Alaskan malamute chondrodysplasia--VII. Isolation and characterization of copper, zinc and iron binding proteins in Canis familiaris.

1. Three low molecular weight (12,000, 10,000 and 7,000) metal binding proteins have been isolated from the livers of normal and chondrodysplastic Alaskan Malamutes. 2. Comparison studies between kennel (mixed breed) dogs and both adult and immature Alaskan Malamutes suggested that the disturbance in trace mineral metabolism found in the Malamutes is almost entirely reflected in the 12,000 mol. wt species. 3. The major copper-inducible protein (10,000 mol. wt) observed in kennel dogs was not found to be inducible in Malamutes and contained constant ratios of both copper and zinc to protein in metal binding proteins isolated from the livers of both normal and dwarf Malamutes. 4. The copper and zinc found in the UM2 concentrates (mol. wt greater than 2000) of immature Malamutes showed very little affinity to the proteins and these metals were found chiefly in a peptide fraction which apparently serves as a reservoir from which the storage proteins obtain the metals that they bind. 5. Regression analysis indicated a statistically significant correlation between both copper and zinc concentrations and the carbohydrate concentration in the proteins investigated.     

Studies of the copper-binding proteins in Menkes and normal cultured skin fibroblast lysates

Proteins of approximately 10,000 daltons (presumably metallothionein) and greater than 75,000 daltons bound 64Cu when this metal was added to fibroblast lysates. Treatment with either 2-mercaptoethanol or the disodium salt of ethylenediamine tetraacetic acid demonstrated that the high molecular weight copper-binding proteins in lysates prepared from both normal and Menkes fibroblasts exhibited a relatively low affinity for copper compared to the 10,000 dalton protein(s). No difference was detected in the affinity of the low molecular weight protein(s) of normal and Menkes fibroblast lysates for copper. The amount of 64Cu bound to the 10,000 dalton protein(s), however, was approximately two to three times greater in lysates prepared from Menkes fibroblasts than from normal fibroblasts. Mixing experiments indicated that the increased binding of 64Cu to the 10,000 dalton protein(s) in lysates of Menkes fibroblasts did not result from the deficiency of a factor that effects the cleavage of copper from this protein(s), from the presence of a soluble inhibitor, or from the lack of an activator. In addition, the use of lysates, rather than whole cells, demonstrated that the observed differences in copper binding between the normal and the Menkes fibroblasts were not caused by an abnormality in the membrane transport of copper in the mutant cells. Thus the findings suggest that the increased accumulation and the reduced efflux of copper previously observed in cultured Menkes fibroblasts result either from an increased amount of the 10,000 dalton copper-binding protein(s) or from an increased capacity of this molecule(s) for copper.     

H-Pro-[3H]Leu-Gly-NH2: Uptake and Metabolism in Rat Brain

The uptake and metabolism of H-Pro-[3H]Leu-Gly-NH2 ([3H]PLG) in rat brain was investigated by reverse-phase paired-ion high pressure liquid chromatography. Following in vitro incubation of [3H]PLG with rat brain subcellular preparations, the microsomal-cytosol fraction was about twice as active in degrading PLG as the crude mitochondrial-synaptosomal fraction. For both enzyme preparations the pH optimum was found at pH 7-7.5. The major labeled metabolite was [3H]leucine, whereas 3H]labeled Leu-Gly-NH2 as the only labeled peptide intermediate was found in trace amounts. After intravenous injection of [3H]PLG the uptake of unmetabolized peptide in the brain appeared to be very low: 0.008% and 0.001% of the administered dose/g tissue at 2 and 5 min after injection respectively, while at longer survival times intact peptide was below the detection limit. Compared with the intravenous route of administration, intracerebroventricular injection of [3H]PLG yielded much higher brain concentrations of unmetabolized PLG. Following both routes of administration, the metabolite profile was in agreement with that obtained after in vitro incubation. However, the in vivo experiments also showed considerable incorporation of [3H]leucine liberated from [3H]PLG into proteins. Both the in vitro and in vivo results indicate that the initial cleavage of PLG in rat brain occurs at the NH2-terminus and that the dipeptide intermediate H-Leu-Gly-NH2 is subsequently hydrolyzed to its constituent amino acids very rapidly.     

