High Molecular Weight Protein Fraction Research Articles

Two models for screening chelating agents for cadmium removal.

The effectiveness of some chelating agents to mobilize cadmium from Chinese hamster ovary cells after chronic exposure (20 hr), as well as from cytosolic metallothionein, was studied. In the first protocol, the most effective substance was 2,3-dimercaptopropanol, followed by 2,3-dimercaptopropane-1-sulfonate and 2,3-dimercaptosuccinic acid, whereas CaNa3-diethylenetriamine pentaacetic acid X 5 H2O showed less effect. Simultaneous incubation of cells with cadmium and the chelating agent resulted in a different order of effectiveness: CaNa3 DTPA prevented cadmium uptake almost totally, 2,3-mercaptopropanol by 75% and 2,3-dimercaptopropane-1-sulfonate by 35%. Neither CaNa3-diethylenetriamine pentaacetic acid X 5 H2O nor 2,3-dimercaptosuccinic acid had altered the distribution of cadmium between the cytosolic protein fractions after a 2 hr incubation of cells, whereas after this period, 2,3-dimercaptopropanol had removed all cadmium from metallothionein, and 2,3-dimercaptopropane-1-sulfonate about 50%. None of the chelating agents had reduced the amount of Cd bound to high molecular weight proteins. In the cell-free system, 2,3-dimercaptopropanol and 2,3-dimercaptopropane-1-sulfonate were equally effective and removed all cadmium from metallothionein within ten minutes. CaNa3-diethylenetriamine pentaacetic acid X 5 H2O, however, even after 60 min, had removed only 50% of the cadmium. The remaining cadmium was found distributed to the high molecular weight and lower molecular weight protein fractions.

Collagen biosynthesis by a breast carcinoma cell strain and biopsy fragments of the primary tumour

Collagen biosynthesis was assayed in tissue fragments and in cultured neoplastic cells derived from primary ductal infiltrating carcinoma of the human breast. Neoplastic cells “in vitro” produce 3–4% of collagen with respect to the high molecular weight protein fraction. The neosynthesized collagen is mainly composed of α 1 (I) chains, which may be assembled as homotrimer molecules, as indicated by their resistance to pepsin digestion. In tissue fragments, (where neoplastic and host stromal cells coexist), the collagen percentage increases up to 15–20% and more than one polypeptide chain is produced. Present data suggest that neoplastic cells “in vivo” contribute to the deposition of collagen components, actively synthesizing a certain amount of the type I-trimer, which is a significant component of the “scirrhous” stroma ( Minafra et al. 1984; Pucci Minafra et al, 1985). This phenomenon is interpreted as one of the numerous interrelationships occurring at the cell-matrix interface during the malignant growth.

Structural similarities among the high molecular weight protein fractions of oilseeds

Data on the physico-chemical properties of proteins from soybean, groundnut, sesame seed, sunflower seed, safflower seed, mustard seed, rapeseed and cotton seed are fairly extensive. An examination of the available data on high molecular weight proteins suggests that there are similarities in many of their properties. In this report the similarity in amino acid composition, size and shape, molecular weight, secondary structure, subunit composition, association-dissociation at high and low pH, stability towards denaturants, hydrolysis by enzymes and quaternary structure of the high molecular weight proteins is discussed. Based on these similarities a model has been proposed for the associationdissociation, denaturation and reassociation behaviour of the high molecular weight proteins of oilseeds.

Chemical characterization of peach kernel oil and protein: Functional properties, in vitro digestibility and amino acids profile of the flour

Kernels of peach fruits contain 54·5% and 27·5% oil and protein, respectively, but ash and total carbohydrates were quite low. Total lipids contained 98% triglycerides; sterols and polar lipids constituted 0·41% and 1·1% respectively. Fatty acid composition revealed the presence of oleic (63·8%) and linoleic (15·4%); there was 20·7% of saturated fatty acids. The defatted flour showed good fat absorption and emulsification properties but water absorption and foaming properties were slightly low. The solubility of protein was high at both acidic and alkaline pH with a minimum solubility at pH 4·3. Albumins were the major protein fractions (60%) followed by non-protein nitrogen (17%); globulins and glutelins constituted 9·5% and 8·2%, respectively, and prolamines only 6·0%. The protein was highly digested by pepsin-pancreatin followed by pancreatin and pepsin and the lowest digestibility was by trypsin. The protein is rich in lysine, leucine, isoleucine, valine, threonine, basic and acidic amino acids but poor in methionine. PAGE showed five bands with different relative mobilities and they contained both high and low molecular weight protein fractions.

