heparin-Sepharose Chromatography Research Articles

The amyloid beta-protein precursor and its mammalian homologues. Evidence for a zinc-modulated heparin-binding superfamily.

The Alzheimer beta-amyloid precursor protein (APP) contains an ectodomain zinc binding site that has been reported to modulate the heparin affinity and protease-inhibitory properties of the molecule. This motif, GVEFVCCP, is highly conserved in amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2), as well as in the Drosophila and Caenorhabditis elegans APP-like proteins (APPL and APL-1). To determine whether the function of this domain is preserved in the human APP-like proteins, the effect of zinc in modulating the elution profile of these proteins upon heparin-Sepharose chromatography was studied. Both APLP1 and APLP2 bound heparin-Sepharose and had NaCl elution profiles similar to that of APP. As previously reported for APP, zinc increased the recovery of APLP1 and APLP2 upon heparin-Sepharose chromatography. APP, APLP1, and APLP2 all bind zinc-chelating Sepharose, indicating that the zinc binding motif may be functionally conserved in these proteins. Additionally, APP, APLP1, and APLP2 migrate at higher molecular sizes (approximately 40 kDa) on SDS-polyacrylamide gel electrophoresis than their predicted molecular sizes. We report data that compare the physicochemical properties of APP to its novel APLP homologues and indicate that these molecules behave as a family of zinc-modulated, heparin-binding proteins.

A novel hepatocytic transcription factor that binds the alpha-fetoprotein promoter-linked coupling element.

We recently characterized a promoter-linked coupling element (PCE) in the rat alpha-fetoprotein (AFP) gene required for strong transcriptional stimulation by distant enhancers (P. Wen, N. Crawford, and J. Locker, Nucleic Acids Res. 21:1911-1918, 1993). In this study, oligonucleotide gel retardation and competition experiments defined the PCE as a 12-bp binding site, TGTCCTTGAACA, an imperfect inverted repeat from -166 to -155 near the AFP promoter. A factor that bound this site (PCF) was abundant in HepG2 nuclear extracts and detectable in extracts from several other AFP-producing hepatocarcinoma cell lines and fetal liver. Hepatocytic cell lines that did not express AFP, nonhepatocytic cell lines, adult liver, and fetal brain did not show the factor. Experiments excluded the possibility that PCF activity was due to binding of glucocorticoid receptor or an AP1-like factor that bound overlapping sites. Competition experiments with several mutant oligonucleotides determined that the optimum PCF binding site was TGTCCTTGAAC(A/T). Mutations decreased binding or totally abolished binding activity. In expression plasmids, PCE mutations strongly reduced gene expression. UV cross-linking to a PCE probe identified peptide bands near 34 kDa. PCF was purified by heparin-Sepharose chromatography followed by affinity binding to oligomerized PCE DNA. The product resolved as a complex of three peptides (PCF alpha 1, PCF alpha 2, and PCF beta, 32 to 34 kDa) on sodium dodecyl sulfate-acrylamide gels. The peptide sizes and gel patterns are unlike those of any of the well-described hepatic transcription factors, and the binding site has not been previously reported. PCF thus appears to be a novel transcription factor.

A novel hepatocytic transcription factor that binds the alpha-fetoprotein promoter-linked coupling element.

We recently characterized a promoter-linked coupling element (PCE) in the rat alpha-fetoprotein (AFP) gene required for strong transcriptional stimulation by distant enhancers (P. Wen, N. Crawford, and J. Locker, Nucleic Acids Res. 21:1911-1918, 1993). In this study, oligonucleotide gel retardation and competition experiments defined the PCE as a 12-bp binding site, TGTCCTTGAACA, an imperfect inverted repeat from -166 to -155 near the AFP promoter. A factor that bound this site (PCF) was abundant in HepG2 nuclear extracts and detectable in extracts from several other AFP-producing hepatocarcinoma cell lines and fetal liver. Hepatocytic cell lines that did not express AFP, nonhepatocytic cell lines, adult liver, and fetal brain did not show the factor. Experiments excluded the possibility that PCF activity was due to binding of glucocorticoid receptor or an AP1-like factor that bound overlapping sites. Competition experiments with several mutant oligonucleotides determined that the optimum PCF binding site was TGTCCTTGAAC(A/T). Mutations decreased binding or totally abolished binding activity. In expression plasmids, PCE mutations strongly reduced gene expression. UV cross-linking to a PCE probe identified peptide bands near 34 kDa. PCF was purified by heparin-Sepharose chromatography followed by affinity binding to oligomerized PCE DNA. The product resolved as a complex of three peptides (PCF alpha 1, PCF alpha 2, and PCF beta, 32 to 34 kDa) on sodium dodecyl sulfate-acrylamide gels. The peptide sizes and gel patterns are unlike those of any of the well-described hepatic transcription factors, and the binding site has not been previously reported. PCF thus appears to be a novel transcription factor.

