Low Molecular Weight Binding Proteins Research Articles

Generation of an S100B homozygous knockout pluripotent stem cell line (WAe009-A-94) by the CRISPR/Cas9 system

S100 calcium binding protein beta (S100B) is an S-100 low molecular weight binding protein that regulates intracellular processes. This protein is involved in myocardial contractility and calcium handling capacity. In this study, a human embryonic stem cell (hESC) line with homozygous S100B knockout (S100B-KO) was generated using the CRISPR/Cas9 editing system. This S100B-KO hESC line maintained normal cell morphology and karyotype, expressed pluripotency markers, and could differentiate into cells of all three germ layers.

PIXE-electrophoresis shows starving collembolan reallocates protein-bound metals

One of multiple functions of metalloproteins is to provide detoxification to excess metal levels in organisms. Here we address the induction and persistence of a range of low to high molecular weight copper- and zinc binding proteins in the collembolan species Tetrodontophora bielanensis exposed to copper- and zinc-enriched food, followed by a period of recovery from metal exposure, in absence and presence of food. After 10days of feeding copper and zinc contaminated yeast, specimens were either moved to ample of leaf litter material from their woodland stand of origin or starved (no food offered). The molecular weight distribution of metal binding proteins was determined by native polyacryl gel electrophoresis. One gel was stained with Comassie brilliant blue and a duplicate gel dried and scanned for the amount of copper and zinc by particle-induced X-ray emission. Specimens exposed to copper and recovered from it with ample of food had copper bound to two groups of rather low molecular weight proteins (40-50kDa) and two of intermediate size (70-80kDa). Most zinc in specimens from the woodland stand was bound to two large proteins of about 104 and 106kDa. The same proteins were holding some zinc in metal-exposed specimens, but most zinc was found in proteins <40kDa in size. Specimens recovered from metal exposure in presence of ample of food had the same distribution pattern of zinc binding proteins, whereas starved specimens had zinc as well as copper mainly bound to two proteins of 8 and 10kDa in size. Thus, the induction and distribution of copper- and zinc-binding proteins depend on exposure conditions, and the presence of low molecular weight binding proteins, characteristic of metallothioneins, was mainly limited to starving conditions.

Differential effect of fasting on IGF-BPs in serum of young and adult rats and its implication to impaired skin GAG content.

During fasting or aging of animals there is a decreased content of skin glycosaminoglycans (GAGs). It has been found that the skin of adult rats contains about 60% of GAGs found in the skin of young animals. Fasting of both groups of animals (young and adult) resulted in decrease of GAG content. However, GAG content in the skin of fasted young rats decreased by 30% and in fasted adult rats by 15% only, compared to fed animals, respectively. The mechanism for the phenomena is not known. We considered insulin-like growth factor-I (IGF-I) as a potential candidate involved in regulation of GAG biosynthesis in both experimental models of animals. Adult rat sera were found to contain about 75% of IGF-I recovered from young rat sera. Fasting of both groups of animals resulted in dramatic decrease in serum IGF-I levels to about 50% of initial values. Since IGF-I activity and IGF-I serum half-life depends on the level of specific IGF-binding proteins (IGFBPs) we determined (i) relationship between main groups of IGFBPs, namely high molecular weight binding proteins (HMWBPs) and low molecular weight binding proteins (LMWBPs) and (ii) the amounts of IGF-I bound to respective proteins in the sera of all experimental animals. Control young rat serum was found to contain about 90% of HMWBPs and about 10% of LMWBPs as determined by ligand binding assay. In contrast, control adult rat serum contained about 60% of HMWBPs and about 40% of LMWBPs. Fasting of both groups of animals resulted in significant increase in serum levels of LMWBPs. Control young rat serum was found to contain about 8% IGF-I bound to LMWBPs while serum of control adult rats contained 18% IGF-I bound to these proteins. In sera of fasted young animals however, about 75% of the bound IGF-I was recovered from LMWBPs (about 60% of total serum IGF-I) while in sera of fasted adult animals only about 56% of the bound IGF-I was recovered from LMWBPs (about 50% of total serum IGF-I). Evidence was provided that during fasting of both groups of animals there is a significant decrease in serum BP-3 and dramatic increase in serum BP-1 concentrations, compared to respective controls. However, the concentration of BP-1 in serum of fasted young rats was increased by about 60 fold while in serum of fasted adult rats only by about 10 fold, compared to respective control animals. Negative correlation between skin GAG content and LMWBPs derived IGF-I during fasting of young (r = -0.943, p < 0.001) and adult ( r = -0.571, p < 0.01) rats was found. The data presented suggest that the effects of aging and fasting on decreased skin GAG content may be due to induction of LMWBPs that are known to (i) inhibit IGF-I dependent function and (ii) increase clearance of IGF-I from circulation. However, the effects of fasting are distinct in respect to young and adult rats suggesting that mechanisms involved in regulation of IGF-I bioactivity during aging are more complex that during fasting.

