Zn-binding Protein Research Articles

Distinctive expression of a zinc-binding protein in rice callus grown in medium with high zinc concentration

We investigated the growth, contents of water-soluble protein and free amino acids of the callus of japonica rice (Oryza sativa L. cv. Nipponbare) cultured in liquid N6 medium containing a high concentration of zinc. Furthermore, we determined the N-terminal amino acid sequence of a Zn-binding protein expressed in large quantities in the callus. The addition of Zn stimulated the growth of the callus and increased the Zn concentration. The callus subjected to the high-Zn treatment (hereafter referred to as CHZn) contained a larger amount of soluble proteins and a smaller amount of free amino acids than the control callus. Zinc-binding proteins were separated by affinity chromatography. The SDS-PAGE pattern of these proteins showed a distinctive protein band of about 29 kDa. Especially, CHZn contained larger quantities of 29 kDa protein than the control callus. Twenty-seven N-terminal amino acids of the protein were sequenced as DYAPMTLTIVNNCPYPVWPGIQANSGH. Results of homology search to the amino acid sequences from the nr-aa database and the dbEST database suggested that this 29 kDa protein may be a novel zinc-binding protein and that the protein may regulate the concentration of free zinc in the cytoplasm of callus cells through its binding to zinc ions.

Concentration of metallothionein in the liver of chicken which were given cadmium (Cd) into their feed

Metallothionein is a metal-binding protein which always detected in the liver of chicken, especially essential element binding protein such as Zn-thionein. Zinc binding protein is usually used for enzyme activity in normal physiology of the animal. The aim of this study was to detect the effect of Cd given into feed on Zn-thionein contain in the liver of broiler chicken. Forty-five broiler chicken were divided into three groups, which was 15 animals in each group. Group 1 as a control group, Group 2 was given 100 mgCd/kg of feed, and group 3 was given 200 mgCd/kg of feed respectively. Body weight of five chicken per group were measured every day and liver samples of five animals per group were collected at day 7 , 14 and 21 after treatment respectively for analysis of Cd and Zn total concentrations and Cd and Zn binding protein. The result indicated that body weight of chicken in the treatment groups were lower than the control group. Zinc binding protein was highest in the control group after 21 days of treatment (2.35 ug/g), but it was low in the two treatment groups were 1.30 and 0.64 ug/g respectively, in which Cd-thionein was formed. On the other hand total Zn concentration were almost similar between treatment, 20.95 ug/g for the control group and 21.24 ug/g for group 2, and 25.37 ug/g for group 3 respectively (P>0.05). It can be concluded that Cd contamination in feed can cause decreased of Zn-thionein within 21 days of treatment, causing retardation of the animals. Key words: Cadmium, zinc, metallothionein, liver, chicken, feed

Identification of the zinc-binding protein specifically present in male rat liver as carbonic anhydrase III

A zinc (Zn)-binding protein that is present specifically in the livers of male adult rats was detected by HPLC with in-line detection by mass spectrometry (ICP MS). The Zn-binding protein was purified on Sephadex G-75 and G3000SW HPLC columns, and was identified as carbonic anhydrase III (CAIII) based on the amino acid sequence of a peptide obtained on lysyl endopeptidase digestion. CAIII is expressed as one of the major Zn-binding proteins in the livers of male rats in an age-dependent manner, a comparable amount of Zn to that of copper, Zn-superoxide dismutase (Cu,Zn-SOD) being bound to CAIII at 8 weeks of age. Castration at 4 or 8 weeks of age was shown to reduce Zn bound to CAIII to 47.5% of the sham-operated control level, suggesting that the sex-dependent expression of CAIII is partly regulated by a sex hormone, androgen. The concentration of CAIII in the livers of Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease, was also estimated as Zn bound to CAIII and shown to be lower than that in Wistar rats before the onset of hepatitis. The concentration of CAIII was decreased specifically by repeated injections of cupric ions without the Cu,Zn-SOD concentration being affected.

