboxymethyl-cellulose or double stranded DNA-cellulose chromatography. In contrast to the molecular weights reported for mitochondrial topoisomerases in other systems, the native leukemia enzyme has a molecular weight of 132,000 daltons as determined by gel permeation chromatography in buffer containing 0.4 M KCl. It also exhibits a sedimentation coefficient of 7.1 S when centrifuged through a 10–30% glycerol gradient in this high salt buffer. The enzyme is presumably a type I topoisomerase analogous to those found in rat liver and $\text{Xenopus}$$\text{laevis}$ mitochondria.     

RNA in the degenerating silk gland of Bombyx mori

Nucleic acids in the degenerating posterior silk gland of Bombyx mori were analysed during the period from larval maturation to early pupal stage, by methylated albumin column chromatography and sucrose density gradient centrifugation. During the first half of the period, the amount of RNA decreased rapidly, but no accumulation of degradation products was detected. The ratio $\text{26}\text{S}\text{17}\text{S}$ rRNA decreased slightly. A decrease of sRNA-like polynucleotide (∼4S RNA) was faster than that of rRNA. During the latter half of the period, rRNA continued to decrease, while ∼4S RNA increased in content. This probably resulted from the degradation of rRNA. There was a significant fall in the ratio of $\text{26}\text{S}\text{17}\text{S}$, suggesting that rRNA, at least in part, was degraded by the scheme of 26 S→17 S→∼ 4S. The possibility that a part of rRNA may be released outside the tissue without complete decomposition is discussed.     

The content of water and potassium in fat cells

The distribution spaces at equilibrium for ³H₂O, [¹⁴C]urea and $\text{3-O-[}{}^{14}\text{C]-}\text{methylglucose}$ were measured in white fat cells using centrifugation through silicone oil at 2500 × g; no significant differences were observed. l-[¹⁴C] Glucose added immediately before the centrifugation was used as a marker for the extracellular water space. The calculated intracellular water content of the cells after the centrifugation through oil (e.g. ³H₂O space minus l-[¹⁴C] glucose space) is an unbiased measure of the water content of the cells in suspension as judged by the following criteria: (1) The intracellular distribution space for $\text{3-O-[}{}^{14}\text{C}\text{]}$methylglucose at equilibrium (methylglucose space minus l-glucose space) was not different from that calculated from a methylglucose wash-out curve. (2) The intracellular content of l-[¹⁴C]glucose (half time of efflux about 60 min) in cells preloaded during incubation of the tissue with collagenase was not different in cells recovered by (a) centrifugation through oil at 2500 × g, (b) centrifugation through oil at 600 × g, (c) centrifugation at 600 × g in the absence of oil and (d) filtration on Millipore filters.The intracellular content of water determined on cells from single rats weighing 120–150 g was 2.75 ± 0.55 μl/100 μl fat cells (± S.D., n = 30). The intracellular content of potassium, determined on cells from the same rats, was 252 ± 62 nmols/100 μl fat cells (± S.D., n = 30). The concentration of potassium in the intracellular water was calculated as 104 ± 15 mM (± S.D., n = 30).     

Changes in structural integrity of heart DNA from aging mice

The state of the structural integrity of the DNA from mouse myocardial cells has been investigated by utilizing both CsCl density gradient sedimentation and digestion by S₁ endonuclease from $\text{Aspergillus}$$\text{orzae}$. The DNA from myocardial cells of young mice sedimented in a narrow peak at the expected density of 1.701 g/cm³, while the DNA from the heart cells of senescent mice became broadly distributed in CsCl gradients, banding even more multimodally in alkaline sucrose gradients. This mode of sedimentation indicates that old mouse DNA becomes partially fragmented. When the native DNA of myocardial cells from 6, 20 and 30 month old mice was treated with single-strand specific S₁ endonuclease, it was the DNA from the senescent mice that showed a progressive increase in sensitivity to digestion by the enzyme. The results indicate that the heart DNA of aging mice develops single-stranded gaps in addition to a breakdown into differently sized fragments.     

Binding properties of 23S,25-dihydroxyvitamin D3: an in vivo metabolite of vitamin D3

23$\text{S}$,25-Dihydroxyvitamin D₃ was isolated from the plasma of vitamin D₃-toxic pigs. An ultraviolet absorbance spectrum confirmed its purity. The configuration of the 23-hydroxyl group was determined to be S by comparison of the natural product with synthetic 23$\text{R}$,25- and 23$\text{S}$,25-dihydroxyvitamin D₃ by high-pressure liquid chromatography. The affinity of both 23$\text{S}$,25- and 23$\text{R}$,25-dihydroxyvitamin D₃ for the plasma vitamin D binding protein was similar to vitamin D₃. Thus, with respect to the plasma vitamin D binding protein, 23$\text{S}$,25-dihydroxyvitamin D₃ is the least potent, naturally-occurring, dihydroxylated vitamin D₃ metabolite known.     