Interaction of high-molecular-weight basic fibroblast growth factor with endothelium: biological activity and intracellular fate of human recombinant M(r) 24,000 bFGF.

The single-copy gene of human basic fibroblast growth factor (bFGF) encodes four co-expressed isoforms, with an apparent molecular weight (M(r)) of 24 kD, 22.5 kD, 22 kD, and 18 kD, co-translated from a single mRNA. As a tool for the study of the role exerted by the different bFGF isoforms in the biology of endothelial cells, human recombinant 24-kD bFGF was produced and purified from transformed Escherichia coli cells. To this purpose, the novel CUG start codon present in human bFGF cDNA and responsible for the synthesis of 24-kD bFGF was mutagenized to the classic AUG start codon. Transient expression of the mutagenized cDNA in simian COS-1 cells, followed by immunolocalization and subcellular fractionation, resulted in the synthesis of high levels of 24-kD bFGF, which localizes in the cell nucleus as an intact protein. When the same 24-kD bFGF cDNA was expressed in E. coli, the recombinant protein was purified to homogeneity by heparin-Sepharose and ion-exchange chromatography. Recombinant 24-kD bFGF was similar to recombinant 18-kD bFGF in receptor-binding activity and in inducing cell proliferation, plasminogen activator production, and chemotactic movement in cultured endothelial cells. In agreement with the in vitro observations, 24-kD bFGF and 18-kD bFGF exerted a similar angiogenic response when assayed in vivo in the rabbit cornea. Experiments performed with the radiolabeled molecule demonstrated that 24-kD bFGF has an intrinsic ability to bind to high-affinity receptors when added to endothelial GM 7373 cell cultures. Receptor-bound 24-kD bFGF is internalized within the cell and associates with the nucleus with kinetics similar to 18-kD bFGF. Internalized 24-kD bFGF is first processed to the 18-kD form via a chloroquine-insensitive pathway and then to smaller fragments into the lysosomal compartment. At variance with the data obtained in transfected COS-1 cells, only limited amounts of exogenous internalized 24-kD bFGF associates with the nucleus in the intact form, mostly of the nuclear-bound molecule being represented by the processed 18-kD protein and by smaller degradation products. In conclusion, human recombinant 24-kD bFGF exerts a biological response in endothelial cells similar to 18-kD bFGF both in vitro and in vivo. Our data point to a different intracellular behavior of the high-molecular-weight bFGF isoform when added exogenously to cultured cells or when produced endogenously in transfected cells.

Penicillin-binding protein 7/8 of Escherichia coli is a DD-endopeptidase.

Penicillin-binding protein 7 (PBP7) and its proteolytic degradation product PBP8 are shown to be soluble proteins, which can be set free from whole cells of Escherichia coli by an osmotic shock. The proteins are loosely associated with the membranes and are totally released into the supernatant in the presence of 1 M NaCl. Partial purification of PBP8 was accomplished by hydroxyapatite, heparin-Sepharose and MonoS chromatography. Murein meso-diaminopimelate-D-alanine DD-endopeptidase activity was demonstrated for both PBP7 and PBP8, which specifically hydrolyse the DD-diaminopimelate-alanine bonds in high-molecular-mass murein sacculi but fail to cleave these bonds in isolated dimeric muropeptides. The enzyme is inhibited by the 'penem' beta-lactam antibiotic CGP31608 at a concentration of 0.25 micrograms/ml by 50%. Thus besides PBP4 and the mepA gene product, a third endopeptidase exists in E. coli.

