A study of copper treatment and tissue copper levels in the murine congenital copper deficiency, mottled.

The injection of copper chloride overcomes the lethality and pigment deficiency in the brindled ($\text{Mo}$^br) mouse mutant but copper levels remain depressed in the liver and brain, and a further accumulation occurs in the kidney. The copper-dependent synthesis of brain noradrenaline returns to normal but the activity of brain cytochrome c oxidase, although increased, remains depressed. Significant changes in tissue copper content of female brindled heterozygotes are reported and in each case, the changes exceed those expected on the basis of X-inactivation. The significance of these results to the development of a satisfactory treatment regime for this disease is discussed.     

The distribution of copper in neonatal mottled mutant mice after exposure to copper and penicillamine

Tissue copper levels of brindled ($\text{Mo}$^br) mice and normal litter-mates after single and repeated dosing with CuCl₂ and/or D-penicillamine are examined, together with a study of the cytosol distribution of copper after CuCl₂ treatment. The results confirm that the mutant mouse kidney is capable of extensive copper accumulation in association with the low MW copper-binding protein. Deficient tissues such as brain, heart and spleen are able to sequester sufficient of the exogenous copper to raise their levels to the normal control level, whereas mutant liver levels, even after copper treatment, remain below normal, indicating that the $\text{Mo}$ gene affects the ability of the liver to retain copper.     

The fraction of phosphatidylinositol that activates the (Na+ + K+)-ATPase in rabbit kidney microsomes is closely associated with the enzyme protein

1. Extensive treatment of rabbit kidney microsomes with phosphatidylinositol-specific phospholipase C under various conditions never resulted in more than 75% hydrolysis of the substrate. 2. The non-degraded fraction of the phosphatidylinositol (10–12 nmol per mg microsomal protein) could be recovered only by an acidic extraction procedure. 3. The $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-ATPase}$ activity found in those membranes was not affected by this treatment. 4. Complete degradation of phosphatidylinositol could be easily achieved when the phospholipase was applied to rat liver microsomes which do not contain any detectable $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-ATPase}$ activity. 5. It is concluded that in rabbit kidney microsomes a close association exist between the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-ATPase}$ and that fraction of the phosphatidylinositol that is directly involved in the maintenance of its activity.     

Met-tRNAfMet binding in murine dystrophy

Dystrophic mice of the C57B1 $\text{dy}{}^{\mathrm{2J}}\text{dy}{}^{\mathrm{2J}}$ strain and of the ReJ 129 $\text{dy}\text{dy}$ strain and littermate controls were used to prepare met-tRNA^fMet binding factors. The tissues were homogenized and fractions were obtained which contained ribosomes. The binding factors were assayed by the binding of [³⁵S]methionyl-tRNA to control liver ribosomes. The binding, i.e. eukaryotic initiation factor 2 (elF 2) activity, was measured in brain, liver and muscle and in all of these tissues there was a significant decrease in the dystrophic mice. This decrease in initiation factor activity of hindleg muscle resembled, in the direction of the effect, the decrease in elongation factor activity of hindleg muscle resembled, in the of $\text{dy}\text{dy}$ mice previously reported by our laboratory. Thus these two defects, taken together may help to explain the marked wasting of the muscles. The decrease in brain in both strains provides evidence for nervous tissue involvement in genetic dystrophy.     

The loss of the phoS periplasmic protein leads to a change in the specificity of a constitutive inorganic phosphate transport system in Escherichia coli

The phoS periplasmic protein, implicated in alkaline phosphatase regulation, is shown to be involved in inorganic phosphate (Pi) transport in $\text{E. coli}$. Although $\text{pho}\text{S}{}^{\mathrm{?}}$ cells dependent upon the PST system for Pi transport can grow in minimal medium with 1 mM Pi as source of phosphorus, the affinity of these cells for Pi is greatly reduced; Km = 18 μM compared with Km = 0.4 μM for $\text{phoS}{}^{\mathrm{+}}$ cells. $\text{pho}\text{S}{}^{\mathrm{?}}$ cells dependent upon the PST Pi transport system acquire the ability to accumulate Asi from the medium in contrast to $\text{pho}\text{S}{}^{\mathrm{+}}$ cells which exclude this toxic anion. It would appear that the periplasmic phoS protein is not essential for Pi accumulation but is involved in maintaining the specificity of the PST Pi transport system.     