A low molecular weight zinc-copper lipolytic protein from chick liver

A lipolytic zinc-copper protein has been isolated from the cytosol of chick liver. This material had a molecular weight of 6000 daltons, contained four atoms of zinc and one atom of copper per molecule. The 6000 dalton fraction aggregated at high ionic strength or in the presence of sodium dodecyl sulphate. Lipolytic activity was observed towards triolein, tripalmitin, phosphatidyl choline and retinyl palmitate, and was stimulated by cholate, Ca and high NaCl concentrations, and was inhibited by sulphydryl reagents, inhibitors of serine esterases, alkaline phosphatase and chelating agents. It appears that this copper-zinc protein is distinct from metallothionein which has no lipolytic activity.     

Trace element uptake by L-cells as a function of trace elements in a synthetic growth medium

The concentration of trace elements in L-cells has been studied as a function of the trace metal content of the growth medium. Cells were cultured in synthetic media which contained varying trace amounts of the elements manganese, iron, cobalt, copper, zinc and molybdenum. The cellular concentration of the elements potassium, iron, copper and zinc were then determined. It was found that the cell accumulates trace metals at a different rate than they are made available. Deficiencies in zinc could be “induced” in the cell by increasing the concentration of iron, manganese and cobalt; cellular iron deficiencies were observed at larger medium concentrations of zinc, manganese, copper and cobalt. Trace metal uptake by the cell was seen to parallel the utilization by multicellular organisms.     

The binding of [3H]chlorambucil to nuclear proteins of the Yoshida ascites sarcoma.

The binding of [3H]chlorambucil to nuclear proteins, extracted from Yoshida ascites sarcoma cells at 6 h and 24 h after administration of 3H-labelled drug to tumour-bearing animals, has been examined. Both covalent and non-covalent binding was detected. Considerably more drug was found associated with the proteins isolated from the tumour sensitive to the effects of the drug compared with similar proteins isolated from the tumour with an acquired resistance to the effects of alkylating agents. The two-fold difference in binding to total cell protein is attributed to a higher intranuclear protein binding in sensitive cells. In particular the soluble nuclear sap fraction from sensitive cells bound at least five times as much drug as the corresponding fraction from resistant cells. Low levels of binding to histones were demonstrated compared with that to the non-histone chromatin proteins. Binding to the nuclear sap and non-histone chromatin proteins was principally to high molecular weight protein species; these did not appear to represent aggregation products as scans of stained polyacrylamide gels of the extracted protein fractions were unaltered by the treatment of tumour-bearing animals with chlorambucil. Binding to the nuclear proteins from sensitive cells tended to persist over a 24-h period, whereas it was considerably reduced in resistant cells.     

A model system for [NiFe] hydrogenase maturation studies: Purification of an active site-containing hydrogenase large subunit without small subunit

The large subunit HoxC of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha was purified without its small subunit. Two forms of HoxC were identified. Both forms contained iron but only substoichiometric amounts of nickel. One form was a homodimer of HoxC whereas the second also contained the Ni-Fe site maturation proteins HypC and HypB. Despite the presence of the Ni-Fe active site in some of the proteins, both forms, which lack the Fe-S clusters normally present in hydrogenases, cannot activate hydrogen. The incomplete insertion of nickel into the Ni-Fe site provides direct evidence that Fe precedes Ni in the course of metal center assembly.     

Role of the cytosolic factor in the interaction of [3H] alpha-tocopherol with rat liver nuclei

A protein fraction (Mr = 30-70 kD) specifically binding [3H]alpha-tocopherol was isolated from rat liver cytosol. Using high performance ion exchange chromatography, this fraction was separated into acid and alkaline protein subfractions. Acid proteins make up to 41% of the total protein pool and they bind the label 8 times more intensively than the alkaline ones. Cytosol and its protein fraction with an average molecular mass increase 2.2-2.5-fold the binding of labeled vitamin E to isolated liver nuclei. It is concluded that the cytosolic proteins having a medium molecular mass are involved in tocopherol interaction with the nuclei.     