TSH binding proteins in rat and human serum.

When serum of hypothyroid rats was fractionated on a Sephadex G-100 column, most of the immunoreactive TSH was found as a front running peak, together with the high molecular weight serum proteins. Similarly, a rTSH preparation (10 mU), chromatographed in the presence of 1 ml of normal rat serum also migrated at the front, however, when a high load of TSH (4.4 U) was added to 1 ml of serum, two immunoreactive peaks were found, suggesting the saturation of the front running fraction. Immunoelectrophoresis and autoradiography of rat or human sera containing the respective 125I-labelled TSHs showed binding of the labelled TSH to IgG, alpha-2-macroglobulin, and to a third unidentified protein, migrating near the albumin line. In order to determine if the bound TSH is biologically active, the high molecular weight protein fraction was separated from hypothyroid rat serum by 45% ammonium sulphate precipitation. Immunoreactivity was determined by RIA and the biological activity was determined, in vitro, by the stimulation of 99Tc uptake by FRTL-5 cells. The 45% ammonium sulphate precipitate contained almost all of the immunoreactivity and the bioactivity of the TSH of whole serum. These results indicate that: a) The endogenous circulating TSH in the hypothyroid rat exists mainly in a protein-bound form and this protein-bound TSH contains most of the hormonal bioactivity of the serum. b) Exogenous TSH binds to serum proteins in euthyroid and hypothyroid rats and in humans. There are three protein fractions in these sera that bind TSH, one of which is an immunoglobulin. The occurrence of TSH binding immunoglobulins may involve autoimmune mechanisms.

Distribution and binding pattern of benzo(a)pyrene in rat liver, lung and kidney constituents after oral administration

Orally administered 3H‐benzo(a)pyrene (BP) was persistent in protein fraction of liver, lung and kidney. The radioactivity in this fraction increased with time after administration and accounted for about 50%, 40% and 65% of total radioactivity in liver, lung and kidney, respectively at 48 hr. The BP metabolites binding proteins were located in cytosol and had molecular weights of 40,000–60,000 and 80,000–100,000 as determined by gel filtration and polyacrylamide gel disc electrophoresis. In addition, at 48hr after administration, about 80% of radioactivity in high molecular weight protein fraction was found to be precipitated by trichloroacetic acid treatment. These results suggest that BP metabolites might be transported by and are persistent in these protein fractions of liver, lung and kidney if the intake of BP is continuous. These proteins, therefore, appeared to be closely related to cell toxicity or mutagenicity in these organs as well as DNA.

Chemical and nutritional changes in soybean during germination

Germination of soybeans for 6 days depressed the trypsin inhibitor activity by 32%. Oil content, acid value, iodine value and total unsaturated fatty acids of soybean oil decreased while peroxide value and total saturated fatty acids pronouncedly increased. Percentage of palmitic and linoleic fatty acids were highly affected during germination. Analysis of the hydrocarbon and sterol classes revealed n-tricosane and β-sitosterol to be, respectively, the major components. Marked changes in both hydrocarbon and sterol constituents occurred upon germination. Different periods of soybean germination increased non-protein nitrogen and decreased reducing sugars. However, total ash content decreased due to soaking; thereafter it was constant for the rest of the germination period. Different amino acid components increased due to germination when compared with their content in dry seeds. The protein solubility of germinated soybean was higher in H 2 O, 5% NaCl and 0·02 m NaOH than it was in ungerminated seeds. Water and oil absorption, emulsification and foaming capacities of soybean were increased after germination. A marked improvement in digestibility by pepsin was noticed while tryptic digestibility was still low (38%). PAGE patterns showed drastic changes in both low and high molecular weight protein fractions due to germination.