Possible role of sulphatide in the K+-activated phosphatase activity

A microsomal fraction rich in $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ has been isolated from the outer medulla of pig kidney. $\text{(}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{activated}$ ouabain-sensitive phosphatase activity was studied in this preparation treated with arylsulphatase, an enzyme that specifically hydrolyzes ceramide galactose-3-sulphate. The activity of phosphatase was inactivated in proportion to the amount of sulphatide hydrolyzed. A maximum inactivation of ouabain-sensitive activity was obtained with 60% of the sulphatide content hydrolyzed. The inactivation caused by arylsulphatase was partially reversed by the sole addition of sulphatide. The evidence offered in this paper about sulphatide function in the sodium pump mechanism supports the idea that sulphatides are involved in the K⁺-activated phosphatase, a partial reaction of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$.     

Bicarbonate ion-ATPase in rat liver cell fractions

The distribution of $\text{HCO}{}_3{}^{\mathrm{?}}\text{-}\text{ATPase}$ activity was studied in cell fractions prepared from homogenates of rat liver. The level of mitochondrial contamination in the microsomal fraction depended on the fractionation procedure and on the method of homogenization. With proper care, microsomes with undetectable mitochondrial contamination could be prepared. These microsomes had no detectable $\text{HCO}{}_3{}^{\mathrm{?}}\text{-ATPase}$ activity. Approximately 85 % of the total $\text{HCO}{}_3{}^{\mathrm{?}}\text{-ATPase}$ activity of the post 6000 x g · min supernatant was recovered in the mitochondrialfraction. The properties of this mitochondrial $\text{HCO}{}_3{}^{\mathrm{?}}\text{-}\text{ATPase}$ were not distinguishable from those of the various microsomal $\text{HCO}{}_3{}^{\mathrm{?}}\text{-}\text{ATPase}$ previously described by other investigators.     

Content of adenosine 3'-5'-cyclic monophosphate in the pancreatic islets of mice with a hereditary defect of insulin secretion

Glucose (20 mM) released insulin from pancreatic islets of $\text{C57BL}\text{6J}\text{-}\text{db}{}^{\mathrm{2J}}\text{db}{}^{\mathrm{2J}}$ mice, the response being potentiated by 1 mM 3-isobutyl-1-methylxanthine. Islets of $\text{C57BL}\text{KsJ}\text{-}\text{db}\text{db}$ mice failed to respond to glucose and released only little insulin when challenged with both glucose and methylxanthine. After incubation with 0 or 20 mM glucose alone the islet content of adenosine 3′:5′-cyclic monophosphate did not differ between the two types of mice or between glucose concentrations. 3-Isobutyl-1-methylxanthine increased the islet adenosine 3′:5′-cyclic monophosphate markedly in $\text{6J-}\text{db}{}^{\mathrm{2J}}\text{db}{}^{\mathrm{2J}}$ mice but not significantly in $\text{KsJ-}\text{db}\text{db}$ mice.     

Immunoreactivity of subunits of the (Na+ + K+)-ATPase Cross-reactivity of the α,α+ and β forms in different organs and species

The immunologic cross-reactivity of the α and α+ forms of the large subunit and the β subunit of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from brain and kidney preparations was examined using rabbit antiserum prepared against the purified holo lamb kidney enzyme. As previously reported by Sweadner ((1979) J. Biol. Chem. 254, 6060–6067) phosphorylation of the large subunit of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in the presence of Na⁺, Mg²⁺, and [$\text{γ-}{}^{32}\text{P}\text{]}\text{ATP}$ revealed that dog and, very likely, rat brain contain two forms of the large subunit (designated α and α+) while dog, rat, and lamb kidney contain only one form (α). The cross-reactivity of the α and α+ forms in these preparations was investigated by resolving the subunits by SDS-polyacrylamide gel electrophoresis. The separated polypeptides were transferred to unmodified nitrocellulose paper, and reacted with rabbit anti-lamb kidney serum, followed by detection of the antigen-antibody complex with ¹²⁵I-labeled protein A and autoradiography. By this method, the α and α+ forms of rat and dog brain, as well as the α form found in kidney, were shown to cross-react. In addition, membranes from human cerebral cortex were shown to contain two immunoreactive bands corresponding to the α and α+ forms of dog brain. In contrast, the brain of the insect Manduca sexta contains only one immunoreactive polypeptide with a molecular weight intermediate to the α and α+ forms of dog brain. The β subunit from lamb, dog and rat kidney and from dog and rat brain cross-reacts with anti-lamb kidney ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ serum. The mobility of the β subunit from dog and rat brain on SDS-polyacrylamide electrophoresis gels is greater than the mobility of the β subunit from lamb, rat or dog kidney.     

