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.     

Distribution of metals during digestion by cutthroat trout fed benthic invertebrates contaminated in the Clark Fork River, Montana and the Coeur d'Alene River, Idaho, U.S.A., and fed artificially contaminated Artemia

The concentrations of essential amino acids in three, undigested invertebrate diets collected from the Clark Fork River (CFR) for cutthroat trout were similar to each other, but were c. 25–75% less than Artemia that were exposed to a mixture of arsenic, copper, cadmium, lead and zinc in the laboratory. The Artemia diet appeared less palatable and the texture, quantity and appearance of the intestinal contents differed between fish fed the Artemia and CFR diets. The Pb% in the fluid fraction of the intestinal contents was greater for the Artemia (29%) than for the CFR diets (10–17%), and the Cu% in the amino acid plus metal fraction of the intestinal contents was greater for the Artemia (78%) than for two of the three CFR diets (67% and 70%). Intestinal contents of fish fed invertebrate diets collected from various sites on the Coeur d'Alene River (CDA), Idaho, were similar in texture, quantity, and appearance. For fish fed the CDA diets, differences in the distribution of metals among fractions of the digestive fluids appeared to be related to concentrations of metals in the invertebrate diets. Pb% was lowest of all metals in the fluid portion of the intestinal contents. However, >80% of all metals in the hind gut were associated with the particulate fraction where they may still be available for uptake through pinocytosis.     

An in vitro study of interactions between insulin-mimetic zinc(II) complexes and selected plasma components

The speciations of some potent insulin-mimetic zinc(II) complexes of bidentate ligands: maltol and 1,2-dimethyl-3-hydroxypyridinone with (O,O) and picolinic acid with (N,O) coordination modes, were studied via solution equilibrium investigations of the ternary complex formation in the presence of small relevant bioligands of the blood serum such as cysteine, histidine and citric acid. Results show that formation of the ternary complexes, especially with cysteine, is favoured at physiological pH range in almost all systems studied. Besides these low molecular mass binders, serum proteins among others albumin and transferrin can bind zinc(II) or its complexes. Accordingly, the distribution of zinc(II) between the small and high molecular mass fractions of the serum was also studied by ultrafiltration. Modelling calculations relating to the distribution of zinc(II), using the stability constants of the ternary complexes studied and those of the serum proteins reported in the literature, confirmed the ultrafiltration results, namely, the primary role of albumin in zinc(II) binding among the low and high molecular mass components of the serum.     

Changes in zinc metabolism following exercise in human subjects

Changes in zinc (Zn) availability in muscle tissue that influence muscle performance in vitro have been observed. The effect of exercise of plasma Zn levels and urinary excretion of Zn was observed in sever untrained volunteers following brief intensive exercise and in seven trained volunteers after more prolonged road-running exercise. With brief exercise, plasma Zn decreased predominantly in the more loosely bound albumin fraction. Prolonged exercise resulted in a greater plasma Zn decrease of 30%. Urinary Zn excretion increased transiently with minimal effect on daily losses. However, weight loss by sweating was significant, and sweat Zn losses were greater than those in the urine. Exercise resulted in changes in Zn metabolism that may influence performance.     

Activation of fibroblast procollagenase by mast cell proteases.

Proteases capable of activating procollagenase from gingiva and from fibroblast and macrophage monolayer cultures were harvested from homogenates of canine tumor mast cells. The mast cell proteases lysed casein and Azocoll but not native collagen. In low salt concentrations the enzymes existed at high molecular weight complexes, which were dissociated by increasing the salt concentration above 1.0 M (NaCl, KCl). Gel filtration in 1.4 M KCl separated the protease activity into three peaks, all of which activated procollagenase. Two of the enzymes showed substrate specificities (hydrolysis of p-tosyl-L-arginine methyl ester and benzoyl-tyrosine ethyl ester) and reactive center reactivities similar to pancreatic trypsin and chymotrypsin. Based on gel filtration, apparent molecular weights of 160 000 (p-tosyl-L-arginine methyl ester esterase), 90 000 (main procollagenase activator) and 36 000 benzoyl-tyrosine ethyl ester esterase) were determined. Activation of procollagenase resulted in a 18-20 000 decrease of the molecular weight. The activation was directly related to the amount of activator added within certain limits. Further addition of activator resulted in proteolytic inactivation of collagenase.     