Subtherapeutic erythropoietin and insulin-like growth factor-1 correct the anemia of chronic renal failure in the mouse

Chronic renal failure (CRF) is associated with a hyporegenerative anemia, which is caused primarily by inadequate production of erythropoietin (EPO) by the diseased kidneys and is responsive to exogenous EPO administration. Little is known about compensatory mechanisms that might supervene in anemia with low levels of EPO. Multiple investigations in vitro suggest an important role for insulin-like growth factor-1 (IGF-1) as well as EPO in erythropoiesis. Recently, both EPO and IGF-1 in vitro have been found to stimulate erythroid colony forming units in the mouse. However, no studies have examined the effect of IGF-1, singly and in combination with EPO, in CRF in vivo. This study examined mice with surgically-induced renal failure of six weeks duration that were treated for three weeks with the combination of subtherapeutic doses of both EPO and IGF-1. The single administration of each cytokine caused no significant change in hemoglobin in all CRF mice. In marked contrast the combined administration of the two cytokines produced a striking rise in hemoglobin, resulting in anemia correction in the majority of animals. The response to the combination therapy was comparable to the maximal response obtained with a single EPO dose (10 U) in a dose-finding study. Although the data are limited to utilizing one dose of each cytokine and one preparation of IGF-1, the large increase in hemoglobin observed with the combination therapy indicates that these two cytokines work in concert to stimulate erythroid precursors in CRF. In addition, untreated CRF mice showed markedly increased serum levels of low molecular weight binding proteins for IGF-1, potentially reducing the bioavailability of IGF-1. These findings taken together suggest that the anemia of CRF may represent both an EPO and a functional IGF-1 deficient state.

Exercise‐induced changes in insulin‐like growth factors and their low molecular weight binding protein in healthy subjects and patients with growth hormone deficiency

Serum concentrations of insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), the low molecular weight form of IGF binding protein (IGFBP-1), insulin, C-peptide and GH were determined in six healthy subjects and four patients with GH deficiency during 30 min of moderate physical exercise on the cycle ergometer. The load corresponded to 60% of individual maximal oxygen uptake. IGF-1 and IGF-2 were determined by radioimmunoassays developed with antibodies isolated from immunized hens eggyolk after separation by automated acid gel filtration of serum samples prior to assay. Significant increases in the serum concentrations (mean +/- SEM) of IGF-1 (157 +/- 24 to 196 +/- 29 micrograms l-1, P less than 0.05) and IGF-2 (451 +/- 37 to 678 +/- 85 micrograms l-1, P less than 0.01) were seen in the healthy subjects after 10 min of exercise. The mean percentage increase was 26 +/- 5% for IGF-1 and 50 +/- 11% for IGF-2. No relation to the GH release was found. In GH-deficient patients the mean IGF-2 concentration rose 48 +/- 17% from basal 216 +/- 63 micrograms l-1 to a peak concentration of 324 +/- 115 micrograms l-1 (P less than 0.01) after 30 min, while the 38 +/- 20% rise of IGF-1 from basal 36 +/- 13 micrograms l-1 to a peak concentration of 55 +/- 27 micrograms l-1 was not significant. The serum IGFBP-1 concentration did not change during exercise, while insulin and C-peptide concentrations, as well as blood glucose, decreased in both healthy subjects and GH-deficient patients.(ABSTRACT TRUNCATED AT 250 WORDS)