UPTAKE OF [14C]ASPARTIC ACID AND [14C]GLUTAMIC ACID BY RETINAL SYNAPTOSOMAL FRACTIONS

Abstract— Uptake systems for [¹⁴C]aspartate and [¹⁴C]glutamate were characterized in two distinct synaptosomal fractions solated from rabbit retina. The P, synaptosomal fraction was highly enriched in large photoreceptor cell synaptosomes but contained very few conventional sized synaptosomes from amacrine, horizontal or bipolar cells. In contrast, the P₂ synaptosomal fraction contained numerous conventional sized synaptosomes and was virtually free of photoreceptor cell synaptosomes. Both synaptosomal fractions took up [¹⁴C]aspartate and [¹⁴C]glutamate with high affinity [ K _m= 1–2μM). Uptake characteristics were similar to those described for high affinity uptake systems in brain synaptosomes, i.e. saturation kinetics; temperature and Na⁺ dependence. Although the presence of a high affinity uptake system is not a definitive criterion for demonstration of functional neurotransmitter systems, it is an important and necessary prerequisite and can thus be considered as supportive evidence for the involvement of asparate and glutamate in neurotransmission in rabbit retina.     

Fractionation of Soluble Copper- and Zinc-Binding Proteins From Cattle Liver

The distribution of copper and zinc among soluble proteins in liver from normal slaughter cattle was examined after gel filtration of the proteins. Gopper- and zinc-binding proteins were mainly separated into three fractions. Varying amounts of zinc were eluted in a fourth fraction of molecular weight less than 2,000. A clear relationship was noted between the amount of copper bound to the low molecular weight fraction (m.w. ~ 10,000) and the total liver zinc concentration. The high molecular weight protein fraction (m.w. > 65,000) dominated in liver with zinc concentrations below 40 µg/g wet weight and total copper concentrations from 16 to 240 µg/g, while in liver with zinc concentrations above 40 µg/g and copper concentrations ranging from 20 to 107 µg/g, the low molecular weight metallothionein-like fraction dominated.     

Cyclic analogs of bradykinin, containing a carboxyl group]

1 alpha-beta-carboxypropionyl-cyclo(9----1 epsilon)-[Lys1, Gly6]bradykinin (Suc-c[Lys1, Gly6]B), 1 alpha-beta-carboxypropionyl-cyclo(10----1 epsilon)kallidin (Suc-cK), cyclo(10 gamma----1 epsilon)-[Glu10]kallidin (c[Glu10]K) and cyclo(11 gamma----1 epsilon)kallidylglutamic acid (cKG) were synthesized. Suc-c[Lys1, Gly6]B and Suc-cK were prepared by acylating the appropriate cyclopeptides with succinic anhydride. c[Glu10]K and cKG were obtained by the classic peptide synthesis, the cyclization being carried out with 61 and 42% yields, respectively. The protecting groups were then eliminated by catalytic hydrogenation. c[Glu10]K and cKG exerted myotropic action on isolated rat uterus (alpha 0.73 and 0.89, pD2 6.61 and 8.61, respectively). cKG displayed direct myotropic activity with respect to electrically stimulated rat vas deferens and guinea-pig ileum, potentiating the contractions (by 100%) in response to electric stimuli. c[Glu10]K and cKG elicit histamine release in isolated rat mast cells (EC30 4.91.10(-5) and 1.47.10(-6) M, respectively). Both cyclopeptides alter arterial pressure following intravenous administration to anaesthetized rats, cats and dogs and affect heart rate. In all assays cKG is more active than c[Glu10]K. Suc-c[Lys1, Gly6]B and Suc-cK do not possess myotropic, histamine-releasing or hypotensive activity, though they were found to elicit a transient increase of bloodflow in cats and dogs.     

The secret life of extracellular vesicles in metal homeostasis and neurodegeneration

Biologically active metals such as copper, zinc and iron are fundamental for sustaining life in different organisms with the regulation of cellular metal homeostasis tightly controlled through proteins that coordinate metal uptake, efflux and detoxification. Many of the proteins involved in either uptake or efflux of metals are localised and function on the plasma membrane, traffic between intracellular compartments depending upon the cellular metal environment and can undergo recycling via the endosomal pathway. The biogenesis of exosomes also occurs within the endosomal system, with several major neurodegenerative disease proteins shown to be released in association with these vesicles, including the amyloid‐β (Aβ) peptide in Alzheimer's disease and the infectious prion protein involved in Prion diseases. Aβ peptide and the prion protein also bind biologically active metals and are postulated to play important roles in metal homeostasis. In this review, we will discuss the role of extracellular vesicles in Alzheimer's and Prion diseases and explore their potential contribution to metal homeostasis.     