Effect of dietary garlic and onion on biliary proteins and lipid peroxidation which influence cholesterol nucleation in bile

Formation of cholesterol gallstones in gallbladder is controlled by procrystallizing and anticrystallizing factors present in bile. Dietary garlic and onion have been recently observed to possess anti-lithogenic potential in experimental mice. In this investigation, the role of biliary proteins from rats fed lithogenic diet or garlic/onion-containing diet in the formation of cholesterol gallstones in model bile was studied. Cholesterol nucleation time of the bile from lithogenic diet group was prolonged when mixed with bile from garlic or onion groups. High molecular weight proteins of bile from garlic and onion groups delayed cholesterol crystal growth in model bile. Low molecular weight (LMW) proteins from the bile of lithogenic diet group promoted cholesterol crystal growth in model bile, while LMW protein fraction isolated from the bile of garlic and onion groups delayed the same. Biliary LMW protein fraction was subjected to affinity chromatography using Con-A and the lectin-bound and unbound fractions were studied for their influence on cholesterol nucleation time in model bile. Major portion of biliary LMW proteins in lithogenic diet group was bound to Con-A, and this protein fraction promoted cholesterol nucleation time and increased cholesterol crystal growth rate, whereas Con-A unbound fraction delayed the onset of cholesterol crystallization. Biliary protein from garlic/onion group delayed the crystallization and interfered with pronucleating activity of Con-A bound protein fraction. These data suggest that apart from the beneficial modulation of biliary cholesterol saturation index, these Allium spices also influence cholesterol nucleating and antinucleating protein factors that contribute to their anti-lithogenic potential.     

Large doses of zinc oxide increases the activity of hydrolases in rats

The effect of pharmacological doses of zinc oxide (1000; 2500; 5000 mg per kg diet) and two levels of dietary protein on pancreatic and intestinal hydrolase activity in rats were studied. It was hypothesized that ZnO would increase intestinal and pancreatic hydrolase enzyme activity. Male Wistar rats, averaging 64 g body weight, were randomly allocated to dietary treatments (chow diets- meeting all NRC requirements) containing 10% or 15% protein supplemented with additional ZnO (above 100 mg/kg ZnSO(4)) as follows: 0.0; 0.1; 0.25; 0.5% w/w. Water and food were provided ad libitum. Animals were fed the diets for 10 days and body weights were recorded; after decapitation blood and organ samples were collected. Amylase, lipase, trypsin, and total protease activity of pancreatic homogenates and small intestinal contents were determined. ZnO supplementation dose dependently increased the plasma Zn concentration and significantly increased amylase, lipase, trypsin and total protease activity in pancreatic homogenates and small intestinal contents. The statistical analysis showed significant protein and ZnO interaction on the activity of amylase in the pancreas, and amylase, trypsin and total-protease in the small intestinal content. Therefore ZnO at high dietary concentration may influence the digestion of nutrients via increased hydrolase activity.     

Purification and characterization of a low molecular weight zinc binding protein from human placenta

A low molecular weight, native zinc binding, cytosolic protein (LMZP) has been isolated, purified and characterized from human normal term placenta. Gel filtration of heat treated placental cytosol after sequential acetone precipitation (80% ppt) revealed a major zinc binding protein in the range of low molecular weight. This partially purified zinc binding fraction was further fractionated on DEAE-Sephadex A-25. The zinc was eluted in one of the three peak fractions. Further, the purity of zinc binding protein was confirmed on fast protein liquid chromatography (FPLC). The purified placental LMZP was homogenous on SDS-polyacrylamide gel electrophoresis with a single band. Ultraviolet (UV) spectrum of LMZP showed an absorption maximum at 257 nm which disappeared at pH 2. Molecular weight of LMZP as determined by gel chromatography, SDS-polyacrylamide gel electrophoresis and amino acid analysis was 6 kDa. It was calculated that 1 g atom of zinc was bound to 1 mole of the LMZP. Unlike in classical metallothionein, the amino acid composition of placental LMZP revealed the presence of aromatic amino acids, lower content of cysteine and higher content of histidine, glutamic acid and aspartic acid (10, 9 and 5 residues/mole, respectively).     

Human fetal endothelial cells acquire Zinc(II) from both the protein bound and nonprotein bound pools in serum

To help determine physiologically important routes by which zinc (Zn) is acquired by human fetal vascular endothelium, the authors incubated cultured umbilical vein endothelial cells with⁶⁵Zn(II)-tracer labeled human fetal whole serum, ultrafiltrate (containing low molecular mass serum zinc complexes), and dialyzed serum (containing protein-bound zinc). Zinc from whole serum and from both serum fractions entered a rapidly labeled cellular compartment removable by edetic acid (EDTA), representing Zn bound to the outside cell surface, and accumulatively, an EDTA-resistant compartment’probably largely internalized Zn. Entry of Zn into the EDTA-resistant pool from both serum fractions was strongly temperature-dependent, and was not via the EDTA-sensitive pool. Entry from the ultrafiltrate was resolvable into high affinity saturable, and non-(or hardly-) saturable components. Transfer from the dialyzed serum fraction was not significantly saturable, but only partially accounted for by nonspecific pinocytosis. Thus, Zn is obtained by fetal vascular endothelium partly from low molecular mass serum species, probably through at least one carrier-mediated membrane transport system; but also from Zn complexed with serum protein, via at least one metabolism-related route.     

