Low Molecular Weight Peak Research Articles

Accumulation of cadmium by the fourth instar larva of the fly Chironomus thummi

Accumulation and effects of cadmium were investigated in Chironomus thummi larvae exposed to 10, 100 and 250 μg radiolabeled Cd/1 for up to 4 days. (1) After 4 days, average cadmium accumulation was 6.6 ng Cd/mg dry weight (10 μg Cd/1 exposure) and 177 ng Cd/mg dry weight (250 μg Cd/1 exposure). (2) Dissection studies showed that by 32 hr of exposure to both cadmium concentrations, 63.5–81.4% of accumulated cadmium was confined to the posterior midgut epithelium. Light microscope autoradiography similarly showed accumulations of cadmium in posterior midgut epithelium and smaller amounts in fat body and muscle. Little cadmium was associated with Malphigian tubules, haemocoel, anterior midgut or exoskeleton. (3) After exposure to 10 or 250 μg Cd/1, 60–75% of cadmium in ultracentrifuged homogenates of whole animals or dissected guts was associated with the resulting supernatant. When supernatants were further analyzed by gel chromatography, cadmium eluted with both a high and low molecular weight peak. The relative proportions of cadmium in the two peaks varied with concentration and length of exposure. (4) Transmission electron microscopy of posterior midgut cells from animals exposed to cadmium demonstrated frequent mitochondrial lesions. Exposure to high cadmium concentrations caused some posterior midgut cells to undergo generalized structural degeneration.

Transport and Intracellular Distribution of Copper in A Human Hepatoblastoma Cell Line, Hepg2

The uptake of radiocopper by HepG2 cells is a saturable, temperature-dependent and cellular energy-independent process with a Vmax of 7.1 +/- 0.2 pmoles min-1 mg protein-1 and an estimated Km of 3.3 +/- 0.5 microM. The rate of copper uptake is reduced at an equimolar concentration of albumin and is unaffected by zinc at a 10-fold molar excess. Approximately 70% of the newly incorporated radiocopper binds to membranes and organelles, while 30% is recovered in the cytosol. The soluble fraction can be resolved into two copper-binding protein peaks. Incubation of HepG2 with nonisotopic copper results in displacement of radiocopper associated with the proteins contained in the lower molecular weight peak. Exposure of the cells to cycloheximide inhibits the incorporation of the isotope into this fraction.

Cysteine proteinase inhibitors in human placenta

When human placental extract was chromatographed on a Sephadex G-75 column, cysteine proteinase inhibitors with molecular weights of 80 000 and 12 300 were eluted. The high molecular weight peak (CPI-H) was identified as alpha-cysteine proteinase inhibitor. The thermostable low molecular weight peak (CPI-L) inhibited plant proteinases (papain, ficin and bromelain) as well as cathepsins B, H and L isolated from the human placenta. No cross-reactivity was observed between placental CPI-L and serum alpha-CPI.

Composition of Lipopolysaccharide from Pseudomonas syringae pv. morsprunorum and its Digestion by Bacteriophage A7

Purified LPS from a virulent cherry isolate of Pseudomonas syringae pv. morsprunorum was a mixture of smooth and rough molecular species. Mild acid hydrolysis yielded a precipitate of lipid A and a carbohydrate fraction which, by gel permeation chromatography, yielded three peaks of material. The first (high molecular weight) peak was composed almost entirely of a rhamnan, the sidechain polysaccharide. The second peak contained core oligosaccharide and comprised rhamnose, glucose, heptose, 2-keto-3-deoxyoctonate (KDO), phosphate, glucosamine, galactosamine and alanine. The third (low molecular weight) peak contained KDO, phosphate and ethanolamine. Lipid A contained glucosamine, phosphate and the fatty acids 12:0, 3-OH 10:0, 2-OH 12:0 (all ester-linked to glucosamine), and 3-OH 12:0, which was amide-linked. The typing phage A7, which uses LPS as its binding site, was found to possess a rhamnanase which split the sidechains from smooth LPS, releasing them as oligosaccharide.