A protein factor from Bufo marinus erythrocytes cross-bridges microtubules in vitro

A microtubule cross-bridging factor was isolated from erythrocytes of the toad, Bufo marinus. Erythrocytes were lysed and their cytoskeletons disassembled by sonication and high salt extraction. The solubilized proteins were recovered and fractionated using Sephadex G-200 column chromatography. The protein fractions from the column were analysed by SDS-PAGE and pooled into three groups: high molecular weight (HMW) proteins that eluted from the column in the void volume and had a protein composition that included HMW polypeptides; intermediate MW proteins that were shown by SDS-PAGE to contain polypeptides smaller than 120 000 D; and low MW (LMW) proteins that contained polypeptides smaller than 70 000 D. Each group was further fractionated by phosphocellulose (PC) chromatography. The flow-through was recovered, and bound proteins were then eluted by a step gradient of salt (0.2, 0.4, 0.6 and 0.8 M KCl). To assay for microtubule cross-bridging activity, column fractions were incubated with taxol-stabilized mictotubules, formed from PC-purified brain tubulin (PC microtubules). Negatively stained samples were examined in the electron microscope for the reconstitution of microtubule bundles with interconnecting cross-bridges. The HMW protein fraction from the G-200 column contained the cross-bridging factor. When these proteins were further fractionated by PC chromatography only the fraction eluted by 0.2 M KCl induced the formation of microtubule bundles with cross-bridges. No other protein fraction isolated by the described method revealed cross-bridges between microtubules in vitro.

Proteinuria in the dog: a clinicopathological study in 51 proteinuric dogs

In 51 dogs with predominantly massive urinary protein loss, the daily loss was quantified and glomerular and tubulointerstitial lesions from renal biopsies were characterised and graded using histology, immune fluorescence and electron microscopy. The highest median daily urinary protein loss occurred in dogs with membranous glomerulonephritis (median 380 mg kg-1 d-1) and renal amyloidosis (median 257 mg kg-1 d-1). Although in nine febrile dogs the urinary protein loss was transient, both glomerular and tubular lesions were diagnosed in five and seven of these dogs, respectively. The pattern of urinary proteins was determined using sodium dodecyl-sulphate polyacrylamide gel electrophoresis. The albumin fractional clearance (FC) was raised in 46 dogs, whereas the FCS of the low molecular weight (MW) protein fraction (MW less than 66,000) and high molecular weight protein fraction (MW more than 66,000) were raised in 42 and 28 dogs, respectively. Both the high molecular weight protein FC and albumin FC significantly correlated to the grade of glomerular lesions, whereas the low molecular weight protein FC only moderately significantly correlated to the grade of tubular lesions. The selectivity index, calculated as (formula; see text) did not differentiate between the various forms of glomerulopathies. The urinary lysozyme concentration was significantly correlated to the grade of tubular lesions. It is concluded that although quantitative and qualitative measurements of urinary proteins can provide additional clinical information, they do not have a reliable predictive value and histopathological examination of renal tissue is still necessary for the final diagnosis.

Interaction of urea with the high molecular weight protein fraction, carmin, from safflower seed (Carthamus tinctorius)

The effect of urea on the high molecular weight protein fraction, carmin, from safflower seed Carthamus tinctorius L, was investigated by the techniques of sedimentation velocity, viscosity, fluorescence and difference spectral measurements, partial specific volume and circular dichroism. The protein underwent dissociation and denaturation simultaneously in the presence of increasing concentration of urea. The protein dissociated to a 2S component and in the process, the buried and exposed residues of tyrosines and tryptophans were perturbed. At 8 M urea concentration, the protein existed completely as a random coil; nearly 535 mol of urea were bound per mol of protein. The results indicated that the subunits of carmin appears to be held principally by noncovalent interactions and are dissociated/denaturated by increasing concentration of urea, leading to the destabilization of the native oligomer.

Influence of maternal mineral deficiency on the hepatic metallothionein and zinc in newborn rats.