The zinc transporter Slc39a5 controls glucose sensing and insulin secretion in pancreatic β-cells via Sirt1- and Pgc-1α-mediated regulation of Glut2

Zinc levels are high in pancreatic β-cells, and zinc is involved in the synthesis, processing and secretion of insulin in these cells. However, precisely how cellular zinc homeostasis is regulated in pancreatic β-cells is poorly understood. By screening the expression of 14 Slc39a metal importer family member genes, we found that the zinc transporter Slc39a5 is significantly downregulated in pancreatic β-cells in diabetic db/db mice, obese ob/ob mice and high-fat diet-fed mice. Moreover,β-cell-specific Slc39a5 knockout mice have impaired insulin secretion. In addition, Slc39a5-deficient pancreatic islets have reduced glucose tolerance accompanied by reduced expression of Pgc-1α and its downstream target gene Glut2. The down-regulation of Glut2 in Slc39a5-deficient islets was rescued using agonists of Sirt1, Pgc-1α and Ppar-γ. At the mechanistic level, we found that Slc39a5-mediated zinc influx induces Glut2 expression via Sirt1-mediated Pgc-1α activation. These findings suggest that Slc39a5 may serve as a possible therapeutic target for diabetes-related conditions.     

An approach to assessing trace element bioavailability from milk in vitro

Both in vitro and in vivo, the use of a radioisotope can significantly enhance the sensitivity of methods for trace element studies. An essential prerequisite for this approach, however, is that the added (extrinsic) radiolabel equilibrates with the native (cold) element within all compartments of the diet. By using ultracentrifugation, ultrafiltration, and gel filtration chromatography, we have shown that the method is valid for zinc, copper, and manganese when using milks and formulas. For iron, however, extrinsic labeling does not necessarily yield results similar to the native distribution. We have used extrinsic labeling to follow the distribution of Zn, Cu, and Mn between high molecular weight compounds (proteins) and low molecular weight complexes in human and bovine milk after in vitro proteolysis. Peptic digestion at various pHs and pancreatic digestion for varying times were used to mimic digestion in the infant. After limited proteolysis, a large proportion of trace minerals in human milk was found in the low molecular weight fraction, whereas in cow's milk a large proportion was bound to incompletely digested casein. These findings may, at least in part, explain the higher bioavailability of trace elements from human milk compared to cow's milk.     

Effect of nicotinic acid on zinc and iron metabolism

Nicotinic acid has functional groups capable of forming complexes with trace metals. The present study examines the effect of nicotinic acid supplementation on absorption and utilization of zinc and iron. In vitro zinc uptake by human erythrocytes was studied using blood samples of 10 healthy subjects. It was found that 8 moles nicotinic acid or NADP increased ⁶⁵Zn uptake by 38.9% and 43.1% in fasting, and by 70.9% and 28.1% in postprandial conditions. In animal experiments, nicotinic acid supplementation to finger millet based diet resulted in significant enhancement of percent zinc absorption, liver zinc and growth of weanling mice (P < 0.05). When mice were fed with nicotinic acid-deficient, -adequate and -excess synthetic diets for four weeks it was observed that, in comparison with the nicotinic acid-deficient diet, percent zinc absorption, intestinal zinc, percent haeomoglobin and liver iron increased significantly under nicotinic acid-adequate and -excess conditions. The results obtained suggested that nicotinic acid, in addition to its known effect on growth and metabolism, may be playing an important role in enhancing zinc and iron utilization.     

SYNTHESIS OF ACETYLCHOLINE IN DIFFERENT COMPARTMENTS OF BRAIN NERVE TERMINALS IN VIVO AS STUDIED BY THE INCORPORATION OF CHOLINE FROM PLASMA AND THE EFFECT OF PENTOBARBITAL ON THIS PROCESS