Biosynthesis of peptides in the atrial gland ofAplysia californica

1. The atrial gland of the marine molluscAplysia californica is a secretory organ in the wall of the large hermaphroditic duct of the reproductive tract. The gland contains at least three peptides capable of inducing behavioral egg laying when injected into another animal. The goals of the present study were to investigate the biosynthesis and biochemical properties of these atrial gland peptides. 2. When the atrial gland is incubated for 2 h in a medium containing a mixture of3H-amino acids, radiolabel is incorporated into a low molecular weight peptide peak (>10,000 Dalton) and a broad asymmetrical protein peak (16,000–40,000 Dalton). Egg-laying activity is recovered only from the low molecular weight peptide peak. 3. Sephadex G-50 gel filtration of radiolabelled atrial gland extracts reveals two major low molecular weight peaks: the first (peak D) is strongly labelled and has a small but variable optical density profile; the second (peak E) is weakly labelled but has a large optical density peak. Egg-laying activity is concentrated in peak D but small amounts are also present in peak E. 4. Isoelectric focusing of the material in peak D reveals three major products: a basic (pI 9.6) peak containing 31% of the incorporated label, a neutral (pI 6.6) peak containing 19% of the label and an acidic (pI 4.9) peak containing 18% of the radiolabel. A minor peak (pI 7.6) is also occasionally observed. The pI 9.6 peak, which probably contains more than one peptide, induces egg laying when injected into intact animals. 5. These experiments demonstrate that the atrial gland rapidly synthesizes both a heterogeneous group of high molecular weight proteins and a complex mixture of low molecular weight peptides. Protein biosynthesis in the atrial gland is, therefore, more complex than has previously been suggested.

Identification of human semen insulin-like growth factor-I/somatomedin-C immunoreactivity and binding protein.

Human seminal plasma (SP) samples have been tested for insulin-like growth factor-I/somatomedin-C (IGF-I/SM-C) immunoreactivity. Gel chromatography of SP at neutral pH indicated that all immunoreactive IGF-I/SM-C was present at a higher molecular weight than the free peptide from plasma, while in 1 M acetic acid, a major peak of immunoreactivity was seen at an apparently lower molecular weight than the free peptide, together with a peak with molecular weight about 40 000. This latter peak contained a binding protein which, on Scatchard analysis, was shown to have a single class of binding site, Ka = 1.2 X 10(10) L/mol. The low molecular weight peak material was displaced from monoclonal and polyclonal IGF-I/SM-C antibodies parallel to standard preparations. Its elution on gel chromatography later than plasma IGF-I/SM-C was shown by re-chromatography to be due to physical retardation on the column, rather than a lower molecular weight. As acid-ethanol extraction of SP samples failed to remove binding activity, samples from patients were all subjected to acid gel chromatography before assay. The mean IGF-I/SM-C content in 5 normal men was 20.7 +/- 3.7 (SD) ng/ml while that for 4 azoospermic subjects was 9.2 +/- 3.7 ng/ml. A hyposomatotrophic subject with normal sperm density and low serum IGF-I/SM-C had an SP IGF-I/SM-C value in the normal range. This study indicates that human semen contains protein-bound immunoreactive IGF-I/SM-C which is at least in part of testicular origin, and apparently not growth hormone-dependent.

Zinc and cadmium metabolism in euglena gracilis: Metal distribution in normal and zinc-deficient cells

Abstract The effect of cadmium (5 μ/ml) on the growth of Euglena gracilis is moderated by the addition of supplemental zinc levels above the normal 1 μ/ml level found in the growth medium. Gel chromatography reveals that th majority (ca. 85%) of cytosolic cadmium is localized in a cadmium-binding protein (CdBP). The CdBP appears to elute in a position slightly lower in molecular weight than mammalian metallothionein, contains thiol, and upon ion-exchange chromatography elutes in multiple peaks at ionic strengths greater than mammalian metallothioneins. The remaining cadmium is associated with high molecular weight components. Cytosolic zinc is found primarily (> 80%) in a low molecular weight peak distinct from cadmium, with the remainder as high molecular weight species. As the cells become zinc-deficient and cease proliferation, zinc is lost from the low molecular weight peak while, based on cytosolic protein, the zinc content of the high molecular weight peak remains essentially constant.

Distribution of radioactive silver in the subcellular fractions of various tissues of the rat and its binding to low molecular weight proteins.

In view of the electron microscopic evidence that silver does not penetrate cellular barriers, the distribution of radioactive silver in rat blood and subcellular fractions of liver, kidneys, spleen, and forebrain was studied. It was found that 24 h after a single intraperitoneal injection high levels of radioactivity were reached which decreased at different rates in the various tissues studied. In plasma, liver, and kidneys there was an initial rapid loss of radioactivity which was followed by a slower rate of loss. In the blood, forebrain, and spleen the loss of radioactivity was linear and somewhat slower than in the other three tissues. The cytosols of the liver and kidneys contained 60% while those of the forebrain and spleen contained 30% of the total radioactivity found in the tissue homogenates. Gel filtration on Sephadex G-75 showed that all cytosols contained two peaks of radioactivity; a high molecular weight peak which eluted just after the void volume and a low molecular weight peak. The amount of radioactivity in both peaks was, however, much lower in the chromatographic peaks of the forebrain and spleen than that found in those of the liver and kidneys. Furthermore, the spleen had a comparatively very small low molecular weight radioactive peak. In vitro experiments with liver cytosol showed similar results to those found in vivo in that the high molecular weight radioactive peak could be removed by heat. It is concluded that silver does enter cells and that silver thionein exists in the cytosols of forebrain, spleen, kidney, and liver.

