Chromatofocusing in the purification and separation of apo- and holo-(vitamin D-binding protein).

Chromatofocusing was used to purify the vitamin D-binding protein (DBP) from pig plasma in a procedure that consisted of an initial DEAE-cellulose chromatography followed by DEAE-Sephadex chromatography, with final purification by chromatofocusing. The protein was purified 184-fold over its concentration in plasma. When the plasma was labelled with a tracer concentration of [3H]calcidiol, it was apparent that holo- and apo-DBP did not co-chromatograph on chromatofocusing. The separation of these two forms of DBP on chromatofocusing was verified by using purified apo-DBP mixed with either a tracer or a saturating concentration of calcidiol. This separation was consistent with differences observed in their isoelectric points. The ability to separate apo and holo forms of DBP should permit the study of their specific interactions with other binding proteins and help determine the physiological relevance of these interactions.     

The structure of human apolipoprotein E2, E3 and E4 in solution. 2. Multidomain organization correlates with the stability of apoE structure

The stabilities toward thermal and chemical denaturation of three recombinant isoforms of human apolipoprotein E (r-apoE2, r-apoE3 and r-apoE4), human plasma apoE3, the recombinant amino-terminal (NT) and the carboxyl-terminal (CT) domains of plasma apoE3 at pH 7 were studied using near and far ultraviolet circular dichroism (UV CD), fluorescence and size-exclusion chromatography. By far UV CD, thermal unfolding was irreversible for the intact apoE isoforms and consisted of a single transition. The r-apoE3 was found to be less stable as compared to the plasma protein and the stability of recombinant isoforms was r-apoE4相似文献   

Elastase controls the binding of the vitamin D-binding protein (Gc-globulin) to neutrophils: a potential role in the regulation of C5a co-chemotactic activity

The vitamin D-binding protein (DBP) binds to the plasma membranes of numerous cell types and mediates a diverse array of cellular functions. DBP bound to the surface of leukocytes serves as a co-chemotactic factor for C5a, significantly enhancing the chemotactic activity of pM concentrations of C5a. This study investigated the regulation of DBP binding to neutrophils as a possible key step in the process of chemotaxis enhancement to C5a. Using radioiodinated DBP as a probe, neutrophils released 70% of previously bound DBP into the extracellular media during a 60-min incubation at 37 degrees C. This was suppressed by serine protease inhibitors (PMSF, Pefabloc SC), but not by metallo- or thiol-protease inhibitors. DBP shed from neutrophils had no detectable alteration in its m.w., suggesting that a serine protease probably cleaves the DBP binding site, releasing DBP in an unaltered form. Cells treated with PMSF accumulate DBP vs time with over 90% of the protein localized to the plasma membrane. Purified neutrophil plasma membranes were used to screen a panel of protease inhibitors for their ability to suppress shedding of the DBP binding site. Only inhibitors to neutrophil elastase prevented the loss of membrane DBP-binding capacity. Moreover, treatment of intact neutrophils with elastase inhibitors prevented the generation of C5a co-chemotactic activity from DBP. These results indicate that steady state binding of DBP is essential for co-chemotactic activity, and further suggest that neutrophil elastase may play a critical role in the C5a co-chemotactic mechanism.     

Initial characterization of the vitamin D binding protein (Gc-globulin) binding site on the neutrophil plasma membrane: evidence for a chondroitin sulfate proteoglycan.

The vitamin D binding protein (DBP) is a multifunctional plasma protein that can modulate certain immune and inflammatory responses. The diverse cellular functions of DBP appear to require cell surface binding to mediate these processes. Numerous reports have detected DBP bound to the surface of several cell types and would support the concept of a cell surface binding site for DBP. However, direct evidence for such a molecule has been lacking and essentially nothing is known about its basic biochemical properties. In the present study, radioiodinated DBP was used as a probe to characterize biochemically the neutrophil DBP binding site. Radiolabeled DBP binds to and remains associated with the plasma membrane and is not degraded. Quantitation of DBP binding to either intact cells or purified plasma membranes showed nonsaturable (linear) binding with positive cooperativity, possibly suggesting DBP oligomer formation. Solubilization of cell bound 125I-DBP with various nonionic and zwitterionic detergents demonstrated that DBP binds to a membrane macromolecule that partitions to the detergent insoluble fraction. Moreover, this molecule does not associate with the cytoskeleton. Cross-linking of radiolabeled DBP bound to plasma membranes increased the amount of protein that partitioned to the insoluble fraction, and analysis of these complexes by SDS-PAGE revealed that they may be very large since they did not enter the gel. Finally, treatment of plasma membranes with either proteases or chondroitinase ABC completely abrogated membrane binding of DBP, suggesting that the protein binds to a chondroitin sulfate proteoglycan.     