Effect of heavy metals on the uptake of [3H]-L-histidine by the polychaete Nereis succinea

Integumentary uptake of [3H]-L-histidine by Nereis succinea was measured in the presence and absence of selected heavy metals and the amino acid L-leucine in 60% artificial seawater (ASW). The time course of 10 microM [3H]-L-histidine uptake into worms over a 60 min incubation was approximately doubled in the presence of 0.5 microM zinc and when calcium in the incubation medium was reduced from 6 mM to 5 microM the stimulatory effect of zinc on amino acid accumulation was reduced and uptake under the latter conditions was approximately half that of the control. Zinc stimulation of [3H]-L-histidine influx was a hyperbolic function of zinc concentration over the range 0 to 50 microM metal and displayed an apparent activation or affinity constant of 385+/-127 nM Zn(2+). The hyperbolic stimulatory effect of 1 microM Zn(2+) on the time course of 10 microM [3H]-L-histidine uptake was abolished in the presence of 25 microM L-leucine, suggesting that this amino acid shared the same transport system as [3H]-L-histidine and acted as a potential competitive inhibitor. Influx of [3H]-L-histidine was a hyperbolic function of external amino acid concentration and displayed an apparent affinity constant (Km) of 23.71+/-5.02 microM and an apparent aximal velocity (J(max)) of 4701+/-449 pmol/g dry wt.x15 min. Addition of 0.5 microM zinc resulted in a four-fold increase in J(max) and a doubling of K(m), suggesting the effect of the metal was mostly on the rate of amino acid transport. [3H]-L-histidine influx was mildly stimulated by Fe(2+) (0.5 microM), but was unaffected by either Ag(+) or Al(3+) (both at 0.5 microM). These results suggest that [3H]-L-histidine uptake into worm integument may take place by the classical Na(+)-independent L-transport system shared by L-leucine and regulated by exogenous calcium and other divalent metal concentrations.     

Studies on the uptake of [3H]dopamine by synaptic vesicle fraction isolated from bovine brain

1. A synaptic vesicle fraction isolated from bovine caudatolenticular nuclei showed enzymic activities of tyrosine hydroxylase [EC 1.14.16.2] and dopamine beta-hydroxylase [EC 1.14.17.1]. Tyrosine hydroxylase, whose subcellular localization is uncertain, appeared to be associated with the synaptic vesicles. 2. The vesicle fraction took up [3H]dopamine increasingly with time without the aid of ATP (6.3 pmol of [3H]dopamine per mg of vesicle proteins, or 3-4% of the added dopamine, at 30 min). In the presence of ATP, a transient accumulation of the amine was observed, reaching the highest level at 7-10 min (5.6 pmol dopamine/mg protein), and then a rapid release of the amine took place, obeying first-order kinetics with respect to the amine concentration. The amine uptake was strongly inhibited with NEM, regardless of the presence or absence of ATP. 3. The vesicle fraction also exhibited a weak ability to translocate protons inward and ATP-dependently, as monitored by an increase in the fluorescence intensity of ANS. The fluorescence enhancement persisted for at least 30 min and this time-dependent change was not consistent with that of the transient accumulation of [3H]dopamine mentioned above. Since the present vesicle preparation contained a small amount of mitochondrial ATPase (18-20% of the total activity), the proton translocating ability could be attributable to contaminating submitochondrial particles. 4. Therefore these results made it impossible to conclude that the transient uptake of [3H]dopamine by the synaptic vesicles was coupled to ATP hydrolysis.     

Interaction of phenylisothiocyanate with human erythrocyte band 3 protein. II. Topology of phenylisothiocyanate binding sites and influence of p-sulfophenylisothiocyanate on phenylisothiocyanate modification

The two structurally related probes, the apolar phenylisothiocyanate and the polar, water-soluble p-sulfophenylisothiocyanate, were analysed for their topological interaction with human erythrocyte band 3 protein. Upon thermolytic and peptic digestion of labeled erythrocyte ghosts, the membrane-integrated segments of band 3 protein, the 17,000 and 10,000 dalton peptides, were isolated. At 2 mM initial label concentration, 90% of the hydrophobic probe phenylisothiocyanate was recovered in the 10,000 dalton peptide, the remaining amount of label being associated with the 17,000 dalton fragment. Pretreatment of the membranes with 5 mM p-sulfophenylisothiocyanate followed by labeling with 2 mM phenylisothiocyanate results in a consistent reduction in binding of phenylisothiocyanate by 1 mol/mol isolated band 3 protein. p-Sulfophenylisothiocyanate reportedly binds to the 17,000 dalton fragment (Drickamer, K. (1977), J. Biol. Chem. 252, 6909-6917). The interaction of the polar probe with the membrane protein affects binding of phenylisothiocyanate to the 10,000 dalton peptide by the equivalent of 1 mol/mol isolated peptide. The topological interrelation of the membrane-integrated segments is concluded.     