The time course of the incorporation of choline from plasma into a high and a low molecular weight fraction from mouse brain synaptosomes was studied. The fractions were obtained from lysed synaptosomes by gel filtration on Sephadex G-25. An extremely rapid incorporation of radioactivity into acetylcholine was found in both fractions and in the time interval 0.25-9 min after the intravenous administration of labelled choline, higher specific radioactivities of acetylcholine were found in the high molecular weight fraction than in the low molecular weight fraction. However, the specific radioactivity of choline in the high molecular weight fraction was much lower than that of acetylcholine. It was found that barbiturate anaesthesia caused a marked decrease in the labelling of acetylcholine in the high molecular weight fraction while the incorporation into the low molecular weight fraction was affected to a much smaller extent. Acetylcholine of the high molecular weight fraction showed properties similar to those of vesicle-bound acetylcholine. The recoveries of labelled and endogenous acetylcholine and choline from the brain homogenates were calculated in different steps of the fractionation procedure. In the fraction containing lysed synaptosomes the recovery of radioactive acetylcholine was lower than that of endogenous acetylcholine. This may indicate the presence of two types of bound acetylcholine in the synaptosomes. Different models for the intraneuronal synthesis of acetylcholine are discussed and it is proposed that a site of acetylcholine synthesis in vivo may be closely associated with some constituent of the high molecular weight fraction and directly coupled with the storage of the transmitter.     

Transglutaminase-catalysed cross-linking of proteins phosphorylated in the intact glucose-stimulated pancreatic beta-cell

Incubation of intact islets in the presence of [32P]Pi and stimulatory levels of glucose followed by separation of phosphorylated islet proteins by SDS-polyacrylamide gel electrophoresis revealed the presence of a high molecular weight phosphopolymer which did not transverse a 3% (w/v) acrylamide gel. The majority of this phosphopolymer (approx. 70%) was present in the 600 x g sedimented fraction of islet homogenates. Islet homogenates obtained from intact islets previously incubated with [32P]Pi and stimulatory levels of glucose when incubated under conditions that activated the islet transglutaminase resulted in an increase in the amount of phosphopolymer present in the 600 x g sedimented fraction. Inhibitors of transglutaminase activity which are known to inhibit glucose-stimulated insulin release led to a significant reduction in the fraction of phosphopolymer present in the glucose-stimulated intact islet. These findings suggest that protein cross-linking and phosphorylation reactions may be closely linked in the pancreatic beta-cell.     

Zinc binding properties of human prostatic tissue, prostatic secretion and seminal fluid

When studied by gel filtration, zinc in prostatic cytosol (10(5) g, 1 h) was associated with high (greater than 80000), medium (3000-80000) and low molecular weight (less than 3000) molecules in approximately equal proportions. The molecules of high and medium Mr were not secreted into the prostatic fluid where all the zinc was associated with molecules of low Mr (probably citric acid). After ejaculation much of the zinc is redistributed and becomes bound to molecules of high and medium Mr of vesicular origin.     

Rat liver membrane secretory component is larger than free secretory component in bile: evidence for proteolytic conversion of membrane form to free form

Secretory component is a receptor for polymeric immunoglobulins on epithelial cells and hepatocytes that facilitates transport of polymeric immunoglobulins into external secretions. Little is known about the transcellular migration of secretory component-polymeric IgA complexes or the membrane forms of secretory component. We therefore examined rat bile and liver membranes to identify and compare the various molecular species of secretory component. Bile or liver membrane proteins were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. Protein profiles on blots were probed with antisecretory component antiserum, and the immunoreactive bands were visualized by indirect immunoperoxidase staining. Bile collected in the presence of proteolytic inhibitors showed an immunoreactive doublet band (Mr = 82,000 and 78,000) in the molecular weight range of free secretory component. By contrast, free secretory component in bile collected in the absence of proteolytic inhibitors and purified by affinity chromatography migrated as a single protein with an Mr = 70,000. Both components of the free secretory component doublet bound dimeric IgA when blots were probed with human dimeric IgA. Crude liver membranes prepared in the presence of proteolytic inhibitors showed two immunoreactive secretory component-containing bands, Mr = 107,000 and 99,000, whereas membranes prepared without proteolytic inhibitors showed two smaller immunoreactive bands; one of these proteolytically severed proteins comigrated with the 82,000-dalton free secretory component in bile. These results indicate that membrane forms of secretory component are present in rat liver. The observations that the membrane secretory component is larger than biliary free secretory component and yields biliary SC-like forms of secretory component upon proteolysis support the hypothesis that free secretory component in bile is a proteolytic product of larger liver membrane-associated secretory component.     