5-kD Zinc-binding Protein Accumulation in Macrophylla-decline-affected Citrus

Macrophylla-decline (MD)-affected citrus display apparent nutrient deficiencies in a sectorial pattern within the citrus tree canopy. The status of several elements (Ca, Cu, Fe, Mg, Mn, and Zn) was assessed in MD and healthy citrus selected from the same citrus orchards. Leaf and phloem tissues were sampled from mature, reproductive trees. Levels of Ca, Cu, Fe, Mg, and Mn were unaffected by the disorder in leaf or phloem tissues. Zinc was diminished in the leaves of MD citrus, and elevated in the whole phloem tissue (2.57-fold on a dry mass basis). Calcium and Cu were sufficient, while Mg, Fe, and Mn were slightly diminished in the leaf tissue, but phloem levels of these elements were not significantly different from that present in the phloem of healthy trees. Since Zn appeared to be redistributed to the phloem tissue from the leaves, the accumulation of the phloem specific, 5-kD Zn-binding protein (ZBP) was assessed in Macrophylla decline trees relative to healthy trees. The 5-kD ZBP was 4.77-fold greater in the phloem of MD citrus relative to healthy. This appears to account for the 2.4-fold greater level of Zn (on a fresh mass basis) found in the crude phloem extracts of the decline-affected citrus relative to healthy. In the purified ZBP fraction from decline-affected citrus, there was 4.73-fold greater Zn than in the ZBP purified from healthy. However, the ratios of Zn to ZBP were equivalent between MD citrus and healthy citrus, suggesting that phloem Zn accumulation in MD citrus is associated with the 5-kD ZBP.

Isolation and Characterization of the Vascular-specific 22-kDa Zn-binding Protein

A 22-kDa Zn-binding protein (ZBP) was isolated from the phloem tissue and evacuated xylem sap of `Valencia' sweet orange [Citrus sinensis (L.) Osbeck] on rough lemon [C. jambhiri (L.)], as well as Valencia on Rangpur lime [Citrus limonia Osbeck]. Phloem and xylem Zn was associated with the 22 kDa ZBP. The Mr value of this ZBP was estimated to be 19,500 by size exclusion chromatography and 22,800 by SDS-PAGE. This protein was isolated with an isoelectric point of 7.5. Ion exchange chromatography demonstrated that 22-kDa ZBP was highly anionic, requiring 0.43 M NaCl for elution from QAE Sepharose. The 22-kDa ZBP appears unique to citrus, having no cross reaction with protein from several tissues from a range of plant species. Accumulation decreased under Zn-deficient conditions, was enhanced by osmotic stress, and the protein completely disappeared with wounding. Amino acid composition demonstrated that the protein was rich in aspartate, and glutamate; and contained 6 cysteine, and 4 histidine residues. These amino acids may be involved in metal binding. N-terminal amino acid sequencing demonstrated that the 22-kDa ZBP had identity with sporamin A&B precursors, Kunitz-type trypsin inhibitors, and miraculin. It is suggested that the genes that encode these proteins are derived from a common ancestral gene.

Serum protein and zinc levels in patients with thoracic empyema

The element Zn is the metal component or activator of many important enzymes. The tissue concentrations and activities of Zn metalloenzymes direct the rate of protein and nucleic acid syntheses, thereby influencing tissue growth and reperative processes. Most of the serum Zn is normally bound to circulating proteins. Low serum Zn concentrations might result from depletion of Zn-binding proteins. Serum protein and Zn concentrations have been reported to be depressed in patients with acute and chronic diseases. We compare the serum protein and Zn values of patients with thoracic empyema (n = 20) with those of a control group (n = 20). The values obtained in the empyema group were significantly lower than those in the control group before the study. Test group administered 220 mg zinc sulfate (ZnSO4.7H2O) over 20 d and there was a significant increase in the values for serum protein and Zn after the oral administration of the zinc sulfate.

DNA recognition code of transcription factors.