The amino terminal sequence of the developmentally regulated Ch21 protein shows homology with amino terminal sequences of low molecular weight proteins binding hydrophobic molecules

Ch21 protein, a developmentally regulated chick embryo protein of 21,000 apparent molecular weight, was purified from culture medium of hypertrophic chondrocytes. The purification method included a DEAE cellulose chromatography column, a CM cellulose chromatography column and a HPLC molecular sieve column. The amino acid sequence of the amino terminal end of the protein was determined. Computer assisted analysis showed significant homology between this sequence and the amino terminal sequences of proteins that belong to the superfamily of the low molecular weight binding proteins sharing a basic framework for the binding and transport of small hydrophobic molecules. Determination of the amino terminal sequence of the chicken retinol binding protein excluded identity between this protein and the Ch21.

Production of immunoreactive insulin-like growth factor-I (IGF-I) and IGF-I binding proteins by human lung tumours

The production of insulin-like growth factor I (IGF-I) and IGF-I binding proteins (BPs) by human lung tumour cell lines in vitro has been examined and the levels of these substances in the serum of lung cancer patients investigated. While small cell lung cancer (SCLC) cell lines secreted both IGF-I and BPs, non-small cell lung cancer (NSCLC) cell lines secreted BPs only. No evidence of increased serum IGF-I levels was obtained in a cohort of 52 lung cancer patients having SCLC and NSCLC histologies. In contrast, serum levels of low molecular weight BPs were markedly elevated in the majority of lung cancer patients.

A comparison of the biological activity of the recombinant intact and truncated insulin-like growth factor 1 (IGF-1)

A truncated form of insulin-like growth factor 1 (IGF-1), which lacked the aminoterminal tripeptide Gly-Pro-Glu, has been isolated from human fetal and adult brain. This truncated IGF-1 displayed more potent cross-reactivity and biological action on brain cells than IGF-1 isolated from human serum. We now present data on a recombinant DNA-derived truncated IGF-1 lacking the aminoterminal tripeptide. Recombinant truncated IGF-1 was 1,4–5-times more potent than recombinant and natural IGF-1 in displacing [ 125I]IGF-1 from human fetal and adult brain and placenta membranes. These differences were slightly enhanced when truncated IGF-1 was used as radioligand. The relative potencies compared to insulin-like growth factor 2 (IGF-2) in displacing [ 125I]IGF-2 from rat liver membranes were recombinant truncated IGF-1, 0.3% and recombinant IGF-1, 0.2%. Recombinant truncated IGF-1 displayed 100-fold reduced affinity for the low molecular weight binding protein (IGF-BP) isolated from human amniotic fluid when compared to recombinant IGF-1. Likewise, the IGF-BP was 100-fold less potent in inhibiting the receptor binding of recombinant truncated IGF-1 than that of recombinant IGF-1. Recombinant truncated IGF-1 was 4-times more potent than recombinant and natural IGF-1 in stimulating DNA synthesis in fetal rat brain cells. This biological activity of recombinant truncated IGF-1 was not affected by the IGF-BP at concentrations which abolished the biological activity of recombinant IGF-1. The hypothesis that IGF-BP bound intact IGF-1 represents the endocrine form of IGF-1, whereas truncated IGF-1 represents the paracrine or autocrine from of IGF-1, is proposed.