Purification and Characterization of Platelet-Activating Factor Acetylhydrolase from Peritoneal Fluid Obtained from Guinea Pigs after Endotoxin Shock1

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) acetyl-hydrolase is an enzyme that hydrolyzes the acetyl ester of PAF. We purified this enzyme, which accumulated in the peritoneal cavity during endotoxin shock, by ammonium sulfate precipitation, and sequential use of butyl-Toyopearl, heparin-Sepharose, Con A-Sepharose, chelating-Sepharose, and MonoQ column chromatographies. We identified a monomeric polypeptide with a molecular weight of approximately 63 kDa on SDS-PAGE. This molecular weight differs from those of previously described acetylhydrolases. The purified enzyme did not degrade phospholipids with a long chain fatty acyl group at the sn-2 position. In addition, the enzyme activity was not inhibited by either pBPB or quinacrine. Accordingly, this enzyme is distinct from phospholipase A2. In addition, this enzyme also hydrolyzed some oxidatively fragmented phospholipids with PAF-like biological activities such as 1-O-hexadecyl-2-glutaroyl-sn-glycero-3-phosphocholine and 1-O-hexadecyl-2-succinoyl-sn-glycero-3-phosphocholine.

Biochemical Characterization of Two Distinct Angiotensin AT2 Receptor Populations in Murine Neuroblastoma N1E‐115 Cells

The murine neuroblastoma N1E-115 cell line possesses a high density of angiotensin II (AngII) receptors that can be solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. These solubilized binding sites exhibited high affinity for CGP-42112A and not Losartan, indicating that they were of the AT2 subtype. However, displacement of 125I-AngII with the AT2 nonpeptide antagonist PD-123319 resulted in a biphasic curve, suggesting heterogeneity of the AT2 receptor population in N1E-115 cells. In support of this view, separation of two receptor populations was accomplished with heparin-Sepharose chromatography. More specifically, three distinct protein peaks eluted from the heparin-Sepharose column, two of which bound 125I-AngII with high affinity and saturability. One of these binding peaks (peak I) eluted rapidly and represented approximately 80% of the total binding activity, whereas the remaining binding activity was contained within a second peak (peak III) that required the addition of 1.5 M NaCl for its complete elution. Pharmacological analysis revealed that both peaks of binding activity were exclusively AT2 receptors insofar as they exhibited high affinity for CGP-42112A and little or no affinity for the AT1-selective antagonist Losartan. However, whereas the nonpeptidic AT2-selective antagonist PD-123319 completely displaced the binding of 125I-AngII from peak I in a monophasic fashion (IC50 = 9.1 +/- 4.1 nM; mean +/- SEM; n = 3), PD-123319 was much less effective in displacing 125I-AngII from peak III (IC50 = 196 +/- 27 nM; mean +/- SEM; n = 3). Treatment of individual peaks with the reducing agent dithiothreitol caused a large increase in 125I-AngII specific binding in peak III, whereas a decrease in binding was observed in peak I. Moreover, GTP gamma S significantly reduced high-affinity agonist binding in peak I but not peak III, further suggesting heterogeneity in the AT2 receptor family. Finally, immunoblotting studies with polyclonal antisera raised against peak I specifically detected two proteins of 110 and 66 kDa, as is true in crude solubilized membranes, whereas no immunospecific proteins were detected in peak III. These same antisera immunoprecipitated 125I-AngII binding activity in peak I but were ineffective in peak III. Collectively, these results suggest that heparin-Sepharose chromatography can efficiently separate two pharmacologically, biochemically and immunologically distinct populations of AT2 receptors.

Binding of lipoprotein lipase to alpha 2-macroglobulin.