Introduction Over 35 years have passed since the 'central dogma' of molecular biology (DNA makes RNA makes protein) was proposed (Crick, 1958). Despite its remarkable verification, it is being seen increasingly as limited, for if the whole flow of information in a cell were unidirectional, all cells with the same complement of genetic material would have identical function and morphology. The truth is manifestly otherwise. A group of proteins, transcription factors, selects the information used in cells by specifically binding to 'regulatory' DNA sequences. Among other effects, this causes the differentiation of cells. These factors act as the final messenger in a transduction pathway of signals which come from outside the cell. Thus, gene expression can be regulated by the environment. Recognition between a transcription factor and its target DNA is achieved through the physical interaction of the two molecules. Since the structures of both DNA and proteins are determined by their primary sequences, there must be a set of rules to describe DNA-protein interactions entirely on the basis of sequences. The fundamental question is whether these rules are simple and comprehensible, such that the DNA recognition code can be compared with the triplet code which summarizes the rules of how DNA and protein sequences are related in the central dogma. As we review in this paper, a simple code for DNA recognition by transcription factors does seem to exist. In fact, the recognition rules allow us (i) to predict DNA-protein interactions, (ii) to change the binding specificity of an existing transcription factor, and (iii) probably even to design in a rational way a new protein which binds to a particular DNA sequence. The code has been derived from crystal structures of transcription factor-DNA complexes (Table I) and the vast body of biochemical, genetic and statistical information about the binding specificity of transcription factors. Most of the transcription factors discussed here use an a-helix, which binds to the DNA major groove, for recognition. Those proteins which have a 'recognition helix' discussed here fall mainly into four families: probe helix (PH), helix-turnhelix (HTH), zinc finger (ZnF) and C4 Zn binding proteins (C4). There is, in addition, one transcription factor family described that uses a (J-sheet, the MetJ repressor-like (MR) family. [See Table I for members of these and other families. Note that (i) individual Zn fingers are further subdivided into A and B fingers, AF and BF (Suzuki et ai, 1994a), (ii) the PH family includes homeodomain and basic-zipper proteins (Suzuki, 1993) and (iii) the C4 family includes the hormone receptors and the GATA proteins (Suzuki and Chothia, 1994).]

A New Zinc-binding Protein of Scallop,Patinopecten yessoensis, Induced under Zinc-enriched Conditions

A zinc-binding protein was purified to homogeneity from a scallop, Patinopecten yessoensis. The protein was synthesized under zincenriched conditions like metallothioneins (MTs). The protein, however, did not satisfy the criteria for classification as MTs in amino acid composition because the cysteine content was extremely low and asparatic and glutamic acids were the predominant residues. These results suggested that the Zn-binding protein purified in this study was a new inducible protein and involved in zinc storage in the scallop.

749 PB 111 EVIDENCE FOR STRUCTURAL AND COMPOSITIONAL CHANGE IN PHLOEM OF BLIGHT-AFFECTED CITRUS

Citrus blight is a decline disorder of unknown etiology. Its earliest symptom is redistribution of Zn within the tree. The canopy exhibits symptoms of Zn deficiency while Zn accumulates in trunk phloem just above the bud union. Phloem Zn was associated with complexing agents. Zn-binding proteins (ZBPs) were isolated from phloem tissue extracts of mature healthy and blight citrus trees. After purification by ion exchange chromatography (IEC) and size exclusion chromatography, ZBP's from healthy and blight-affected citrus trees {`Valencia' sweet orange [Citrus sinensis (L.) Osbeck] on rough lemon [C. jambhiri (L.)]) were compared for relative abundance. Size exclusion chromatography indicated that the proteins were and 19.5 kD Mr. IEC demonstrated that the 5 kD ZBP was slightly anionic and that the 19.5 kD ZBP was highly anionic. There were 2.5 × times more of the 5 kD ZBP in blight material than in healthy (purified on an equal prot basis). Levels 19.5 kD ZBP were equivalent in blight and healthy phloem tissue extracts, on both fresh weight and total protein bases. The amino acid composition of the 5 kD ZBP was much more complex than would be expected for phytochelatins. Altered phloem structure also was apparent in blight versus healthy trees, with phloem fresh and dry weights increased in the blight-affected citrus. Phloem specific expression of the ZBPs was also apparent.

The LIM motif defines a specific zinc-binding protein domain.