Low molecular weight inhibitors of cathepsins B, H and T in human serum, synovial fluid and CSF

When human plasma was chromatographed on a Sephadex G-75 column, cathepsin B inhibitors with molecular weights of 80,000 and 13,000 were eluted. The high molecular weight peak (TPI-H) contained α-thiol proteinase inhibitor and haptoglobin, both of which inhibited cathepsins B and H. The low molecular weight peak (TPI-L) inhibited cathepsins B, H and T, but had little or no activity against papain, ficin or bromelain. TPI-L from plasma contained two molecular forms of inhibitor, while TPI-L from pooled serum samples contained nine. Serum TPI-L was found to be different from the low molecular weight inhibitors present in lung and many other organs. When rheumatoid synovial fluid was analyzed by gel filtration, the eluate contained TPI-H and TPI-L. Human cerebrospinal fluid eluates contained TPI-L, but not TPI-H.

Molecular Localization of Zinc in Rat Milk and Neonatal Intestine

The molecular localization of zinc in rat milk and pup intestine was determined by gel chromatography. Commerical gels were found to give low recoveries of zinc and also caused anomalous elution patterns. Treatment of the gel with sodium borohydride corrected these problems. With treated gels, all of the zinc in rat milk eluted with high molecular weight proteins (MW greater than or equal to 30,000). In neonatal intestine, zinc eluted in two major peaks with molecular weights 30,000 and 6,000-8,000, respectively. However, incubating intestinal homogenates at 37 degrees for 30 minutes produced a redistribution of zinc with a new major low molecular weight (less than or equal to 1,500) peak. These results demonstrate the importance of sample preparation and chromatographic conditions when studying trace element ligands and also suggest that rat milk is not analogous to human milk in regard to zinc binding.

Low molecular weight nonheme iron and a highly labeled heme pool in the reticulocyte

The ligands that bind iron and heme in reticulocyte and may regulate their distribution are not known. Guinea pig reticulocyte hemolysates were labeled with 59Fe and filtered through molecular weight sieving columns. 59Fe-containing peaks of ferritin, transferrin, and hemoglobin were identified, as well as a low molecular weight peak containing predominantly nonheme iron, and a 17,000 mol wt heme containing peak with a specific activity (59Fe/heme) 20-fold greater than hemoglobin. We note that the low molecular weight peak, the existence of which has been postulated, has been identified for the first time without the addition of a chelating agent. We speculate that the 17,000 mol wt peak is an alpha-chain pool.

Low Molecular Weight Nonheme Iron and a Highly Labeled Heme Pool in the Reticulocyte

Abstract The ligands that bind iron and heme in reticulocyte and may regulate their distribution are not known. Guinea pig reticulocyte hemolysates were labeled with 59Fe and filtered through molecular weight sieving columns. 59Fe-containing peaks of ferritin, transferrin, and hemoglobin were identified, as well as a low molecular weight peak containing predominantly nonheme iron, and a 17,000 mol wt heme containing peak with a specific activity (59Fe/heme) 20-fold greater than hemoglobin. We note that the low molecular weight peak, the existence of which has been postulated, has been identified for the first time without the addition of a chelating agent. We speculate that the 17,000 mol wt peak is an alpha-chain pool.

Inactivation of complement by mouse saliva: Enzymatic degradation of C2, C3, C4, C5, and C7

While searching for lymphocyte surface antigens in mouse saliva we have observed that this secretion prevented the complement-mediated lysis of lymphocyte in a microlymphocytotoxicity test. We show in this report that mouse saliva has a specific action on C2, C4, C3, C5, and C7 components which are inactivated very rapidly while C1, C6, C8, and C9 are not modified after 15 min of incubation. The action of mouse saliva is inhibited by PMSF, TLCK, and the Kunitz bovine trypsin inhibitor, which indicates the role of a tryptic-like enzyme. After fractionation on G-75, the main activity is recovered in a low molecular weight peak which is composed of a group of three to four proteins of 10–25,000 molecular weight. When mouse saliva is injected iv into rats, their complement hemolytic activity is completely abolished in the first 24 hr and then recovers to its normal level on the third day after injection. These preliminary results suggest that mouse saliva may become a useful tool for in vivo decomplementation experiments. This inhibition of the complement reaction is obtained with rat saliva but was never observed with any of the human saliva tested.

