scholarly journals Isolation and partial characterization of the low-molecular-mass zinc/cadmium-binding protein from the testes of the patas monkey (Erythrocebus patas). Distinction from metallothionein

1988 ◽  
Vol 256 (1) ◽  
pp. 131-137 ◽  
Author(s):  
M P Waalkes ◽  
A Perantoni ◽  
A E Palmer
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Metal Binding ◽  
Binding Proteins ◽  
Binding Protein ◽  
Gel Filtration ◽  
Primate Species ◽  
Erythrocebus Patas ◽  
Patas Monkey ◽  
Zinc Cadmium

The mammalian testes are generally quite susceptible to cadmium. A deficiency of metallothionein (MT), a metal-binding protein linked to Cd tolerance, has been observed in rat testes and may explain the sensitivity in rats. Little is known about the metal-binding proteins in primate testes. Thus this study examined the nature of these proteins in a non-human primate species, the patas monkey (Erythrocebus patas). In all cases proteins isolated from testes were compared with authentic MT isolated from the liver of a zinc-treated monkey. A low-molecular-mass Zn/Cd-binding protein was seen in testicular and hepatic cytosol after gel filtration. Neither protein had substantial amounts of associated copper. These proteins could be partially purified from both sources by heat treatment and acetone precipitation. When such extracts were further separated by reverse-phase h.p.l.c., four hepatic forms were isolated, all of which proved to be authentic MT by amino acid analysis. However, only two testicular forms were separated by h.p.l.c., both of which had amino acid compositions quite unlike that of MT, having a much lower cysteine content and amino acids which are absent from MT (leucine and phenylalanine). The testicular protein appeared to be uninducible by Zn treatment. These results suggest that the low-molecular-mass Cd/Zn-binding proteins in the patas testes are not MTs and further support the hypothesis that a MT deficiency may be an important determinate of the marked testicular sensitivity to Cd toxicity.

scholarly journals Cadmium-binding proteins of rat testes. Apparent source of the protein of low molecular mass

1984 ◽  
Vol 220 (3) ◽  
pp. 819-824 ◽  
Author(s):  
M P Waalkes ◽  
S B Chernoff ◽  
C D Klaassen
Keyword(s):  
Amino Acid ◽  
Metal Binding ◽  
Amino Acid Analysis ◽  
Binding Proteins ◽  
Binding Protein ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Breakdown Product ◽  
Heat Stable ◽  
Metal Binding Protein

Fractionation of rat testicular cytosolic proteins by gel filtration indicates three major metal-binding proteins, or groups of proteins, termed testicular metal-binding protein (TMBP) 1, 2 and 3 by order of elution. The major heat-stable, metal-binding proteins in testes is TMBP-2, which has an Mr of approx. 25000. In most tissues, metallothionein (MT) is the major heat-stable, metal-binding protein, but it has an Mr of 6000. This testicular protein (TMBP-2) is much larger than MT, and since polymeric forms of MT have been previously reported, further characterization of TMBP-2 was performed. TMBP-2 was separated into two forms by DEAE-Sephadex A-25 anion-exchange chromatography. Amino acid analysis of both forms of TMBP-2 revealed that they differed markedly from MT, having particularly low cysteine contents. However, amino acid analysis showed that TBMP-2 was strikingly similar to TMBP-3, with an approximate stoichiometric relationship of 4:1. Therefore, experiments were conducted to determine if TMBP-3 could be a breakdown product of TMBP-2. Heat treatment of testicular cytosol in room air before gel filtration resulted in a marked increase in TMBP-3 and loss of TMBP-2. Storing intact testes at −20 degrees C for 2 weeks before processing for gel filtration also resulted in an increase in TMBP-3 and a loss of TMBP-2. Addition of a reducing agent (dithiothreitol) or proteinase inhibitor (N-ethylmaleimide) in processing of samples before gel filtration inhibited the appearance of TMBP-3. Results suggest that the low-Mr Cd-binding protein (TMBP-3) of rat testes results from either proteolytic or oxidative breakdown of a higher-Mr species, or from a combination of such factors.