The effects of maternal Zn, Cu, or Fe deficiencies during late gestation on hepatic levels of metals and metallothionein (MT) and the binding of Zn and Cu to protein fractions were investigated in newborn rats. Timed pregnant rats were fed one of the following diets: Zn deficient (Zn-D), Cu-D, Fe-D, or control from day 12 of gestation until birth. The specific nutritional deficiency status of the dams was confirmed by low plasma levels of the deficient metal. Livers from pups were analyzed for MT, metal content, and metal-protein binding. Maternal Zn-D resulted in a greater than 50% reduction of hepatic MT levels in pups, whereas Cu-D and Fe-D had no significant effects. Pups in each deficient group showed a significant decrease in the hepatic levels of the respective metals. Fractionation of hepatic cytosols from the pups by Sephadex G-75 gel filtration showed that in both Fe-D and Cu-D pups the respective metals were depleted from the high molecular weight protein fraction, whereas in Zn-D pups the Zn was depleted mainly from the MT fraction (Ve/V0 approximately 2). Incorporation of [35S] cysteine into MT fractions was significantly lower in Zn-D pups as compared with control pups. These results indicate that there is a specific effect of the maternal Zn-D on the hepatic storage of Zn as MT in newborn rats.

Intestinal uptake and retention of copper in the suckling rat, Rattus rattus—III. Effects of closure

1. 1. The ileum of suckling rats contains a high level of copper, most of which is concentrated within cytoplasmic vesicles of the enterocytes. Intestinal closure, 20–21 days after birth, results in the replacement of enterocytes by cells devoid of these vesicles and there is a concomitant fall in the level of copper in the ileum. 2. 2. Administration of cortisone acetate (0.5 mg/g body wt) to 5-day-old rats results in premature loss of copper-laden ileal enterocytes and an 80–90% decrease in the ileal copper concentration. 3. 3. The loss of copper is mainly from the soluble fraction of the tissue and is proportionally greater from the high molecular weight-protein fraction rather than from the copper complex.

Association-dissociation of glycinin in urea, guanidine hydrochloride and sodium dodecyl sulphate solutions

The effect of urea, guanidine hydrochloride and sodium dodecyl sulphate on glycinin, the high molecular weight protein fraction from soybean has been investigated by analytical ultracentrifugation. Urea and guanidine hydrochloride dissociate the protein to a ‘2S’ protein through the intermediary 7S and 4S proteins. Howeαer, in sodium dodecyl sulphate the protein directly dissociates to a 2S protein. Analysis of the data by calculation of per cent fraction and S20,w value indicates that dissociation and denaturation of glycinin occur simultaneously in the presence of the aboαe reagents but to different extents.

Serum reduces the TSH content in rat pituitary cells in culture.

Euthyroid rat pituitary cells were plated on a natural extracellular matrix (ECM) and cultured in either a medium supplemented with hormones and growth factors (SM) or in an unsupplemented medium (USM). Hormone supplementation resulted in a marked increase in the number of cells and in a less pronounced increase in the TSH content. Addition of 10% dehormonized serum (DHS) to the medium caused a further increase in the cell number as well as a significant elevation in the GH content but, surprisingly, it caused also a dramatic decrease in the TSH content. Time course experiments revealed that the serum doubled the rate of suppression of the TSH. DHS prepared from rat serum was as effective as that prepared from foetal calf serum and heat-inactivation of the serum did not change its suppressive effect on the production of TSH. This effect was confined to the serum fraction which was precipitable with 45% ammonium sulphate but did not bind to Con-A. It is suggested that a high molecular weight protein fraction of serum reduces the number of thyrotrophs in the culture and/or decreases the rate of TSH production by these cells.