Developments in column chromatography for the separation and characterization of casein micelles

Skim-milk was fractionated by permeation chromatography on CPG-10 (300 nm) at 20 degrees C with synthetic milk serum. The elution profile, which was highly reproducible, showed 3 partly resolved peaks. On treatment with 0.1% glutaraldehyde before chromatography, a similar profile was obtained indicating no appreciable micellar dissociation during chromatography of the milk. The casein composition of the eluent fractions, determined by quantitative chromatography on hydroxyapatite, showed that 12.1% of total casein was associated with the low molecular weight peak. The micelle content of beta- and kappa-casein increased with increasing elution volume, and the alphas/kappa ratio showed a progressive diminution from 4.3 to 2.3. Large differences were found in the colloidal phosphate contents and the weight average diameters of the micelle fractions which clearly show that CPG-10 fractionates on the basis of molecular size.

Zinc Metabolism in Normal, Lectin-stimulated and Leukemic Lymphocytes

Gel filtration of the cytoplasmic fraction of homogenate from human peripheral lymphocytes incubated with <ce:inf>65</ce:inf>Zn gave five separate peaks with molecular weights of > 150,000, 90,000, 65,000, 20 000, and < 1000 daltons. Peaks I–IV consisted of proteins. The low molecular weight (peak V) may consist of nucleotides, polyamines and amino acids. After gel filtration 75–80% of the 65Zn activity was found in peak V. Lectin-induced stimulation of normal lymphocytes revealed a distribution pattern of 65Zn binding similar to that of unstimulated cells. There was only a slightly enhanced incorporation into the protein peaks I–IV. When peripheral lymphocytes of B lymphocyte origin from patients with chronic lymphocytic leukemia were incubated with 65Zn the same peaks were seen as with supernatants obtained from normal lymphocytes. Lectin-induced stimulation of leukemic lymphocytes had no significant effect on the 65Zn distribution pattern.

Factors affecting the relative distribution of high and low molecular weight forms of factor VIII

We have recently shown that in heparinized plasma, approximately 50% of the Factor VIII procoagulant activity has a very low molecular weight (VLMW) of approximately 20,000. The balance of the procoagulant activity is in a high molecular weight form (HMW) which is associated with the carrier protein (VIII:RAG). On the other hand, Factor VIII which has been purified from citrated plasma is known to have a molecular weight of about 10 6 daltons and can be separated after CaCl 2 dissociation into a high molecular weight carrier (VIII:RAG, molecular weight 10 6) and a low molecular weight procoagulant component (LMW, molecular weight 150,000). We have now studied the factors which influence the aggregation of the native VLMW form of Factor VIII. The ratio of VLMW:HMW in heparinized plasma is 1:1 and decreases to 1:5 when CPD is used as anticoagulant. VLMW is not detectable in EDTA plasma. The collection of blood into heparin and graduated amounts of CPD results in an increased proportion of VLMW as the CPD concentration is reduced. The ratio of VLMW:HMW remains constant at 1:1 during storage of the heparinized plasma but decreases to almost 0:1 after 24 hours in citrated plasma. When separated from HMW, the VLMW alone aggregates on storage at −80°C for 72 hours producing forms of intermediate molecular weight although the majority of the activity is in a high molecular weight form. This aggregated VLMW material is devoid of VIII:RAG and dissociates into low and high molecular weight peaks following chromatography with buffer containing 0.25 M CaCl 2. Both cryoprecipitation and PEG precipitation of heparinized plasma shift the VLMW:HMW ratio from 1:1 to 0:1. These results show that the usual conditions of storage and processing of plasma result in the aggregation of the normal VLMW to high molecular weight forms which are artifacts of the isolation process, and are further evidence against the “one molecule” theory of Factor VIII structure.

Discontinuous duplication of both strands of virus 2C DNA.