Structural alterations of double-stranded DNA in complex with the adenovirus DNA-binding protein. Implications for its function in DNA replication.

The Adenovirus DNA-binding protein (DBP) binds to single-stranded (ss) DNA as well as to double-stranded (ds) DNA and forms multimeric protein-DNA complexes with both. Gel retardation assays indicate rapid complex formation for both DNAs. DBP rapidly dissociates from dsDNA, indicating a dynamic equilibrium, whereas the ssDNA-DBP complex is much more stable. We investigated the complex between DBP and dsDNA in more detail. Electron microscopical analysis shows thick filament-like and beaded structures in which the length of the DNA is not significantly altered. Cryo-electron micrographs suggest the presence of interwound protein fibres around the DNA. Ligase-mediated cyclization, but not linear multimerization, of DBP-saturated DNA fragments exceeding the persistence length was severely inhibited. This suggests that DNA may be organized by DBP into a rigid structure. Under those conditions, DBP induces distinct changes in the circular dichroism spectrum of the DNA, indicative of structural DNA changes. No bending or twisting of the complex was observed. Hydroxyl radical footprinting showed that the breakdown pattern of DNA at saturating DBP concentrations is much more regular than the protein-free DNA. This suggests the removal of tertiary structures, which may be related to the effects of DBP on enhanced NFI binding and chain elongation during Adenovirus DNA replication. Using purified proteins in an in vitro replication system, we correlate the structural changes with the effects of DBP on enhancement of NFI-binding as well as on DNA replication.     

Fatty acids bound to vitamin D-binding protein (DBP) from human and bovine sera

Human and bovine vitamin D-binding protein (DBP) have been isolated from serum by a method that does not involve denaturing steps. This method includes Cibacron Blue-Sepharose chromatography, gel filtration, DEAE-Sephadex chromatography and albumin immunoadsorption. Analysis of fatty acids bound to the isolated human and bovine DBP showed molar ratios of fatty acid to protein of 0.4 and 1.3 respectively meanwhile human and bovine albumin have bound 1.8 and 1.5 moles per mol respectively. Most of fatty acids bound to human and bovine DBP are monounsaturated and saturated, mainly oleic and palmitic acids, which together account for 50% of the total of fatty acids in both species. By contrast, polyunsaturated fatty acids represented a minor component, less than 5%.     

Age-related decline in the activites of erythrocyte membrane adenylate cyclase and protein kinase.

The circular dichroism and thermal denaturation properties of chromatin isolated from duck erythrocytes have been carefully examined as has the chromatography of sonicated erythrocyte chromatin on ECTHAM-cellulose. The circular dichroism spectrum and thermal denaturation profile resemble much more closely those of chromatin from liver than has been previously reported by other workers. The chromatography of erythrocyte chromatin on ECTHAM-cellulose gave results that differ dramatically from those obtained from chromatography of f1-containing chromatins on this weak anion exchanger, in that no variations in histone content, circular dichroism spectra or thermal denaturation profiles were observed in the eluted material. Coupled with our earlier finding of no variation in relative content of individual histones (Reeck, G. R. et al. (1974) Eur. J. Biochem. 49, 407–414), we interpret the results of ECTHAM-cellulose chromatography of erythrocyte chromatin to indicate that f2c-depleted regions analogous to the f1-depleted regions found in f1-containing chromatins do not exist in duck erythrocyte chromatin.     

Platelet-derived thrombospondin-1 is necessary for the vitamin D-binding protein (Gc-globulin) to function as a chemotactic cofactor for C5a

The chemotactic activity of C5a and C5a des Arg can be enhanced significantly by the vitamin D-binding protein (DBP), also known as Gc-globulin. DBP is a multifunctional 56-kDa plasma protein that binds and transports several diverse ligands. The objective of this study was to investigate the mechanisms by which DBP functions as a chemotactic cofactor for C5a using neutrophils and U937 cells transfected with the C5aR (U937-C5aR cells). The results demonstrate that U937-C5aR cells show C5a chemotactic enhancement only to DBP in serum, but, unlike mature neutrophils, this cell line cannot respond to DBP in plasma or to purified DBP. Analysis by SDS-PAGE and isoelectric focusing revealed no structural difference between DBP in serum compared with DBP in plasma. However, plasma supplemented with either serum, DBP-depleted serum, or activated platelet releasate provides a required factor and permits DBP to function as a chemotactic cofactor for C5a. Fractionation of activated platelet releasate revealed that the additional factor possessed the properties of thrombospondin-1 (TSP-1). Finally, purified TSP-1 alone could reproduce the effect of serum or platelet releasate, whereas Abs to TSP-1 could block these effects. These results provide clear evidence that TSP-1 is needed for DBP to function as a chemotactic cofactor for C5a.     