Turnover of parenterally administered zinc and cadmium and the redistribution of metallothionein bound zinc in newborn rats

The whole body retention, tissue distribution and protein binding patterns of 65Zn were compared with 109Cd in newborn rats during postnatal development. One-day-old pups received a single injection of either 65Zn (2.5 μCi) or 109Cd (2.5 μCi plus 1 mg Cd/kg as CdCl 2). During the 22 days of age, the whole body retention of 109Cd was higher than that for 65Zn. The biological half times were 466 and 46.3 days for 109Cd and 65Zn, respectively. There were marked differences in tissue deposition of these metals. Both liver and kidney accumulated more 109Cd than other tissues while the 65Zn showed a uniform distribution, with a gradual decrease in radioactivity with age. At the time of weaning, 109Cd had accumulated mainly in liver and kidney whereas, 65Zn was found predominantly in bone and skin. The specific binding of 109Cd to hepatic MT in newborn rats did not change with growth. Although a significant amount of 65Zn initially accumulated in the MT fractions in the liver, it was transferred gradually to high molecular weight protein fractions during development. The administration of these 2 metals had no effect on the body weight, liver weight and total hepatic zinc concentration. However, a significantly high content of MT and zinc in MT fractions was detected in the livers of Cd-treated rats at 22 days of age. The results show the transfer of the essential metal, zinc from hepatic MT to other proteins and the specific binding of cadmium, the non-essential metal to MT during postnatal development in rats.

Ribosomal protein gene expression during drosophila early development: [formula omitted] & [formula omitted]. Dept. of Developmental Genetics & Anatomy, Case Western Reserve University, Cleveland, OH 44106, USA

Crystallin fragments are known to aggregate and cross-link that lead to cataract development. This study has been focused on determination of post-translational modifications (PTMs) of human lens crystallin fragments, and their aggregation properties.Four crystallin fragments-containing fractions (Fraction I [∼3.5 kDa species], Fraction II [∼3.5–7 kDa species], Fraction III [∼7–10 kDa species] and Fraction IV [>10–18 kDa species]), and water soluble high molecular weight (WS-HMW) protein fraction were isolated from water soluble (WS) protein fraction of human lenses of 50–70 year old-donors. The crystallin fragments of the Fractions I–IV were separated by two-dimensional (2D)-gel electrophoresis followed by analysis of their gel-spots by mass spectrometry. The Fractions I–IV were examined for their molecular mass, particle-diameters, amyloid fibril formation, and for their aggregation by themselves and with WS-HMW proteins.Crystallin fragments in Fractions I–IV were derived from α-, β- and γ-crystallins, and their 2D-gel separated spots contained multiple crystallins with PTMs such as oxidation, deamidation, methylation and acetylation. Crystallin fragments from all the four fractions exhibited self-aggregated complexes ranging in Mr from 5.5×105 to 1.0×108 Da, with diameters of 10–28 nm, and amyloid fibril-like formation, and aggregation with WS-HMW proteins.The crystallin fragments exhibited several PTMs, and were capable of forming aggregated species by themselves and with WS-HMW proteins, suggesting their potential role in aggregation process during cataract development.Crystallin fragments play a major role in human cataract development.

Multiple forms of lysozyme in copper stressed mussels ( Mytilus edulis)

In a previous study we monitored fluctuations in the concentration of lysozyme in the hemolymph of copper stressed bay mussels, Mytilus edulis, concurrently by measuring enzymatic activity and by an enzyme-linked immunosorbent assay (ELISA). 1 In a number of instances the data collected by the two techniques from copper stressed mussels yielded contradictory results. In these instances the quantity of lysozyme, determined enzymatically, was significantly greater than the quantities determined by ELISA. These results prompted subsequent investigation into the cause of the discrepancies. In the course of the study a high molecular weight protein fraction (35 000 MW) from mussel hemocytes was isolated. Decreasing the ionic strength and concentration of the fraction caused dissociation into a 14 600 MW fraction characteristically identical to lysozyme. 2 Association to the 35 000 MW form is reversible upon an increase in ionic strength and concentration, and is presumed to be a dimer of lysozyme. 3,4 The ionic strength and pH optima for hydrolysis of suspensions of Micrococcus luteus were determined for the dimer. Polyclonal anti-lysozyme gammaglobulin preparations 1 were used in determinations of the relative immunological reactivity of various lysozyme preparations by immunodiffusion. 5 The monomer form was more favorably precipitated or detectable than the lysozyme in hemocytes. disturbed hemocytes indicate an apparent increase in the extracellular concentration of the dimer as a result of cell damage. The observed low immunological reactivity would allow for the lack of detection by ELISA, and may have interfered with antibody-monomer interactions.