The cysteine-rich protein (CRP) contains two copies of the LIM sequence motif, CX2CX17HX2CX2CX2CX17-CX2C, that was first identified in the homeodomain proteins Lin-11, Is1-1, and Mec-3. The abundance and spacing of the cysteine residues in the LIM motif are reminiscent of a metal-binding domain. We examined the metal-binding properties of CRP isolated from chicken smooth muscle (cCRP) and from a bacterial expression system and observed that cCRP is a specific Zn-binding metalloprotein. Four Zn(II) ions are maximally bound to cCRP, consistent with the idea that each LIM domain coordinates two metal ions. From spectroscopic studies of Co(II)- and 113Cd(II)-substituted cCRP, we determined that each metal ion is tetrahedrally coordinated with cysteinyl sulfurs dominating the ligand types. One metal site within each LIM motif has tetrathiolate (S4) coordination, the second site may either be S4 or S3N1. The LIM motif represents another example of a specific Zn-binding protein sequence.

Zinc status in proliferative response of T lymphocytes.

The in vitro proliferation of T lymphocytes are highly susceptible to Zn deprivation and Cd addition. In order to define Zn status in T cell proliferation, toxicological and biochemical situation of Zn in proliferative response of T cells was investigated by use of mouse spleen cell cultures stimulated by T cell-mitogen, Con A. The inhibitory effect of Cd on T cell proliferation was protected specifically by Zn. The protection by Zn was effective when Zn was added within 16 h after Con A stimulation. Zn addition affected neither Cd content in cells nor induction of Cd-thionein. These findings indicate that Zn-dependent processes expressed before the S phase of cell cycle are critical for T cell proliferation and are targets susceptible to Cd. Moreover, electrophoretical analysis showed two unknown Zn-binding proteins (49 and 100 KD) induced in spleen cells incubated with Con A and radioactive Zn. These Zn-binding proteins may be associated with the Zn-dependent processes critical for T cell proliferation.

Significance of extracellular zinc‐binding ligands in the uptake of zinc by human fibroblasts

Extracellular zinc (Zn)-binding ligands were investigated as vehicles for uptake of Zn by human fibroblasts. The uptake of alpha 2-macroglobulin, a major serum Zn-binding protein proposed to have a function in Zn transport, was less than 1/200 that of the Zn uptake rate. The fibroblast growth medium, BME with 10% FBS, contains several Zn-binding ligands. These were separated into components of MW greater than 30,000 and components of MW less than 30,000 using an Amicon microconcentrator. Cells accumulated Zn from both fractions; however, there was more uptake from the filtrate (MW less than 30,000), containing ligands with low affinity for Zn, hence with greater free Zn concentration. Zn uptake from a number of ligands with a range of affinities for Zn was examined and found to be inversely proportional to the Ka value for the ligands and therefore proportional to the free Zn concentration. When histidine and desferrioxamine, two structurally different Zn-binding ligands were compared, analysis of the concentration curves of calculated free Zn against Zn uptake gave similar Vmax and Km values (+/- S.E.M.) of 373 +/- 6 pmol/micrograms DNA/h and 0.08 +/- 0.004 microM for histidine, and 349 +/- 10 pmol/micrograms DNA/h and 0.06 +/- 0.008 microM for DFO, suggesting that the same transport mechanism was operating in both systems. We conclude that no specific ligands are essential for transport of Zn into fibroblasts, but that "free" Zn is acquired by the cell.

Studies on zinc- and cadmium-bound proteins in bovine kidneys by biochemical and neutron activation techniques

Bovine kidneys were found to contain about 78 ppm Zn and 0.78 ppm Cd. Approximately 45% of Zn and 60% of Cd were present in the cytosol fraction. More than 95% of these two metals were bound to macromolecules. Both Zn- and Cd-protein complexes were observed to be stable between pH 7 and 10.5. Separation and characterization of these proteins were carried out using several chromatographic and electrophoretic techniques in conjunction with instrumental neutron activation analysis (INAA). The results showed the presence of at least four Zn-binding proteins with mol wt greater than 300,000, 260,000, 89,000, and 27,000 and at least three Cd-binding proteins of mol wt greater than 300,000, 32,000, and 13,000.