HUMAN GRANULOSA CELLS SYNTHESIZE LOW MOLECULAR WEIGHT INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN

Human follicular fluid contains insulin-like growth factor I (IGF-I) and its low molecular weight binding protein (IGF-BP). We studied the synthesis of IGF-BP by the granulosa cells obtained after ovarian hyperstimulation for in vitro fertilization. The granulosa cells were cultured for 72 hours in Ham's F-10 medium supplemented with 10% fetal calf serum (FCS). Samples of the culture medium were collected every 24 hours. The IGF-BP concentration in culture medium increased from 1.2 to 2.1 micrograms/l at 48 h and to 3.3 micrograms/l at 72 h. De novo synthesis of IGF-BP was shown by incorporation of labeled methionine into immunoreactive IGF-BP, as detected by SDS polyacrylamide electrophoresis (PAGE) and fluorography. Our results demonstrate synthesis of IGF-BP in the human ovary.

Insulin-like growth factor (IGF-I) and its low molecular weight binding protein in human milk

Insulin-like growth factors (IGF-I and IGF-II) are mitogenic polypeptides bound in serum to specific binding proteins. This study reports the occurrence and concentrations of IGF-I and its low molecular weight IGF binding protein (IGF BP) in human milk and serum after delivery in 41 healthy women. The levels of IGF-I in milk declined between 4 and 92 hours postpartum from 49 ± 17 μg/l to 29 ± 27 μg/l ( p < 0.01). The level of IGF BP also declined from 165 ± 80 μg/l to 97 ± 70 μg/l (p < 0.01), whereas the serum concentration of this protein remained unchanged. A positive correlation was observed between the individual levels of IGF-I and its binding protein in milk (r = 0.49, p < 0.01), but no correlation was found between the individual levels of 34 kDa IGF BP in milk and serum. The relative abundance of IGF BP in the first milk compared to IGF-I is intriguing and its biologic significance remains to be clarified.

Induction of metal binding proteins in striped bass, Morone saxatilis, following cadmium treatment

1. 1. The time course of induction of cytosolic metal binding proteins (MBP) was observed following up to three daily intramuscular injections of cadmium chloride (0.2 mg cadmium/injection). 2. 2. Low molecular weight binding proteins were resolved by gel permeation chromatography on Sephadex G-75. Based on total metal binding capacity, the concentration of crude MBP increased 2.6 fold. This level of induction of MBP was confirmed by polarographic analysis. 3. 3. Initial binding of cadmium to MBP resulted in displacement of zinc, while at later times, zinc associated with MBP increased above control levels. 4. 4. Using 35S-cysteine incorporation, it was shown that the rate of hepatic MBP synthesis was significantly greater than controls and sham injected fish 18 hr after the third cadmium injection. 5. 5. Due to interfering proteins of molecular weights similar to the metal binding proteins one dimensional PAGE was not capable of verifying induction. However, the metal binding proteins were resolved using two dimensional gel electrophoresis.

Insulin-like growth factor-I: specific binding to high and low affinity sites and mitogenic action throughout the life span of WI-38 cells.

Insulin-like growth factor-I (IGF-I) (13 nM) can replace insulin (0.8 microM) in a serum-free medium containing epidermal growth factor (EGF) (16 nM) and dexamethasone (DEX) (140 nM) and stimulate DNA synthesis in young cultures of WI-38 cells, similar to the stimulation of serum-supplemented medium. By contrast, senescent cells become unresponsive to all of these hormones. The effect of IGF-I, EGF, and DEX is synergistic in stimulating multiple rounds of low density cell division. Total specific binding of [125]IGF-I per cell in monolayer culture does not change with age, which indicates, in light of increased cell size with age, an actual decrease in specific binding per micron2 of cell surface area. Binding can be traced to two separate cell proteins. Binding to the alpha subunit of the IGF-I transmembrane receptor may increase slightly with age while the 50% displacement remains unchanged. The remainder of the IGF-I specific binding (five- to thirty-fold more) is to a low molecular weight, cell-associated binding protein whose 50% displacement is 10 times higher, but also remains unchanged with age. Specific binding to the lower affinity sites decreases slightly with age at equal cell densities. IGF-I binding to the alpha subunit of the transmembrane receptor is independent of cell density, while binding to the low molecular weight binding protein is inversely proportional to cell density and may vary by as much as tenfold.

A low molecular weight binding protein for organic anions (Z protein) from human hepatic cytosol: purification and quantitation.