The interaction between bovine lipoprotein lipase (bLPL) and human alpha 2-macroglobulin (alpha 2M) was studied by use of non-denaturing PAGE and gel-permeation, Zn(2+)-Sepharose and heparin-Sepharose chromatography. It was demonstrated that bLPL in vitro binds non-covalently to native alpha 2M, but not to the receptor-recognized form produced by treatment of alpha 2M with chymotrypsin or methylamine. A small amount of bLPL was bound covalently to alpha 2M by disulphide interchange, when incubated together with chymotrypsin or methylamine. Whereas alpha 2M in complex with bLPL still bound to Zn(2+)-Sepharose, bLPL lost the ability to bind to heparin-Sepharose. Preincubation of bLPL with heparin prevented complex-formation with alpha 2M, suggesting that alpha 2M interacts with the heparin-binding domain of bLPL. Experiments in which 125I-bLPL was incubated with human plasma at 20 degrees C demonstrated an 11-17% binding of the labelled lipase to alpha 2M, indicating that this interaction may be of physiological significance.

Nongranular proteolytic enzymes of rat IL‐2–activated natural killer cells. II. Purification and identification of rat A‐NKP 1 and A‐NKP 2 as constituents of the multicatalytic proteinase (proteasome) complex

We have recently described nongranular, cytosolic, high-molecular-weight trypsin-like (A-NKP 1) and chymotrypsin-like (A-NKP 2) proteases of interleukin-2-activated rat natural killer (A-NK) cells. A functional correlation between the inactivation of A-NKP 2 and the inhibition of rat A-NK cell-mediated cytotoxicity was found. Herein we describe the 6,000-fold purification of A-NKP 2 to apparent homogeneity following: isopycnic sucrose gradient fractionation of postnuclear supernatants, molecular sieve chromatography, and heparin-Sepharose chromatography. We also report the novel finding that A-NKP 2 as well as A-NKP 1, derived from either rat A-NK cells or the rat NK leukemic cell line CRNK-16, are constituents of the multicatalytic proteinase (MCP/proteasome) complexes of these cells. Characteristic biochemical, biophysical, and electron microscopic/ultrastructural similarity to the rat liver proteasome was observed. However, Western blot analysis using polyclonal antibodies to the rat liver proteasome clearly indicated differences in the rat hepatic proteasome and the CRNK-16-derived proteasomal subunits. The identification, characterization, and purification of A-NKP 1 and A-NKP 2, described herein, now allow for further investigation of the potential role of these proteasome components in NK cell function. Moreover, the proteasome of NK and A-NK cells can now be compared and contrasted to the granzymes of lytic granules with respect to their role in cell-mediated cytotoxicity.

Micromolar calcium decreases affinity of inositol trisphosphate receptor in vascular smooth muscle.

The mechanism by which Ca2+ inhibits InsP3-induced Ca2+ release from sarcoplasmic reticulum of vascular smooth muscle was investigated. InsP3 binding to sarcoplasmic-reticulum vesicles from dog aortic smooth muscle was inhibited by 51 +/- 6% by 2 microM Ca2+ in the presence of 10 nM [3H]InsP3. Scatchard analysis indicated the presence of two InsP3-binding sites in the absence of Ca2+ (Kd = 2.5 +/- 0.9 and 49 +/- 8 nM InsP3), though the low-affinity site was more prevalent (representing 92 +/- 3% of the total number of binding sites). Ca2+ (2 microM) did not alter InsP3 binding to the high-affinity site (P > 0.05), but increased the Kd of the low-affinity site 3-fold (Kd = 155 +/- 4 nM InsP3; P < 0.001). The possibility that the apparent decrease in InsP3 affinity was caused by Ca(2+)-dependent activation of an endogenous phospholipase C could be excluded, because the Ca(2+)-dependent inhibition of InsP3 binding was completely reversible and insensitive to an inhibitor of phospholipase C. Moreover, Ca2+ did not inhibit InsP3 binding to InsP3 receptor partially purified by heparin-Sepharose chromatography, though another fraction (devoid of InsP3 receptor) restored Ca(2+)-sensitivity of the partially purified InsP3 receptor. Thus Ca2+ binding to a Ca(2+)-sensitizing factor associated with the InsP3 receptor decreases the affinity of the receptor complex for InsP3. This Ca(2+)-sensitizing factor may provide a negative-feedback mechanism for regulating the rise in cytosolic Ca2+ concentration in vascular smooth muscle after hormone activation of the phosphoinositide cascade.