Formation of vitamin D-binding protein-actin and binary and ternary plasma gelsolin-actin complexes in human serum

After the addition of actin to serum, the binding of actin to serum actin-binding proteins was analyzed by the method of immunoblotting using monospecific antibodies against vitamin D-binding protein (DBP) (group-specific component, Gc), human skeletal actin and human plasma gelsolin. When increasing amounts of globular actin were added to serum, actin bound to DBP preferentially. After exhausting DBP, actin began to bind to plasma gelsolin. When equally increasing amounts of filamentous actin were added to serum, actin was bound to both plasma gelsolin and DBP, and then uncomplexed DBP removed one actin molecule from gelsolin-actin 1:2 complex, resulting in a gelsolin-actin 1:1 complex. These results support the theory that the actin-depolymerizing activity of serum is due to the concerted role of plasma gelsolin and DBP.     

Human Calprotectin： Effect of Calcium and Zinc on its Secondary and Tertiary Structures, and Role of pH in its Thermal Stability

Calprotectin, a heterodimeric complex belonging to the S 100 protein family, has been found predominantly in the cytosolic fraction of neutrophils. In the present study, human calprotectin was purified from neutrophils using two-step ion exchange chromatography. The purified protein was used for circular dichroism study and fluorescence analysis in the presence of calcium and zinc at physiological concentrations, as well as for assessment of its inhibitory activity on the K562 leukemia cell line. The thermal stability of the protein at pH 7.0 （physiological pH） and 8.0 （similar to intestinal pH） was also compared. The results of cell proliferation analysis revealed that human calprotectin initiated growth inhibition of the tumor cells in a dose- dependent manner. The intrinsic fluorescence emission spectra of human calprotectin （50 ktg/ml） in the presence of calcium and zinc ions show a reduction in fluorescence intensity, reflecting a conformational change within the protein with exposure of aromatic residues to the protein surface that is important for the biological function of calprotectin. The far ultraviolet-circular dichroism spectra of human calprotectin in the presence of calcium and zinc ions at physiological concentrations show a decrease in the m-helical content of the protein and an increase in [3- and other structures. Our results also show that increasing the pH level from 7.0 to 8.0 leads to a marked elevation in the thermal stability of human calprotectin, indicating a significant role for pH in the stability of calprotectin in the gut.     

Uptake and degradation of vitamin D binding protein and vitamin D binding protein-actin complex in vivo in the rat.

We have labelled the rat vitamin D binding protein (DBP), DBP-actin and rat albumin with 125I-tyramine-cellobiose (125I-TC). In contrast with traditional 125I-labelling techniques where degraded radioactive metabolites are released into plasma, the 125I-TC moiety is trapped intracellularly in the tissues, where the degradation of the labelled proteins takes place. By using this labelling method, the catabolism of proteins can be studied in vivo. In this study we have used this labelling technique to compare the tissue uptake and degradation of DBP, DBP-actin and albumin in the rat. DBP-actin was cleared from plasma at a considerably faster rate than DBP. After intravenous injection of labelled DBP-actin complex, 48% of the radioactive dose was recovered in the liver after 30 min, compared with 14% when labelled DBP was administered. Only small amounts of DBP-actin complex were recovered in the kidneys. In contrast with the results obtained with DBP-actin complex, liver and kidneys contributed about equally in the uptake and degradation of DBP determined 24 h after the injection. When labelled DBP was compared with labelled albumin, the amount of radioactivity taken up by the liver and kidneys by 24 h after the injection was 2 and 5 times higher respectively. In conclusion, liver and kidneys are the major organs for catabolism of DBP in the rat. Furthermore, binding of actin to DBP enhances the clearance of DBP from circulation as well as its uptake by the liver.     