Zinc- and copper-binding proteins in the plasma of winter flounder (Pseudopleuronectes americanus)

1980 ◽  
Vol 58 (4) ◽  
pp. 609-613 ◽  
Author(s):  
P. E. Fletcher ◽  
G. L. Fletcher
Keyword(s):  
Molecular Weight ◽  
Binding Proteins ◽  
Binding Protein ◽  
Gel Filtration ◽  
Protein S ◽  
Egg Yolk ◽  
Winter Flounder ◽  
Zinc Binding ◽  
Copper Binding ◽  
Copper Binding Proteins

Zinc- and copper-binding proteins were isolated from the plasma of winter flounder using gel filtration chromatography. A single copper-binding protein fraction of molecular weight 170 000 was isolated from the plasma of both sexes.In male and female flounder over 95% of the plasma zinc was associated with a zinc-binding protein(s) with a molecular weight of 76 000. In male flounder the remaining zinc appeared to be bound to a protein(s) of molecular weight 186 000. In female flounder the remaining 5% of the zinc was associated with two zinc-binding fractions with apparent molecular weights of 186 000 and 340 000 – 370 000.Extracts of plasma vitellogenin and egg yolk proteins revealed significant quantities of zinc and copper. It is hypothesized that the female specific zinc-binding protein (340 000 – 370 000) was vitellogenin.

scholarly journals Increasing Ceftriaxone Resistance and Multiple Alterations of Penicillin-Binding Proteins among Penicillin-Resistant Streptococcus pneumoniae Isolates in Taiwan

2007 ◽  
Vol 51 (9) ◽  
pp. 3404-3406 ◽  
Author(s):  
Cheng-Hsun Chiu ◽  
Lin-Hui Su ◽  
Yhu-Chering Huang ◽  
Jui-Chia Lai ◽  
Hsiu-Ling Chen ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Amino Acid ◽  
Binding Proteins ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Penicillin Binding Proteins

ABSTRACT The rate of nonsusceptibility of penicillin-resistant Streptococcus pneumoniae strains to ceftriaxone increased significantly in Taiwan in 2005. Approximately 90% of the ceftriaxone-nonsusceptible isolates were found to be of four major serotypes (serotypes 6B, 14, 19F, and 23F). Seven amino acid alterations in the penicillin-binding protein 2B transpeptidase-encoding region specifically contributed to the resistance.

scholarly journals Heparin- and Zn2+-binding proteins from boar seminal plasma.

1999 ◽  
Vol 46 (4) ◽  
pp. 935-939 ◽  
Author(s):  
D Hołody ◽  
J Strzezek
Keyword(s):  
Amino Acid ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Seminal Plasma ◽  
Binding Proteins ◽  
Reproductive Tract ◽  
Female Reproductive Tract ◽  
Heparin Binding ◽  
Sds Page ◽  
Protein Components

Low molecular mass, heparin-binding proteins from seminal plasma play an important role in gametes interaction whereas plasmatic Zn2+-binding proteins stabilize chromatin and plasmalemma structures and protect spermatozoa in the female reproductive tract. By means of affinity chromatography the heparin- and Zn2+-binding proteins were isolated from boar seminal plasma and both preparations were analyzed by reverse HPLC. Most of the proteins bound to heparine and Zn2+-ions were classified as spermadhesins. Three fractions binding exclusively Zn2+ were isolated. They differ in amino-acid composition, content of glucosamine and content of protein components revealed by SDS/PAGE.

Physicochemical properties of a novel α-L-arabinofuranosidase fromRhizomucor pusillusHHT-1

2001 ◽  
Vol 47 (8) ◽  
pp. 767-772 ◽  
Author(s):  
A KM Shofiqur Rahman ◽  
Shinya Kawamura ◽  
Masahiro Hatsu ◽  
M M Hoq ◽  
Kazuhiro Takamizawa
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Rhizomucor Pusillus ◽  
Terminal Amino ◽  
Metal Cofactors

The zygomycete fungus Rhizomucor pusillus HHT-1, cultured on L(+)arabinose as a sole carbon source, produced extracellular α-L-arabinofuranosidase. The enzyme was purified by (NH4)2SO4fractionation, gel filtration, and ion exchange chromatography. The molecular mass of this monomeric enzyme was 88 kDa. The native enzyme had a pI of 4.2 and displayed a pH optimum and stability of 4.0 and 7.0–10.0, respectively. The temperature optimum was 65°C, and it was stable up to 70°C. The Kmand Vmaxfor p-nitrophenyl α-L-arabinofuranoside were 0.59 mM and 387 µmol·min–1·mg–1protein, respectively. Activity was not stimulated by metal cofactors. The N-terminal amino acid sequence did not show any similarity to other arabinofuranosidases. Higher hydrolytic activity was recorded with p-nitrophenyl α-L-arabinofuranoside, arabinotriose, and sugar beet arabinan; lower hydrolytic activity was recorded with oat–spelt xylan and arabinogalactan, indicating specificity for the low molecular mass L(+)-arabinose containing oligosaccharides with furanoside configuration.Key words: α-L-arabinofuranosidase, enzyme purification, amino acid sequence, Rhizomucor pusillus.