Role of nuclear proteins on [H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

Met5]-enkephalin-like peptides of the adrenal medulla: release by nerve stimulation and functional implications

In adrenal chromaffin cells, various molecular forms of polypeptides cross-reacting with [met5]-enkephalin antisera are stored in granules together with catecholamines and soluble proteins. Splanchnic nerve stimulation increases the release of enkephalin-like peptides from the adrenal gland into the adrenal vein. The release of these peptides appears to be mediated by a cholinergic nicotinic receptor. Fractionation of plasma extracts on Bio-gel P-2 shows the presence of only low molecular weight peptides in the resting condition. The low molecular weight fraction contains mainly [met5]-enkephalin and [leu5]-enkephalin. When the splanchnic nerve is stimulated high and low molecular weight peptides are released and the amount of low molecular weight peptides in plasma is increased. The content of enkephalin-like peptides in adrenal venous plasma was similar in control and reserpinized dogs. Splanchnic nerve stimulation increased the peptide content but not the epinephrine content of plasma in reserpinized dogs. This also caused a fall of arterial blood pressure which could be prevented by pretreatment with naloxone. A decrease in blood pressure was also directly elicited by the injection of [met5]-enkephalin-[arg6-phe7]. The duration of the hypotensive effect of this peptide was prolonged by prior injection of captopril.     

Early changes in the synthesis of proteins with affinity for single-stranded DNA during the onset of transformation in NRK cells.

Early alterations in the synthesis of proteins which bind to single-stranded DNA have been examined following the onset of transformation in NRK cells transformed by a heat-sensitive mutant (ts339) of Rous sarcoma virus. Transformation was initiated by shifting quiescent cultures from nonpermissive to permissive temperatures. Cultures were prelabelled with [3H]leucine for several generations at the non-permissive temperature, and with [35S]methionine at times after shift to the permissive temperature. Cytosol extracts were passed through sequential columns of double-stranded and single-stranded DNA bound to cellulose. Within the first hour of transformation there was an increase in the synthetic rate of proteins binding tightly to single-stranded DNA, but not to double-stranded DNA. More loosely bound protein fractions showed no such early synthetic increase. Electrophoresis of the fraction eluted from single stranded DNA-cellulose with 2 M NaCl demonstrated the presence of a major protein of 93 000 daltons, which comprised more than 0.1% of the cytosol protein. The synthesis of the 93 000 dalton protein increased continuously over the first 4 h interval after the onset of transformation. The synthetic rate of a 35 000 dalton protein, a major DNA-binding polypeptide found in mammalian cells, began to increase after a 1-h lag, following the onset of transformation. The protein fraction containing the 93 000 dalton protein had considerable unwinding activity, depressing the melting temperature of poly(dA-dT) by 39 degrees C. The protein fraction containing the bulk of the 35 000 dalton protein did not have unwinding activity. Transformation-induced DNA synthesis was measured in cells made permeable to deoxyribonucleoside triphosphates at times after shift to the permissive temperature. It was determined that synthesis of DNA began within the first 1--2 h after the onset of transformation. We conclude that the early transformation-associated synthesis of SS93 and perhaps other proteins binding to single-stranded DNA may be related to early transformation-associated changes preparatory to DNA replication and subsequent growth.     

Role of nuclear proteins on [3H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

Effects of temperature oscillations on the distribution of 65Zn on cytoplasmic proteins

1. Oscillating temperatures cause an increase in 65Zn uptake into the digestive gland of M. edulis when compared with the effects of constant temperatures. 2. The increased uptake is associated with a change in the distribution of 65Zn onto cytoplasmic proteins. There is a fall in the percentage of metal on the greater than 70 K dalton fraction and a rise in the 3 K dalton fraction. 3. A possible hypothesis is produced to explain these effects in terms of heat lability of metal-protein ligands.