Apparent deficiency of metallothionein in the Wistar rat prostate

The high affinity metal-binding protein metallothionein (MT) is thought to detoxify cadmium (Cd) but appears to be deficient in several known targets of Cd carcinogenesis. The rat ventral prostate (VP) was recently identified as one of these target tissues. The nature of the Cd-binding proteins in the prostate has not been well defined, and thus this study attempted to define the nature of these proteins in the Wistar rat. A Zn-, Cd-binding protein fraction in the low-molecular-weight range was seen by gel filtration in cytosol from either dorsal prostate (DP) or VP. These prostatic proteins eluted with a relative elution volume similar to that of authentic MT, and were extractable by heat treatment and sequential acetone precipitation, a technique originally developed for purification of MT. Preparations of such partially purified prostatic protein were further purified using a reverse-phase HPLC technique developed for MT isoform isolation. One form was detected from the VP while the DP displayed five separate forms, eluting in a range like that of the two isoforms of rat MT. However, on the basis of amino acid content, none of these prostatic forms were classifiable as MTs, due to the absence of cysteine, a very common amino acid in MT. Unlike MT which is devoid of aromatic amino acids, the prostatic proteins also contained significant amounts of tyrosine and phenylalanine. The prostatic proteins also contained much more glutamate than MT. Cadmium treatment, which is known to cause a marked induction of MT, did not alter levels of this protein in the prostate, while markedly increasing hepatic MT. The present results indicate that these low-molecular-weight Cd-, Zn-binding proteins present in the rat prostate are not MTs and provide a further correlation between MT deficiency and sensitivity to the carcinogenic effects of Cd.

The Effect of Valproic Acid on 65Zn Distribution in the Pregnant Rat

The effect of valproic acid on the distribution of gavaged 65Zn in maternal and embryonic tissue of Sprague-Dawley rats was examined 24 h after gavaging of the drug on d 13 of pregnancy. Valproic acid treatment resulted in a significantly higher retention of 65Zn in maternal liver and lower amounts in uterus, placenta and embryos than in controls. Compared to controls, gel chromatography of maternal liver from valproic acid-treated dams showed higher 65Zn counts associated with a protein peak of molecular weight of 6,500, the approximate molecular weight of the Zn-binding protein metallothionein. These results support the idea that the teratogenicity of valproic acid is in part due to an induction of embryonic Zn deficiency secondary to a drug-induced sequestering of Zn into maternal liver that results in a decrease in maternal plasma Zn and subsequent reduction in embryonic Zn uptake.

Purification and characterization of a nonmetallothionein Zn-binding protein from spotted seatrout ( Cynoscion nebulosus) oocytes

There is a paucity of information on the function of metal-binding proteins in the regulation of essential metals in vertebrate oocytes. The present investigation was conducted to determine whether metal-binding proteins, such as metallothionein (MT), are present in the soluble fraction of spotted seatrout oocytes as well as in developing eggs. Gel-filtration analysis of the cytosol revealed the presence of three zinc-binding protein fractions in whole ovarian tissues, unfertilized eggs and 12-h fertilized eggs. In all cases, the major zinc-binding protein fraction had a low molecular weight (6000–10 000) similar to mammalian hepatic MT. However, less zinc was associated with this fraction in the fertilized eggs compared to unfertilized eggs, suggesting a physiological role for metal-binding proteins in developing vertebrate eggs. In addition, the fraction from whole ovarian tissue was further purified by DEAE-Sephadex chromatography and gel-permeation chromatography. The ovarian protein was found to be distinct from MT based on amino acid analysis. This study also provides evidence for homology among gonadal metal-binding proteins isolated from different vertebrate species.

Strain dependence in mice of resistance and susceptibility to the testicular effects of cadmium: Assessment of the role of testicular cadmium-binding proteins

The nature of the cadmium (Cd)-binding proteins in the mouse testes is unknown, although some studies have implied metallothionein (MT) is responsible for the marked strain dependence of Cd-induced testicular necrosis in mice. This study attempted to define the role of MT in strain-dependent Cd resistance using NFS (susceptible) and BALB/c (resistant) mice. In all cases, testicular proteins were compared to hepatic MT isolated after treatment with zinc (Zn). A low-molecular-weight ( M r) Zn-, Cd-binding protein was detected in testicular and hepatic cytosol from both strains after gel filtration. These proteins were extractable by heat treatment and sequential acetone precipitation. When such extracts were further purified with reversephase HPLC, two forms of authentic MT were shown by amino acid analysis from both NFS and BALB/c livers. However, of two testicular forms separated by HPLC from NFS and BALB/c mice, neither could be classified as MT based on amino acid composition. Methylation of the MT-I gene was also studied in testicular and hepatic DNAs isolated from control mice or from mice made resistant to Cd-induced testicular necrosis by Zn treatment. Major differences in methylation between tissues were seen, as the testicular gene of both NFS and BALB/c mice was highly methylated, a condition often linked to genetic quiescence. Zn treatment had no effect on MT-I gene methylation in testes of either strain, although rendering the NFS strain resistant to Cd. Zn treatment did not alter levels of this testicular protein in either strain while causing a marked induction of hepatic MT in both. These results indicate the low- M r Cd-, Zn-binding proteins present in the testes of both resistant and susceptible mice are not MTs and further that the MT gene may not be expressed in either strain.