scholarly journals Characterization of a glutathione S-transferase and a related glutathione-binding protein from gill of the blue mussel, Mytilus edulis

1995 ◽  
Vol 305 (1) ◽  
pp. 145-150 ◽  
Author(s):  
P J Fitzpatrick ◽  
T O B Krag ◽  
P Højrup ◽  
D Sheehan
Keyword(s):  
Amino Acid ◽  
Mytilus Edulis ◽  
Blue Mussel ◽  
Binding Protein ◽  
Protein A ◽  
Gel Filtration ◽  
Cumene Hydroperoxide ◽  
Gill Tissue ◽  
Glutathione S Transferase ◽  
Sequencing Studies

The major isoenzyme of glutathione S-transferase (GST 1) was purified to homogeneity from cytosolic extracts of Mytilus edulis gill tissue by GSH-agarose affinity chromatography followed by Mono Q ion-exchange f.p.l.c. This enzyme was particularly active with 1-chloro-2,4-dinitrobenzene, ethacrynic acid and cumene hydroperoxide as substrates. Immunoblotting and amino acid sequencing studies indicate that the enzyme belongs to the Pi class of GSTs. A related protein which binds to GSH-agarose was also purified. This GSH-binding protein did not immunoblot with GST antisera and showed no detectable catalytic activity with GST substrates although its N-terminal sequence was similar to Mu-class GSTs. Gel-filtration chromatography indicated that GST 1 is a dimer and the GSH-binding protein a monomer. Mass spectrometry and SDS/PAGE indicate subunit molecular masses of 24 kDa (GST 1) and 25 kDa (GSH-binding protein), respectively. Both proteins have amino acid compositions typical of GSTs.

Binding of Al by protein in plasma of patients on maintenance hemodialysis.

1985 ◽  
Vol 31 (8) ◽  
pp. 1314-1316 ◽  
Author(s):  
M Cochran ◽  
D Patterson ◽  
S Neoh ◽  
B Stevens ◽  
R Mazzachi
Keyword(s):  
Molecular Mass ◽  
Binding Protein ◽  
Equilibrium Dialysis ◽  
Gel Filtration ◽  
Human Albumin ◽  
Hemodialysis Patients ◽  
Single Peak ◽  
Molar Ratio ◽  
Maintenance Hemodialysis ◽  
Mass Fractions

Abstract Gel filtration of plasma from hemodialysis patients, with use of reagents and apparatus with carefully minimized background Al concentrations, reproducibly showed a single peak for Al, corresponding exactly to the elution position of transferrin. The Al/transferrin molar ratio in adjacent fractions was constant (mean 0.126, SE 0.006) in replicate experiments. In contrast, the association of Al with albumin varied. Using both equilibrium dialysis and gel-filtration techniques, in the presence and absence of calcium or phosphate, we could demonstrate no significant binding of Al by human albumin at Al concentrations of 1 to 12 mumol/L. We saw no Al peak in pooled, concentrated, low-molecular-mass fractions of plasma gel-filtered on Sephadex G-50. Evidently, transferrin is the sole Al-binding protein in plasma of hemodialysis patients.

scholarly journals Structural features of the low-molecular-mass human salivary mucin

1992 ◽  
Vol 287 (2) ◽  
pp. 639-643 ◽  
Author(s):  
M S Reddy ◽  
L A Bobek ◽  
G G Haraszthy ◽  
A R Biesbrock ◽  
M J Levine
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Structural Features ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Mrna Species ◽  
Salivary Mucin