Overexpression,purification, and structural analysis of the hydrophobic E5 protein from human papillomavirus type 16

The E5 proteins of human papillomavirus (HPV) are highly hydrophobic transmembrane proteins that display weak transforming activity. The HPV E5 proteins are localized largely to intracellular membranes, such as the Golgi apparatus and endoplasmic reticulum, but also appear in the plasma membrane. Infection with HPV16 is the cause of over 90% of human cervical cancers. HPV E5 is known to interact with growth factor receptors and gap junction proteins and is believed to play a role during the initiation of neoplasia. The structure of HPV E5 and the mechanism of its interactions with growth factor receptors remain largely unknown. In the present studies, the E5 protein of HPV16 was cloned into the pBAD/TOPO vector fused to an N-terminal thioredoxin leader and a C-terminal His-tag, and expressed in Escherichia coli. The identity of the protein was confirmed by immunoblotting using antibodies against a V5-epitope tag engineered into the protein. Due to formation of high molecular mass superaggregates of the protein, two chromatography steps were employed for its purification: (1) gel filtration chromatography to separate the superaggregated protein from other soluble proteins and (2) Ni-chelate affinity chromatography in the presence of detergent. The superaggregates of the E5-fusion protein were broken down to monomers and various oligomers by sonication in the presence of 0.2% SDS. The purified E5-fusion protein was then reconstituted into lipid vesicles and initial structural analysis of the protein was performed using circular dichroism spectroscopy.     

Metal ion affinity adsorption of a Zn(II)-transport protein present in maternal plasma during lactation: Structural characterization and identification as histidine-rich glycoprotein

A high-affinity Zn(II)-binding protein has been purified to homogeneity (880-fold) from the plasma of lactating women by a single affinity adsorption step on columns of tris(carboxymethyl)ethylenediamine (TED)-agarose loaded with Zn(II) ions. Purity was evaluated by high-performance reverse-phase (phenyl) chromatography and by silver staining after SDS-polyacrylamide gradient gel electrophoresis. The mass of denatured Zn(II)-binding protein was estimated by SDS-polyacrylamide gradient gel electrophoresis to be 75 kDa under both reducing and nonreducing conditions; by matrix-assisted uv laser desorption time-of-flight mass spectrometry the purified protein mass was determined to be 66 kDa. The amino acid composition revealed a high content of His (13 mol%) and Pro (12 mol%). N-terminal amino acid sequence analysis (50 residues) identified the purified protein as histidine-rich glycoprotein (HRG). Immunoblots demonstrated the absence of fragments in the purified product. An enzyme-linked immunosorbent assay was developed; a 75% recovery of intact HRG from the immobilized Zn(II) ion affinity column was documented. The circular dichroism spectra for the purified human HRG in the far uv (260-178 nm) were similar to those published for human and rabbit serum HRG. These results demonstrate that TED-immobilized Zn(II) ions can be used as a new and efficient method for the isolation of structurally intact human plasma HRG.     

A gel-based method for purification of apolipoprotein A-I from small volumes of plasma

We present here a gel-based method for rapid purification of apolipoprotein A-I (apoA-I) from small volumes of human plasma. After isolation of high density lipoprotein from plasma, the apoA-I protein was separated by electrophoresis and the apoA-I band excised from the gel. The apoA-I was then eluted from the gel strip, concentrated, and delipidated ready for use. The structure and function of the gel-purified apoA-I protein was compared against apoA-I purified by the traditional size-exclusion chromatography method. The α-helical content of the gel-purified apoA-I as determined by circular dichroism was similar to chromatography-purified apoA-I. The functional activity of gel-purified apoA-I, as determined by cholesterol efflux assays in primary human fibroblasts and RAW264.7 macrophages, was also comparable with chromatography-purified apoA-I. This method is a valid alternative for apoA-I purification with some advantages over traditional chromatography purification including a much reduced plasma volume requirement, less time and cost, and a higher percentage protein recovery. The method is particularly suitable for applications requiring the purification of apoA-I from multiple human or animal samples of interest.     

Effects of carbohydrate depletion on the structure, stability and activity of glucose oxidase from Aspergillus niger

Glucose oxidase from Aspergillus niger was purified to homogeneity by hydrophobic interaction and ion-exchange chromatography. Approx. 95% of the carbohydrate moiety was cleaved from the protein by incubation of glucose oxidase with endoglycosidase H and alpha-mannosidase. Cleavage of the carbohydrate moiety effected a 24-30% decrease in the molecular weight and a reduction in the number of isoforms of glucose oxidase. No significant changes were observed in the circular dichroism spectra of the deglycosylated enzyme. Other properties, such as thermal stability, pH and temperature optima of glucose oxidase activity and substrate specificity were not affected. However, removal of the carbohydrate moiety marginally affected the kinetics of glucose oxidation and stability at low pH. From these results it appears that the carbohydrate chain of glucose oxidase does not contribute significantly to the structure, stability and activity of glucose oxidase.     