Iron content and degree of lipid peroxidation in liver mitochondria isolated from iron-loaded rats

1.The content of non-heme iron and the degree of lipid peroxidation were measured in liver mitochondria isolated from rats injected with either Jectofer (an iron-sorbitol-citric acid complex) or iron-nitrilotriacetate. 2. The sedimentation profiles of the mitochondria from controls and iron-treated rats as revealed by analytical differential centrifugation, indicated single population of mitochondria with $\text{s}{}_{\mathrm{4,B}}$ values of 13200± 560 S and 14200±590 S for controls and iron-loaded animals, respectively. In contrast, the sedimentation profiles of the acid phosphatase activity and the non-heme iron revealed marked polydispersities with at least three populations of particles for both controls and iron-loaded animals. 3. The mitochondria and iron-rich lysosomes were separated by density-gradient centrifugation in an isotonic medium of Percoll and sucrose. With this technique, the amount of non-heme iron in a mitochondrial fraction by differential centrifugation decreased from 69±28 nmol/mg protein to 5.6±1.1 nmol/mg protein and from 19.3±5.6 nmol/mg protein to 3.3±0.6 nmol/mg protein for Jectofer and iron-nitrilotriacetate injected rats, respectively. For control rats the amount of mitochondrial non-heme iron was about 2.7 nmol/mg protein both before and following density gradient centrifugation. The extra amount of non-heme iron still present in the purified mitochondrial fraction from iron-loaded rats, as compared to controls, was further characterized by the reactivity towards bathophenanthroline sulfonate. The results suggest that the extra iron was due to a small amount of either ferritin or hemosiderin still contaminaning the mitochondrial fraction. The amount of mitochondrial heme iron was the same in iron-loaded rats and controls. 4. The degree of lipid peroxidation in the mitochondria was estimated from the amount of malondialdehyde. The thiobarbituric acid method used for the quantitation of malondialdehyde was modified so that it was insensitive to variable amounts of iron present in the samples. No difference in the degree of lipid peroxidation was observed between the mitochondria from iron-loaded rats and controls. 5. In contrast to recent proposals (Hanstein, E.G. et al. (1981) Biochim. Biophys. Acta 678, 293–299), the present study showed that the amounts of non-heme iron and the degrees of lipid peroxidation are the same in mitochondria isolated from iron-loaded and control animals.     

Differences in aminoacylation in vivo and in vitro of lysine isoaccepting tRNAs from virus-transformed cells

When murine sarcoma virus-transformed cells are labeled with [³H]lysine $\text{in}\text{vivo}$ for various periods, 5 of 6 isoaccepting lysine tRNAs separable by RPC-5 chromatography are aminoacylated in 1 hr to the same extent that they are aminoacylated $\text{in}\text{vitro}$. The sixth isoacceptor, tRNA₆^Lys, is not aminoacylated $\text{in}\text{vivo}$ to a measurable extent in 1 hr, although it is present in the tRNA prepared from the cells. All six isoacceptors are aminoacylated with [³H]lysine $\text{in}\text{vivo}$ when the labeling period is 2 or 3 hr. These results further show that $\text{in}\text{vitro}$ correlations of the amount of tRNA₄^Lys with cell division accurately reflect the situation $\text{in}\text{vivo}$. Results of differential centrifugation indicate that tRNA₆^Lys occurs in mitochondria.     

Adenosine 5'-monophosphate-removing system in fructose-1,6-bisphosphatase assay mixture: a new approach

An improved procedure for the isolation of mitochondria in high yields from normal and oxygen-deficient myocardium is described. The heart muscle is digested with Nagarse and homogenized simultaneously using a Polytron tissue homogenizer. Mitochondria are isolated by differential centrifugation, and othe subcellular fractions are carefully rinsed to maximize mitochondrial yields. Yields of 28 to 33 mg of mitochondrial protein/g wet wt of heart were obtained from normal (nonperfused and control perfused) hearts and from oxygen deficient (ischemic and autolyzed) hearts. This represents a recovery of 52 to 61% of the total mitochondrial content of the tissue. These mitochondria are functionally intact, with respiratory control ratios of 5.0 to 7.6 and $\text{ADP}\text{O}$ ratios of 2.34 to 2.66. The lysosomal content of the mitochondrial preparations was not increased by this procedure. This method is especially suitable for the preparation of mitochondria in high yield from a single heart, but can also be used to obtain high yields of mitochondria from larger quantities of myocardial tissue.     