Vitamin-A-dependent zinc-binding protein and intestinal absorption of Zn in chicks.

It was demonstrated that the development of experimental avitaminosis A in chicks led to secondary zinc deficiency. The balance of Zn in the chick became negative, while the Zn content of various tissues decreased. Thus in vitamin-A-deficient chicks the serum Zn content was 1258 (SD 26.3) micrograms/l which was considerably lower than 1652 (SD 97.8) micrograms/l in controls. Zn absorption was considerably reduced throughout the entire small intestine of vitamin-A-deficient chicks and most markedly in the ileal region. Within 72 h after retinyl acetate administration Zn absorption was fully restored in this region of the intestine. The 65Zn-binding capacity of soluble proteins, present in the supernatant fraction of ileal-mucosa homogenates of vitamin-A-deficient chicks, was found to increase 2.6 times by 72 h after the administration of a single dose of retinyl acetate. A vitamin-A-dependent Zn-binding protein (ZnBP), absent in vitamin-A-deficient chicks, was isolated from the ileal mucosa after their repletion with vitamin A. Competitive-binding studies (calcium, cadmium, copper) showed the protein to be highly specific for Zn ions. The molecular weight of ZnBP was 83 kDa. The association constant of the protein-Zn complex was 0.8 X 10(6)/mol. The protein was acidic with approximately 20% of its amino acid residues belonging to dicarboxylic acids. ZnBP was found to be a glycoprotein, and it contained hexose as a carbohydrate component. It is suggested that ZnBP is involved in the binding of Zn in the ileal mucosa of chicks.

Essential metal contents and metallothionein-like protein in testes of mice after cadmium administration

To study the testicular injury by Cd from the viewpoint of essential element levels, male mice were injected with Cd subcutaneously at a dose of 30 μmol/kg body weight singly and the concentrations of 8 elements (Na, Mg, P, K, Ca, Fe, Zn, and Cd) in the testis were measured for 6 weeks. The most pronounced alteration was an increase of Ca content with time after the injection; the experimental group had 270 times more Ca than the control despite a decrease of the testis weight to one-third of the control at 4 weeks. K content decreased markedly and Fe remained at an elevated level throughout the experimental period. Decreased Mg, P, and Zn levels started to be restored after 2 weeks. Metallothionein (MT)-like Zn-binding proteins were studied in the cytosol fraction with a high performance liquid chromatograph-atomic absorption spectrophotometer. MT-like proteins were observed at a higher concentration in the control than in the Cd-injected group. No indications of new induction of MTs or MT-like proteins could be detected.

Physiological zinc-binding proteins of medium molecular weight in the rat gut.

1. Gel filtration on Sephadex G 75 was used to separate the medium-molecular-weight zinc-binding proteins from the soluble fractions from the duodenal and jejuno-ileal segments of the rat gut at 30 min after the intragastric administration of a tracer dose of 65Zn. These proteins were resolved by ion-exchange chromatography on DEAE cellulose. 2. In both the duodenum and jejuno-ileal segment an appreciable fraction of the total soluble Zn was bound in a protein fraction that resembled metallothionein (MT) in its behaviour on gel filtration. These fractions, however, were not homogeneous, but contained several medium-molecular-weight Zn-binding proteins. In the duodenum, but not in the jejuno-ileal segment, two of these proteins appeared to be the isometallothioneins, ZnMT-I and ZnMT-II. 3. These results suggest a possible role for MT in the binding of newly-absorbed Zn in the duodenal mucosal cells. They also show that gel filtration alone is insufficient for the identification of MT in the intestine.