Basic fibroblast growth factor and its receptor messenger ribonucleic acids are expressed in human ovarian epithelial neoplasms

Our purpose was to determine whether basic fibroblast growth factor is present in, and synthesized by, human ovarian epithelial neoplasms and to evaluate the expression of gene for the basic fibroblast growth factor receptor. The synthesis of basic fibroblast growth factor and its receptor was investigated in seven primary human ovarian epithelial neoplasms. Neoplastic tissues were homogenized and the cytoplasmic extracts purified by heparin-sepharose chromatography with a linear salt gradient of 0.6 to 3 mol/L sodium chloride in Tris-hydrochloric acid. The in situ synthesis of basic fibroblast growth factor and its receptor was demonstrated by polymerase chain reaction. Total ribonucleic acid was reverse transcribed and then amplified with two oligonucleotide primers specific for the bovine and human basic fibroblast growth factor gene and its human receptor gene. As assessed by both bioassay and radioimmunoassay a peak of basic fibroblast growth factor-like activity was present in all tumors in the chromatographic fractions eluted with 3 mol/L sodium chloride. The mitogenic effect on bovine adrenocortical endothelial cell proliferation varied from 35% to 153% above control cultures. Levels of basic fibroblast growth factor-like immunoreactivity were between 4 and 33 ng/ml. Qualitatively similar results were obtained after purifying the cytoplasmic extract of dispersed human ovarian tumor cells. The mitogenic effect was completely abolished by a specific neutralizing anti-basic fibroblast growth factor antibody. Single major deoxyribonucleic acid bands of the expected size (354 and 661 bp) were detected in all tumors studied. The identify of this material with the human basic fibroblast growth factor sequence was confirmed by restriction enzyme analysis. These data demonstrate that both basic fibroblast growth factor and its receptor are present in and synthesized by human ovarian tumor cells. Thus basic fibroblast growth factor might stimulate their abnormal proliferation through an autocrine mechanism.

SbvI restriction endonuclease from Streptococcus bovis

Restriction endonuclease SbvI, an isoschizomer of HaeIII, has been isolated from rumen amylolytic bacterium Streptococcus bovis II/1. SbvI was purified from cell extract by phosphocellulose chromatography and heparin-Sepharose chromatography. The recognition sequence of SbvI was identified by digestion of pBR322, pUC9 and lambda-DNA and comparing the cleavage patterns obtained with computer-derived data. SbvI recognizes the 4-bp palindrome, 5'-GGCC-3' and cleaves DNA after the second G in the sequence, producing blunt ends.

The identification of gonadotrophin surge inhibiting factor and its role in the regulation of pituitary gonadotrophin secretion

Evidence from several laboratories suggests that the ovaries of rats, pigs, monkeys and women produce a non-steroidal factor which inhibits or attenuates the mid-cycle gonadotrophin surge. This substance is present in follicular fluid and we have called it gonadotrophin surge inhibiting factor (GnSIF). Utilizing a rat pituitary cell bioassay, we have monitored GnSIF activity during purification from porcine follicular fluid (PFF). Rat pituitary cells in short-term culture were incubated with GnSIF for 48 h, washed and then exposed to gonadotrophin releasing hormone (GnRH) plus GnSIF for 4 h. GnSIF activity was defined as suppression of GnRH-stimulated LH secretion over 4 h, and inhibin activity was expressed as suppression of basal follicle stimulating hormone (FSH) secretion over 48 h. Approximately 5 l of PFF was fractionated through heparin-Sepharose, Q-Sepharose, Mono-S, hydroxylapatite, and gel permeation chromatography steps. This fractionation removes all detectable inhibin and follistatin immunoactivity and bioactivity. The purified GnSIF inhibits GnRH-stimulated LH secretion with little or no effect on basal FSH release. In summary, we have obtained a purified preparation of GnSIF which contains undetectable inhibin and follistatin. As such, GnSIF appears to be distinct from other known gonadal proteins regulating reproductive function, and may participate along with the inhibins, activins and follistatins in the gonadal regulation of pituitary gonadotrophin secretion.

The ovarian modulation of gonadotrophin releasing hormone-induced luteinizing hormone secretion in women.