Affinity separation of human plasma gelsolin on Affi-Gel Blue

Human plasma gelsolin was specifically eluted with 1 mM adenosine 5'-triphosphate from an Affi-Gel Blue column. Since the ionic strength of sodium chloride required to elute the protein from the dye column was much higher than that of 1 mM adenosine 5'-triphosphate, the binding of plasma gelsolin with the dye-ligand appeared to be biospecific. Taking advantage of this affinity interaction, we have developed a revised purification method of human plasma gelsolin. The purification included ammonium sulfate precipitation, diethylaminoethyl-Sepharose chromatography, Affi-Gel Blue chromatography, and Phenyl-Sepharose chromatography. The method allowed a reproducible purification of the protein to apparent homogeneity, producing a 331-fold purification with a yield of 6%.     

Purification and characterization of the carboxyl-terminal transactivation domain of Vmw65 from herpes simplex virus type 1.

A glutathione S-transferase fusion to the COOH-terminal acidic transactivation domain of Vmw65 from herpes simplex virus type 1 was overexpressed in Escherichia coli and isolated by affinity chromatography on glutathione-Sepharose. Following cleavage of the fusion protein with thrombin, the transactivation domain was purified to homogeneity by ion exchange chromatography yielding approximately 0.6 mg of protein/liter of bacterial culture. Equilibrium sedimentation analysis showed the purified polypeptide to be monomeric; however, it displayed aberrant electrophoretic and chromatographic properties. Contrary to secondary structure predictions, circular dichroism spectroscopy demonstrated that this transactivation domain was devoid of significant alpha-helical structure at physiological conditions. The polypeptide, however, became notably more structured under hydrophobic conditions or at low pH, suggesting that it was sensitive to its environment. Near-UV circular dichroism suggested that phenylalanyl and tyrosyl residues were under influence from tertiary structure.     

A study of tubulin dimer conformation by near-UV circular dichroism

The near-UV circular dichroism properties of tubulin dimer have been measured for different preparative methods. Tubulin dimer was obtained from assembly compenent microtubule protein by gel filtration, or phosphocellulose ion-exchange chromatography in the presence of magnesium. Tubulin dimer prepared by the protocol of Weisenberg R.C. and Timasheff, S.N. (1970) Biochemistry 9, 4110–4116, was found to be markedly different due to some apparently irreversible change in conformation. We conclude that the removal of microtubule-associated proteins by phosphocellulose ion-exchange chromatography in the presence of magnesium can be performed without affecting the conformation of native tubulin dimer as judged by near-UV circular dichroism.     

Are cardiovascular and sympathoadrenal effects of human "new pressor protein" preparations attributable to human coagulation beta-FXIIa?

"New pressor protein" (NPP) derived from normal human plasma is an extra renal enzyme that shares strong sequence homology with human coagulation beta-FXIIa. Under our bioassay conditions, human NPP (10-20 microl plasma equivalent/ approximately 300 g rat iv) can raise the systolic blood pressure (SBP) by 40-50 mmHg, the diastolic blood pressure (DBP) by 15-20 mmHg, and the heart rate (HR) by 70-90 beats/min. Plasma epinephrine (of adrenal medullary origin) and norepinephrine rise by about 50- and 10-fold, respectively. Because beta-FXIIa is not normally associated with pressor properties, we endeavored to substantiate that the hypertensive effects of impure NPP preparations used in our experiments are attributable to their content of beta-FXIIa. We carried out comparisons with highly purified (>90%) commercial human beta-FXIIa and found that by gel filtration (Sephadex G-100 and G-75), NPP bioactivity appeared in the approximately 30-kDa elution zone, consistent with the molecular mass of beta-FXIIa. Retention time using fast-protein liquid chromatography anion exchange chromatography was identical. Molecular mass and comigration were confirmed by SDS-PAGE gel electrophoresis, and the recovered approximately 30-kDa protein bands yielded beta-FXIIa fragments identified by mass spectrometry. Matched doses of the NPP preparations produced dose-response curves very similar to those elicited by beta-FXIIa with respect to increments of SBP, DBP, and HR, whereas plasma catecholamine increments were generally comparable. We propose that beta-FXIIa is substantially, if not exclusively, responsible for the observed effects of our NPP preparations and that this points to a novel axis connecting the FXII coagulation cascade and the sympathoadrenal gland to other cardiovascular regulatory mechanisms.