When Bacillus subtilis infected with virus 2C was labeled with short pulses of tritiated uracil (a specific precursor of the hydroxymethyluracil replacing thymine in 2C chromosome) and the cell lysate was fractionated in alkaline sucrose gradients, most of the radioactivity was found within a lowmolecular-weight peak of DNA (“Okazaki pieces”) of about 2×106 daltons. After longer pulses, segments of large size and full strands were the main labeled products. A shift from low to high-molecular-weight nascent DNA took place slowly during the eclipse phase, and rapidly during the maturation phase. This suggested a lagging of the ligase behind the polymerase at early stages. During the entire replication cycle, pulse-labeled DNA was present within the replication fork in its native form with no involvement of proteins. Furthermore, the amount of lowmolecular-weight pulse-labeled DNA hybridizing with the L strand was higher than that annealing with the H strand. To explain this finding it is suggested that the polymerization or the ligation of small segments is faster for one strand than for the other. Finally, the DNA fraction which is resistant to single-strand specific nuclease was about 10% when alkali-denatured Okazaki pieces were neutralized at 4C and was increased about five-fold upon selfannealing. It is concluded that both strands of 2C DNA duplicate in a discontinuous fashion through the formation of Okazaki pieces, which are held by hydrogen bonds initially and subsequently by phosphodiester linkages. It is proposed also that ligase action is faster for the L strand than for the H strand, and that this phenomenon might be related to both the polarity of chain growth and the asymmetry of transcription of 2C chromosome.

RABBIT POLYMORPHONUCLEAR LEUKOCYTE CHEMOTACTIC FACTOR GENERATED IN VIVO BY BACTEROIDES FRAGILIS LIPOPOLYSACCHARIDE

By chromatographic separation on Sephadex gels a peptide, termed the lipopolysaccharide-induced chemotactic factor (LPS-CF), has been isolated from inflammatory exudate. The exudate was obtained from Teflon chambers implanted subcutaneously in rabbits 3 h after LPS from Bacteroides fragilis ss. fragilis had been injected. Three chemotactic peaks were eluted by fractionation of the exudate on Sephadex G-200 columns; one major peak with molecular weight of approximately 16,000 and two minor peaks with molecular weights of approximately 68,000 and 7,000. Refiltration of the major peak on G-75 showed the same elution profile as that found on G-200 columns. By addition of 8 M urea to the elution fluid only the major and the low molecular weight peaks appeared. The molecular weight of the major chemotactic peak was calculated to 16,000 on Sephadex gels, and also using SDS-polyacrylamide gel electrophoresis and equilibrium centrifugation. The chemotactic factor was quite heat-stable and was also non-dialyzable, and freezing and thawing as well as storage at 4 degrees C for several weeks did not impede its activity. This chemotactic factor is probably identical to the cytotaxic fragment split off from C5 upon interaction with LPS.

Demonstration of an endogenous, competitive inhibitor(s) of [ 3H] diazepam binding in bovine brain

An endogenous inhibitor(s) of [ 3H] diazepam binding to synaptosomes has been demonstrated in bovine brain. The inhibitory activity of crude extracts is heat stable, dialyzable, and not affected by ether extraction. Three distinct peaks of inhibitory activity were resolved using Sephadex G-25 chromatography. The lowest molecular weight peak (<700 daltons) had the highest specific inhibitory activity and its inhibition of [ 3H] diazepam binding was competitive. A similar low molecular weight fraction was not observed in either muscle or liver suggesting that it may be unique to brain. Thin layer chromatography of the Sephadex G-25 fractions revealed a discrete band of inhibitory activity in the two low molecular weight peaks.

The mitogenic effect of lipopolysaccharide: Metabolic processing of lipopolysaccharide by mouse lymphocytes

Bacterial lipopolysaccharide (LPS), from S. enteritidis, consisted of two components, a heavy fraction with a molecular weight > 2 × 10 6 and a light fraction with a molecular weight < 40,000. Assuming the specific radioactivity to be the same for the two fractions, the ratio of heavy: light fractions was 4.6; both fractions were mitogenic to splenocytes of nude mice, but neither was active with lymphocytes from the genetically low responding strain C 3H/HeJ. When splenic lymphocytes from nude mice or from strains shown to be high responders to LPS were pulsed with labeled LPS for 1 hr the cell-bound label was progressively unloaded into the medium and recovered in the supernatant, reaching an asymptotic value of 70–80% unloading in about 6 hr. Chromatography of the unloaded material on agarose columns revealed that the ratio of heavy: light fractions declined progressively, reaching a value of 1.45 in 12 hr. Cells from high responders were incubated for 1 hr with purified heavypeak material, and the low molecular weight peak, unloaded from the cells after 12 hr, was purified by gel filtration and was concentrated by lyophilization; this fraction was found to be highly mitogenic when tested with splenic lymphocytes of the low LPS responder strain C 3H/HeJ. Thymic lymphocytes from high responder strains could not be stimulated by this fraction. Populations of thymocytes and splenocytes from the low responders bound and unloaded the LPS as did the high responders, but the ratio of heavy: light fractions remained constant during the unloading. Thus, only cells capable of showing a vigorous blastogenic response to LPS are able to process the molecule into a form which induces a high response from cells of the low responder strain. We hypothesize that an activated form of the low molecular weight species is in fact the active mitogen.