Fractionation of vanadium complexes in serum, packed cells and tissues of Wistar rats by means of gel filtration and anion-exchange chromatography

Male Wistar rats were intraperitoneally injected with [(48)V]vanadium tracer to (1) investigate the distribution of vanadium over different tissues and (2) study the distribution of vanadium over the proteins and peptides in serum, packed cells and homogenates of tissues by means of liquid chromatography experiments (size exclusion, ion exchange). Target organs were primarily kidney, bone, spleen and liver. In serum we found that vanadium was mainly bound to transferrin; however, a small amount was also bound to albumin. Besides these two complexes, a significant part of vanadium occurred as readily exchangeable ("free") vanadium. In packed cells, vanadium is mainly bound to hemoglobin and to two abundant low molecular mass complexes. The chromatograms of tissues (kidney, liver, testes, spleen and lung) show similar high molecular mass complexes (vanadium co-elutes with ferritin, transferrin and hemoglobin). Between the low molecular mass complexes there are similar peaks for spleen, testes and kidneys on the one hand, and liver and lung on the other hand, albeit the differences are small. In the case of lung, there is an additional low molecular mass peak.     

Presence of immunoreactive atrial natriuretic peptide in follicular fluid, ovary and ovarian perfusates

Immunoreactive atrial natriuretic peptide (ir-ANP) was measured in the follicular fluid of pig ovarian follicle, and rabbit ovarian homogenates and perfusates using a specific radioimmunoassay (RIA). Serial dilution curves made with the extracts of follicular fluid, ovarian homogenates and perfusates using SepPak C18 cartridges were parallel with the RIA standard curve. On gel filtration chromatography and reverse phase HPLC, all extracted materials showed high and low molecular weight forms of ir-ANP. The amount of ir-ANP in rabbit ovary was 40.70 +/- 0.39 pg/mg and that in follicular fluid of pig ovarian follicle was 18.88 +/- 2.49 pg/ml.     

Identification of a mucosal form of enteropeptidase in triton X-100 extracts of porcine duodenal mucosa

Porcine enteropeptidase (EC 3.4.21.9) purified from acetone powders of fresh duodenal fluid shows a molecular weight, as determined on Ultragel AcA-34, of 190000. Enteropeptidase has been solubilised from pig intestinal mucosa using 1% (v/v) Triton X-100. When Triton X-100 extracts of freeze-dried mucosa after partial fractionation on DEAE-cellulose were chromatographed on Sephadex G-200, the bulk of the activity eluted in the void volume rather than with an expected Ve/V0 ratio of about 1.24 corresponding to a molecular weight of around 200000. Gel filtration of aqueous mucosal extracts obtained in the absence of Triton X-100 showed two regions of enzymic activity in approximately equal proportions, one in the void volume, and the other with the expected Ve/V0 ratio of 1.24, whereas the Triton X-100 extracts of the residue from the above extract showed the presence of only the macromolecular species of enteropeptidase. This species was excluded from Sepharose 4B. It was confirmed that aminopeptidase was also extracted by Triton X-100 in a molecular form which was excluded from Sepharose 4B. The results suggest that Triton X-100 extracts enteropeptidase with a membrane component attached and in agreement with this it was found that proteolysis rapidly converted the macromolecular form to a stable smaller molecular species corresponding in size to that found in solution in the duodenal fluid. There was full recovery of the enzymic activity following this conversion. Papain and trypsin brought about an almost complete conversion to the smaller form of enteropeptidase whereas chymotrypsin, pancreatin and an intestinal peptidase preparation were only partially effective. It is concluded that membrane bound enzymes such as enteropeptidase and aminopeptidase are bound to the intestinal brush border membrane in a similar manner and are not actively secreted into the lumen but rather are largely released or solubilised by the combined action of the bile and pancreatic secretions.     

Heavy metal composition of polysomal fractions following cadmium challenge

Endogeneous levels of zinc and copper were found to be 1.2±0.1×10⁻² and 0.3±0.1×10⁻² μg/A260 unit, respectively, in polysomal fractions from control animals; cadmium, however, was undetectable. In experimental animals (injected with cadmium) zinc, copper, and cadmium were found in polysomal fractions isolated by two different methods. One hour after a cadmium injection there was a rise in both the zinc and copper content of the polysomal fractions, which then declined steadily to below control levels by 16 h. Neither zinc nor cadmium were dialyzable from these fractions by a TRIS buffer; however, addition of 0.01M EDTA to the buffer resulted in removal of 75% of the zinc and all of the detectable cadmium. The addition of cadmium (CdCl₂) to control supernatants (adjusted to the cadmium concentration present in supernatants 6 h after in vivo exposure) resulted in metal binding to polysomal fractions in levels comparable to those observed after in vivo exposures to the metal. When cadmium was added in the form of cadmium thionein, a smaller fraction of the metal was isolated with the polysomal fraction. Cadmium bound to polysomal fractions in vivo (24 h after exposure) was sensitive to release by protease digestion, but insensitive to release by ribonuclease digestion.