Human Z protein from liver was purified to homogeneity. The protein has a molecular weight of 11,000 an an isoelectric point of pH 5.8. Circular dichroism spectra of Z protein-bilirubin (unconjugated and diglucuronide) complexes revealed two ellipticity extrema, a negative peak at 460 nm, and a positive peak at 410 nm. Human serum albumin had a higher affinity for bilirubin than did Z protein. Fluorescence studies showed the approximate association constants of this protein and bilirubin, bromosulfophthalein, and indocyanine green were 10(6) M-1, 10(5) M-1, and 10(6) M-1, respectively. Immunofluorescence studies revealed that Z protein was localized in the cytoplasm of hepatocytes, proximal tubular epithelium, and epithelial cells of the small intestine. Radioimmunoassay studies were done to assess the amount of Z protein in controls and in various liver diseases. The highest concentrations of Z protein were found in the liver, kidney, heart muscle, and small intestine, in that order. In cases of acute and chronic hepatitis, hepatic Z protein concentrations were generally decreased, whereas serum Z protein concentrations were increased. In contrast, both serum and hepatic Z protein concentrations were decreased in cases of constitutional hyperbilirubinemia.

Binding of juvenile hormone, methoprene and hydroprene to haemolymph proteins of larvae of the southwestern corn borer, Diatraea grandiosella

The binding of tritiated juvenile hormone I, and the juvenile hormone mimics, methoprene and hydroprene (alkyl-3,7,11-trimethyl-2,4-dodecadienoates), to proteins of the haemolymph of larvae of the southwestern corn borer, Diatraea grandiosella, was examined in vitro. The techniques of analytical isoelectric focusing, disc electrophoresis and gel filtration were employed. Juvenile hormone was shown to bind to a high affinity low molecular weight protein in the haemolymph of diapausing larvae. This protein has an apparent dissociation constant of about 3.1 × 10 −7 M at 4°C, an isoelectric point of about 4.85, and a molecular weight of about 3 × 10 4 . Juvenile hormone was also bound to at least one low affinity high molecular weight protein fraction. High juvenile hormone binding activity was found in the haemolymph of newly diapaused larvae. Methoprene and hydroprene bound selectively to a different protein fraction present in the haemolymph of pre-diapausing larvae. This protein fraction had a low relative mobility when subjected to electrophoresis at pH 8.9. A preliminary study of the binding of tritiated juvenile hormone I to proteins of the haemolymph of pre-diapausing larvae of the southern cornstalk borer, Diatraea crambidoides, was also conducted in vitro. A low molecular weight binding protein with identical electrophoretic properties to that of D. grandiosella was present. The high molecular weight binding protein fraction of the two species, however, had different electrophoretic properties.

Prevention by copper of cadmium sequestration by metallothionein in liver

Following chronic CdCl 2 administration to rats, more than 98% of the metal in liver supernatant is bound to the low molecular weight binding protein, metallothionein. Simultaneous administration of high doses of Cd and copper salts result in an increase in toxicity which is accompanied by a failure of Cd sequestration by metallothionein in vivo. This may be due to an aggregation of metallothionein which has been observed in the presence of copper in vitro.

Synthesis of juvenile hormone binding proteins by the fat body of Manduca sexta

The hemolymph of fourth instar Manduca sexta larvae contains both a relatively low molecular weight protein and a large molecular weight lipoprotein that are capable of binding the C 18 juvenile hormone (JH). The low molecular weight binding protein (BP) has a much greater affinity for JH than the lipoprotein. A variety of organs were incubated in vitro and both the media and homogenate supernatants were analyzed in order to determine the site of synthesis of the BP. By obtaining antibodies against the BP, immunoprecipitation and immunoelectrophoretic studies could be conducted along with polyacrylamide slab gel electrophoresis and double labeling experiments. The data strongly suggest that the fat body is the insect's source of JH-BP and that the BP is rapidly released from the fat body. That is, the fat body synthesizes BP but does not store it in significant quantities. The same appears to be true of the specific lipoprotein capable of binding JH.