On the effect of rfe mutation on the biosynthesis of the 08 and 09 antigens of E. coli.

From phosphomannose isomerase-less mutants of $\text{E. coli}$ strains 08 and 09, $\text{rfe}{}^{\mathrm{?}}$ derivatives were constructed by recombination with a $\text{Salmonella rfe}{}^{\mathrm{?}}$ donor. In contrast to membranes from the parent $\text{E. coli}$ strains, those from the $\text{rfe}{}^{\mathrm{?}}$ recombinants did not synthesize the 08 or 09 mannan from GDP mannose $\text{in vitro}$. They could, however, be restored to biosynthetic activity with butanol extracts from the $\text{E. coli rfe}{}^{\mathrm{+}}$ bacteria. This indicated that the $\text{rfe}$ mutation affects the synthesis of a hydrophobic acceptor.     

Defective PAPS-synthesis in epiphyseal cartilage from brachymorphic mice

Activity levels of sulfotransferases, requisite for the sulfation of chondroitin sulfate proteoglycan, were measured in cell-free homogenates prepared from neonatal epiphyseal cartilage of normal C57B1/6J or homozygous brachymorphic mice. In the presence of [³⁵S]-PAPS only or [³⁵S]-PAPS plus an exogenous sulfate acceptor, comparable amounts of ${}^{35}\text{SO}{}_4{}^{\mathrm{2?}}$ were incorporated into chondroitin sulfate by the normal and mutant types of cartilage. In contrast, the mutant cartilage catalyzed the conversion of only 30% of the ${}^{35}\text{SO}{}_4{}^{\mathrm{2?}}$ into chondroitin sulfate as compared to normal mouse cartilage when synthesis was initiated from ATP and H₂³⁵SO₄. These results suggest that the production of an undersulfated proteoglycan which has previously been reported in brachymorphic mice (Orkin, R.W. $\text{et}\text{al}$. (1976) Devel. Biol. $\text{50}$, 82–94) may result from a defect in the synthesis of the sulfate donor PAPS.     

Reciprocal changes in Ca2+/protein activator-dependent and-independent cyclic AMP phosphodiesterase during the development of chick embryos.

The changes in hepatic $\text{Ca}{}^{\mathrm{2+}}\text{protein}$ activator-dependent and independent cAMP phosphodiesterase activity during the development of chick embryo were investigated. Nearly all of the cAMP phosphodiesterase found in the liver during the early embryonic stage was the $\text{Ca}{}^{\mathrm{2+}}\text{protein}$ activator-dependent enzyme; however, this enzyme progressively diminished and the independent enzyme increased proportionately with embryonic development. In the adult liver, little or no $\text{Ca}{}^{\mathrm{2+}}\text{protein}$ activator-dependent enzyme activity could be observed. These results indicate that the concentration of intracellular cAMP may be regulated by these two enzymes in compliance with the development of the chick.     

Purification from brain of an intrinsic membrane protein fraction enriched in (Na+ + K+)-ATPase

A microsomal fraction from canine brain gray matter has been extracted with the detergent sodium dodecyl sulfate to partially purify the membrane bound $\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-stimulated}$ adenosine triphosphatase. Phospholipid, glycolipid, and a family of other glycoproteins are also enriched by the procedure; it is proposed that the product is an intrinsic membrane protein fraction. 6–8-fold purification of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ is obtained without solubilizing the enzyme and without irreversibly altering its turnover number. Final specific activities are 350–400 μmol of ATP hydrolyzed/h per mg protein. The stimulation and reversible inactivation of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ by dodecyl sulfate were examined for information relevant to the mechanism of action of the detergent.     

Photoconversion and regeneration of active protochlorophyll(ide) in mutants defective in the regulation of chlorophyll synthesis