Effect of pH, carbamylation and other hemoglobins on deoxyhemoglobin S aggregation inside intact erythrocytes as detected by proton relaxation rate measurements.

Measurement of the transverse water proton relaxation rate has been used to study the effect of pH, carbamylation, and other hemoglobins on the aggregation of deoxyhemoglobin S inside intact erythrocytes. Upon complete deoxygenation, cyanate-treated ($\text{S}\text{S}$) erythrocytes and erythrocytes heterozygous with respect to hemoglobin S ($\text{A}\text{S}$, $\text{C}\text{S}$, and $\text{S}\text{D}$) have high transverse water proton relaxation rates very similar to the values obtained with homozygous ($\text{S}\text{S}$) erythrocytes. These results suggest extensive intermolecular interactions between deoxyhemoglobin S molecules and a resultant increase in the correlation time for the small fraction of “irrotationally bound” water. When the transverse relaxation rate in deoxygenated ($\text{S}\text{S}$) erythrocytes was measured as a function of pH, the maximum rate was observed between pH 7.0 and 7.5. Upon increasing the pH beyond this range the observed relaxation rate decreases as does the number of sickled cells. Upon decreasing the pH, the observed transverse relaxation rate also decreases but the ratio of values from $\text{deoxy}\text{oxy}$ ($\text{S}\text{S}$) erythrocytes remains in the normal range of 4–6 and the number of sickled cells does not change. Therefore, the deoxyhemoglobin S aggregate inside sickled erythrocytes, as observed by water proton relaxation rates, is not altered by carbamylation or by the presence of nongelling hemoglobins. In addition, the enhancement of the relaxation rates as a function of pH is consistent with the number of sickled forms observed.     

Determination of sedimentation coefficients of subcellular particles of rat liver homogenates. A theoretical and experimental approach

A simple and convenient method is described for the determination of the average sedimentation coefficients ($\overline{S}?values$) of subcellular particles in a homogenous solution by velocity sedimentation in the preparative ultracentrifuge (swinging-bucket rotor). Theoretically the method is based on the distribution of intact organelles between sediment and supernatant and the convergence of their sedimentation at high centrifugal effects. The data have been treated according to the rate equation of parallel sedimentation in horizontal cylindrical tubes which has been converted to a linear function. By measuring activities of marker enzymes in the sediments, the theory has been confirmed experimentally in the determination of $\overline{S}?values$ and the distribution between supernatant and sediment of intact mitochondria, lysosomes and peroxisomes of rat liver homogenates. The effect of particle concentration on the $\overline{S}?values$ of mitochondria has been determined.     

Protein synthesis in mitochondrial and microsomal fractions from rat brain and liver after acute or chronic ethanol administration

Incorporation of C¹⁴ Leucine was determined $\text{in vitro}$ or $\text{in vivo}$ in isolated mitochondria and microsomes of rat brain and liver after acute or chronic ethanol administration $\text{in vivo}$.The protein synthesis in mitochondrial and microsomal preparation was inhibited respectively by chloramphenicol and cycloeximide, specific inhibitors for the two systems tested. The experimental data demonstrate that the $\text{in vitro}$ protein synthesis in both systems, mitochondrial and microsomal, is strongly affected only after chronic treatment which produces significant activation at the mitochondrial and microsomal level in the liver and an inhibition on the same systems of the brain.The data for $\text{in vivo}$ protein synthesis instead show strong inhibition after acute administration, except for brain mitochondria, which are practically unaffected, while after chronic treatment no significant alterations are observed.     

Conformational states of enkephalins in solution.

Chicken erythrocyte chromatin was partially digested with micrococcal nuclease and separated into multimeric subunit fractions by gel permeation chromatography. The fractions were characterized by their Svedberg constant, diffusion coefficient, circular dichroism, and electrophoresis pattern of the extracted DNA. The molecular weight dependence of the sedimentation coefficient was found to be S_20,w = .011 × M^.554. The molecular weight dependence of $\text{rmf}\text{f}{}_{\mathrm{o}}$ is best represented in the Kirkwood theory by either a helical superstructure $\text{or}$ a flexible coil $\text{with}\text{attractive}\text{interactions}$ between nucleosome units. The dimer calculations of $\text{f}\text{f}{}_{\mathrm{o}}$ suggest that the core particles are separated by spacer regions which contribute up to ～20% of the frictional properties of the molecule.     