Partitioning of copper among copper-binding proteins in the mussel Mytilus edulis exposed to soluble copper

Partitioning of copper among copper-binding proteins was evaluated in digestive glands of Mytilus edulis exposed to soluble copper. Groups of mussels were held in flow-through bioassay systems and exposed to either approx. 1 (control) or 25 ..mu..g Cu/L for as long as 21 weeks. At 3-week intervals, groups of 25 mussels were removed and the digestive glands were analyzed for copper-binding proteins by gel-permeation chromatography and atomic absorption spectrometry. Chronic exposure to copper resulted in increased amounts of copper in the low molecular-weight (LMW) protein fraction, which contains metallothionein-like proteins, and in the high molecular-weight (HMW) protein fraction, which contains metalloenzymes. Concentrations of copper in the LMW protein fraction increased and then appeared to plateau with long exposure times, whereas those in the HMW protein fraction continued to increase with exposure time.

The Chemical Form of Cadmium in Subcellular Fractions Following Cadmium Exposure

Wistar rats were given drinking water containing 250 ppm Cd for 12 months. After excision of the kidney and liver, the organs were subfractionated into nuclear, mitochondrial, microsomal and cytosol fractions, and the chemical forms of Cd in the subcellular fractions were examined. Although approximately 90% of the total Cd was present in the cytosol, in the form of metallothionein, 3-5% was also present in the mitochondrial fraction and 5-7% in the microsomal fraction from both organs. By Sephadex G-75 gel filtration, after solubilizing the particulate fractions with sodium deoxycholate, approximately 89% of Cd in the microsomal fraction and 94% in the mitochondrial fraction eluted with the same retention time as that of metallothionein in both liver and kidney, while the remainder was found in a high molecular weight protein fraction. The Cd that eluted with the high molecular protein fraction may be involved in dysfunction in subcellular organelles. For estimating the toxicity of Cd associated with the high molecular weight fraction, rat liver microsomes and CdCl2 were mixed with 1% of sodium deoxycholate and the protein-Cd complex produced was isolated by eluting with Sephadex G-75. This complex had a strong toxicity toward the alcohol dehydrogenase activity (SH-enzyme), and the Ki values of the Cd-protein complexes decreased with increased amount of Cd bound to the microsomal protein fraction. The above results suggest that loosely bound Cd increased in the case of higher Cd/protein and plays a toxic role in the living cells.

The chemical form of cadmium in subcellular fractions following cadmium exposure.

Wistar rats were given drinking water containing 250 ppm Cd for 12 months. After excision of the kidney and liver, the organs were subfractionated into nuclear, mitochondrial, microsomal and cytosol fractions, and the chemical forms of Cd in the subcellular fractions were examined. Although approximately 90% of the total Cd was present in the cytosol, in the form of metallothionein, 3-5% was also present in the mitochondrial fraction and 5-7% in the microsomal fraction from both organs. By Sephadex G-75 gel filtration, after solubilizing the particulate fractions with sodium deoxycholate, approximately 89% of Cd in the microsomal fraction and 94% in the mitochondrial fraction eluted with the same retention time as that of metallothionein in both liver and kidney, while the remainder was found in a high molecular weight protein fraction. The Cd that eluted with the high molecular protein fraction may be involved in dysfunction in subcellular organelles. For estimating the toxicity of Cd associated with the high molecular weight fraction, rat liver microsomes and CdCl2 were mixed with 1% of sodium deoxycholate and the protein-Cd complex produced was isolated by eluting with Sephadex G-75. This complex had a strong toxicity toward the alcohol dehydrogenase activity (SH-enzyme), and the Ki values of the Cd-protein complexes decreased with increased amount of Cd bound to the microsomal protein fraction. The above results suggest that loosely bound Cd increased in the case of higher Cd/protein and plays a toxic role in the living cells.