Seedlings carrying mutations in regulatory genes for protochlorophyll(ide) synthesis accumulate protochlorophyll(ide) in darkness in amounts exceeding the wildtype level. Thus, +/tig-d¹² and $\text{tig-b}{}^{24}\text{tig-b}{}^{24}$accumulate 2-fold, $\text{tig-o}{}^{34}\text{tig-o}{}^{34}$ 5- to 6-fold, and $\text{tig-d}{}^{12}\text{tig-d}{}^{12}$ 15-fold more protochlorophyll(ide) than the wild type.The amount of photoconvertible protochlorophyll(ide) accumulated in darkness is the same in all genotypes, despite the large differences in total protochlorophyll(ide) content, indicating a constant number of photoconversion sites.When briefly illuminated leaves are returned to darkness, regeneration of active protochlorophyll(ide) from the pool of inactive protochlorophyll(ide) takes place in wild-type and mutant leaves. Compared to the wild type, the rate of protochlorophyll(ide) activation during 4- and 10-min dark periods is higher in +/tig-d¹², $\text{tig-b}{}^{24}\text{tig-b}{}^{24}$, and $\text{tig-o}{}^{34}\text{tig-o}{}^{34}$, but lower in $\text{tig-d}{}^{12}\text{tig-d}{}^{12}$.There was no indication that the accumulation of protochlorophyll(ide) influences the conversion sites of the protochlorophyll(ide) holochrome, as the kinetics of photoconversion of initially active protochlorophyll(ide) in leaves with the genotypes +/+, +/tig-o³⁴, and $\text{tig-o}{}^{34}\text{tig-o}{}^{34}$ are similar or identical.     

Comparison of red cell and kidney (Na+ + K+)-ATPase at 0°C

Human red cell and guinea pig kidney $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ were phosphorylated at 0°C. Using concentrations of ATP ranging from 10^?6 to 10^?8 M, ATP-dependent regulation of reactivity is observed with red cell but not kidney $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ at 0°C. In particular, with the red cell enzyme only, the following are observed: (i) the ratio of enzyme-bound ATP (E·ATP, measured by the pulse-chase method of Post, R.L., Kume, S., Tobin, T., Orcutt, B. and Sen, A.K. (1969) J. Gen. Physiol. 54, 306s-326s) to steady-state level of total phosphoenzyme (EP) decreases with decrease in ATP concentration and (ii) the apparent turnover of phosphoenzyme (ratio of Na⁺-stimulated ATP hydrolysis to level of total EP at steady state) also varies as a function of ATP concentration. In addition, when EP is formed at very low ATP (0.02 μM), and then EDTA is added, rapid disappearance of a fraction of EP occurs, presumably due to ATP resynthesis, only with the red cell enzyme. These differences in behaviour of the red cell and kidney enzymes are explained on the basis of the observed predominance of K⁺-insensitive EP in red cell, but K⁺-sensitive EP in kidney $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ at 0°C.     

The permeability of the lysosomal membrane to small ions

The permeability of the lysosomal membrane to small anions and cations was studied at 37°C and pH 7.0 in a lysosomal-mitochondrial fraction isolated from the liver of untreated rats. The extent of osmotic lysis following ion influx was used as a measure of ion permeancy. In order to preserve electroneutrality, anion influx was coupled to an influx of K⁺ in the presence of valinomycin, and cation influx was coupled to an efflux of H⁺ using the protonophore $\text{3-tert-}\text{butyl-5,2′-dichloro-4′-nitrosalicilylanilide}$. Lysosomal lysis was monitored by observing the loss of latency of two lysosomal hydrolases.The order of permeability of the lysosomal membrane to anions was found to be $\text{SCN}{}^{\mathrm{?}}\text{> I}{}^{\mathrm{?}}\text{> CH}{}_3\text{COO}{}^{\mathrm{?}}\text{> Cl}{}^{\mathrm{?}}\text{≈ HCO}{}^{\mathrm{?}}{}_3\text{≈ P}{}_{\mathrm{i}}\text{> SO}{}_4{}^{\mathrm{2?}}$ and that to cations $\text{Cs}{}^{\mathrm{+}}\text{> K}{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{> H}{}^{\mathrm{+}}$. These orders are largely in agreement with the lyotropic series of anions and cations.The implications of these findings for the mechanism by means of which a low intralysosomal pH is produced and maintained are discussed.     

Metabolite concentrations in the liver of the adult and developing guinea pig and the control of glycolysis in vivo.

A range of metabolite concentrations have been determined in the liver of the adult and fetal guinea pig during the latter half of gestation. Adenine nucleotides showed little change during development of the fetal liver and the only major difference from the adult was a low ADP concentration. The hexose phosphates, particularly fructose 1,6-diphosphate, were higher and the triose phosphates in the glycolytic pathway after glyceraldehyde 3-phosphate were lower in the fetal liver. Cytosolic $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratios were comparable in both adult and fetal livers as were cytosolic $\text{NADP}{}^{\mathrm{+}}\text{NADPH}$ ratios for the last 15–20 days of gestation. The metabolite concentrations have been used to indicate that glycolysis in the fetal guinea pig liver is controlled largely by hexokinase, glyceraldehyde 3-phosphate dehydrogenase, and pyruvate kinase.