Cholesterol enrichment of normal mitochondria in vitro: a model system with properties of hepatoma mitochondria.

It is known that rat hepatoma mitochondria contain higher levels of endogenous cholesterol than do mitochondria from normal liver. In the experiments described here, normal liver mitochondria were enriched with cholesterol by a solid-state transfer process $\text{in}\text{vitro}$ and some of their enzymic properties were compared with literature values reported for hepatoma mitochondria. Striking parallels were seen. The data indicate that normal mitochondria, enriched with cholesterol $\text{in}\text{vitro}$, may create an interesting model system for examining some metabolic characteristics of tumor mitochondria.     

Synthesis of the four isomers of 5-hydroxy-PGI1

We wish to report here the syntheses of (5$\text{S}$, 6$\text{R}$)-5-hydroxy-, (5$\text{R}$, 6$\text{R}$)-5-hydroxy-, (5$\text{R}$, 6$\text{S}$)-5-hydroxy-, and (5$\text{S}$, 6$\text{S}$)-5-hydroxy-PGI₁ and their methyl ester derivatives. Treatment of (5$\text{R}$, 6$\text{S}$)-5-epoxy- and (5$\text{S}$, 6$\text{R}$)-5-epoxy-PFG_1α methyl esters with acid washed silica gel afforded (5$\text{R}$, 6$\text{R}$)-5-hydroxy- and (5$\text{S}$, 6$\text{S}$)-5-hydroxy-PGI₁ methyl esters; correspondingly, silica promoted cyclization of (5$\text{S}$, 6$\text{S}$)-epoxy- and (5$\text{S}$, 6$\text{R}$)-5-epoxy- PGF₁ methyl esters yielded (5$\text{S}$, 6$\text{R}$)-5-hydroxy- and (5$\text{R}$, 6$\text{S}$)-5-hydroxy- PGI₁ methyl esters. Alternatively, the 5-hydroxyl group was introduced into the PGI₁ skeleton $\text{via}$ reaction of the 5-mercuric halides with sodium borohydride in the presence of oxygen. Stereochemical assignments were based on their mode of synthesis and ¹H nmr shift differences.     

A mitochondrial defect in brown adipose tissue of the obese (ob/ob) mouse: reduced binding of purine nucleotides and a failure to respond to cold by an increase in binding.

Atractyloside-insensitive binding of purine nucleotides is reduced in brown adipose tissue mitochondria of the obese ($\text{ob}\text{ob}$) mouse. Exposure of the $\text{ob}\text{ob}$ mouse to 4°C does not induce the usual increase in binding. Atractyloside-insensitive binding of purine nucleotides is believed to be a measure of the heat-producing proton conductance pathway in brown adipose tissue mitochondria. It is, therefore, suggested that the impaired thermogenesis of the $\text{ob}\text{ob}$ mouse is due to a defect in this pathway in the mitochondria of the brown adipose tissue, the major thermogenic tissues in rodents. The greater metabolic efficiency which would result from a reduced operation of this pathway might be the basis for the obesity in the $\text{ob}\text{ob}$ mouse.     

Electron paramagnetic resonance studies of membrane proteins in hepatic microsomes

Hepatic microsomal membranes, prepared under various conditions that yield either ‘intact’ or ‘disrupted’ microsomal vesicles, have been labeled via the sulfhydryl groups of intrinsic membrane proteins using nitroxide analogs of $\text{N-}\text{ethylmaleimide}$. Electron paramagnetic resonance spectra revealed the presence of two dominant classes of bound label corresponding to differing degrees of immobilization, the ratio of which were quantitated using a parameter designated the ‘$\text{W/S}$’ ratio. For latent microsomes, the value of this parameter was determined to be $\text{0.65 ± 0.02}$ and was influenced by factors such as label/protein ratio, incubation period, nitroxide structure, temperature and pH. The $\text{W/S}$ ratio was also sensitive to the degree of membrane integrity as revealed by the latency of mannose 6-phosphate activity of glucose-6-phosphohydrolase. In addition, membrane disruption resulted in a corresponding decrease in the order parameter for nitroxide-labeled fatty acids intercalated within the lipid bilayer. The $\text{W/S}$ ratio was observed to be dependent upon the method of microsome preparation yielding values of $\text{1.02 ± 0.02}$ for ‘hypertonically disrupted’ vesicles and $\text{1.28 ± 0.02}$ for ‘mechanically disrupted’ vesicles. Microsomal marker enzymes such as cytochrome $\text{P-450}$ and FAD-containing monooxygenase retained significant levels of functionally following nitroxide incorporation.