The low-molecular-mass human salivary mucin has at least two isoforms, MG2a and MG2b, that differ primarily in their sialic acid and fucose content. In this study, we characterize further these isoforms, particularly their peptide moieties. Trypsin digests of MG2a and MG2b yielded high- and low-molecular-mass glycopeptides following gel filtration on Sephacryl S-300. The larger glycopeptides from MG2a and MG2b had similar amino acid compositions and identical N-terminal sequences, suggesting common structural features between their peptides. An oligonucleotide probe generated from the amino acid sequence of the smaller glycopeptide from MG2a was employed in Northern-blot analysis. This probe specifically hybridized to two mRNA species from human submandibular and sublingual glands. A cDNA clone selected from a human submandibular gland cDNA expression library with antibody generated against deglycosylated MG2a also hybridized to these two mRNA species. In both cases, the larger mRNA was polydisperse, and the hybridization signal was more intense in the sublingual gland. In addition, the N-terminal amino acid sequence of the larger glycopeptide was found to be part of one of the selected MG2 cDNA clones.

scholarly journals Relationship between biotin-binding proteins from chicken plasma and egg yolk

1978 ◽  
Vol 175 (2) ◽  
pp. 629-633 ◽  
Author(s):  
R D Mandella ◽  
H W Meslar ◽  
H B White
Keyword(s):  
Binding Proteins ◽  
Elevated Temperatures ◽  
Binding Protein ◽  
Gel Filtration ◽  
Egg Yolk ◽  
Egg White ◽  
Similar Molecular Weight ◽  
Precipitin Line ◽  
Ion Exchange Column ◽  
Biotin Binding

The plasma of laying hens contains a specific biotin-binding protein that appears to be identical with an egg-yolk biotin-binding protein. Both proteins are saturated with biotin and require elevated temperatures to effect the exchange of [14C]biotin for the protein-bound vitamin. The heat-exchange curve in each case is the same and differs sharply from that of avidin, the egg-white biotin-binding protein. On Sephadex G-100 gel filtration, plasma and yolk biotin-binding proteins were each eluted slightly ahead of avidin (mol.wt. 68,000), suggesting that they are of similar molecular weight. Plasma and yolk biotin-binding proteins required the same ionic strength to be eluted from a phosphocellulose ion-exchange column. Both the plasma and yolk biotin-binding proteins had a pI of 5; avidin has a pI of 10. Plasma biotin-binding protein cross-reacted with antiserum to yolk biotin-binding protein and showed a precipitin line of identity with purified yolk biotin-binding protein. It is suggested that biotin-binding plays an important role in mediating the transport of the vitamin from the bloodstream to the developing oocyte.

scholarly journals Cadmium-binding proteins of rat testes. Characterization of a low-molecular-mass protein that lacks identity with metallothionein

1984 ◽  
Vol 220 (3) ◽  
pp. 811-818 ◽  
Author(s):  
M P Waalkes ◽  
S B Chernoff ◽  
C D Klaassen
Keyword(s):  
Metal Binding ◽  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Or Groups

Cadmium-binding proteins in the cytosol of testes from untreated rats were separated by Sephadex G-75 gel filtration. Three major testicular metal-binding proteins (TMBP), or groups of proteins, with relative elution volumes of approx. 1.0 (TMBP-1), 1.7 (TMBP-2) and 2.4 (TMBP-3) were separated. Elution of Zn-binding proteins exhibited a similar pattern. TMBP-3 has previously been thought to be metallothionein (MT), and hence this protein was further characterized and compared with hepatic MT isolated from Cd-treated rats. Estimation of Mr by gel filtration indicated a slight difference between MT (Mr 10000) and TMBP-3 (Mr 8000). Two major forms of MT (MT-I and MT-II) and TMBP-3 (TMBP-3 form I and TMBP-3 form II) were obtained after DEAE-Sephadex A-25 anion-exchange chromatography, with the corresponding subfractions being eluted at similar conductances. Non-denaturing polyacrylamide-gel electrophoresis on 7% acrylamide gels indicated that the subfractions of TMBP-3 had similar mobilities to those of the corresponding subfractions of MT. However, SDS (sodium dodecyl sulphate)/12% (w/v)-polyacrylamide-gel electrophoresis resulted in marked differences in migration of the two corresponding forms of MT and TMBP-3. Co-electrophoresis of MT-II and TMBP-3 form II by SDS/polyacrylamide-gel electrophoresis revealed two distinct proteins. Amino acid analysis indicated much lower content of cysteine in the testicular than in the hepatic proteins. TMBP-3 also contained significant amounts of tyrosine, phenylalanine and histidine, whereas MT did not. U.v.-spectral analysis of TMBP-3 showed a much lower A250/A280 ratio than for MT. Thus this major metal-binding protein in testes, which has been assumed to be MT is, in fact, a quite different protein.

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