Ovine pituitary cell culture and perfusion bioassays were used to investigate gonadotrophin surge-attenuating factor (GnSAF) bioactivity in vitro. GnSAF is present in steroid-free human follicular fluid (HFF) from superovulated women and specifically attenuates gonadotrophin releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion. HFF produced dose-dependent suppression of GnRH-induced LH and basal FSH secretion, the former reflecting GnSAF bioactivity, the latter inhibin bioactivity. Heparin-Sepharose chromatography of HFF removed inhibin bioactivity, but not GnSAF bioactivity, indicating that these are distinct entities. Ultrafiltration of HFF suggests that GnSAF has a bioactive form of 10-30 kDa. Ovine pituitary perfusion demonstrated that GnSAF bioactivity in HFF is matched in the serum of superovulated and pregnant women. In addition, HFF and serum suppress both the initial and reserve pools of GnRH-induced LH secretion and greatly reduce the self-priming effect of GnRH in ovine pituitary tissue perfusion. In-vitro and in-vivo GnSAF bioactivity is consistent with a role for GnSAF in the gonadal feedback regulation of pituitary response to GnRH.

Conditioned Media from a Renal Cell Carcinoma Cell Line Demonstrates the Presence of Basic Fibroblast Growth Factor

In a previous report, we demonstrated the isolation and purification of a heparin binding growth factor from human renal carcinoma, and suggested that this growth factor may play a role in the neovascularity and growth of the tumor.In this report, we demonstrate that the growth of the renal cell carcinoma cell line RC29 is stimulated by the addition of exogenous fibroblast growth factor (FGF), epidermal growth factor (EGF) and transforming growth factor alpha (TGFα). Also, media conditioned by this cell line was able to stimulate growth of the A431 vulvar tumor cell line, known for its high concentration of EGF receptors, 3T3 fibroblasts, human umbilical vein (HUV) cells and RC29 cells.Using heparin-sepharose chromatography and then SDS polyacrylamide gel electrophoresis (PAGE), we were able to demonstrate several proteins in the conditioned media of the RC29 cell line. Using Western blot analysis, we detected that at least one of the proteins expressed in this conditioned media was FGF and that it belongs to the basic, not acidic, family of fibroblast growth factors. These findings suggest that renal tumors may express growth factors that may play a direct role in maintaining their unrestricted proliferation.

Development of polyclonal antibodies against angiotensin type 2 receptors.

Murine neuroblastoma N1E-115 cells are a useful system in which to study neuronal angiotensin II (AngII) receptors. N1E-115 cells possess both type 1 (AT1) and type 2 (AT2) AngII receptor subtypes, as does mammalian brain. AT2 receptors in brain or N1E-115 cells can be solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. In the present study, heparin-Sepharose chromatography was used to partially purify solubilized N1E-115 membranes to produce an enriched population of AT2 receptors. Subsequently, an eluted peak, containing the majority of AT2 binding activity, was used as an immunogen in the development of protein-directed polyclonal antibodies. The antibodies specifically detected immunoreactive proteins of approximately 110 and 66 kDa in both solubilized N1E-115 cells, as well as the original protein material that eluted from the heparin-Sepharose column, whereas no such immunoreactivity was detected in a kidney epithelial cell line that lacks any specific 125I-labeled AngII (125I-AngII) binding activity. Moreover, the antibodies immunoreacted with affinity-purified AT2 receptors. These antibodies were also able to immunoprecipitate AT2 receptors from solubilized N1E-115 cells, as revealed by the pharmacologic profile of 125I-AngII binding to the precipitated protein. Similarly, the antibodies were able to immunoprecipitate a 66-kDa protein that had been covalently crosslinked with 125I-AngII by use of the homobifunctional crosslinker dithiobis(succinimidyl propionate). Collectively, these results demonstrate the development of a specific AT2 receptor antibody that may be used to further characterize this receptor subtype at both the cellular and molecular levels.

Human very low density lipoprotein structure: interaction of the C apolipoproteins with apolipoprotein B-100

Very low density lipoproteins (VLDL) are a heterogenous population of particles differing in size and composition. Heparin-Sepharose chromatography yields three VLDL subfractions. Two subfractions, VLDLNR-1 and VLDLNR-2, which are not retained by heparin, contain little or no detectable apolipoprotein (apo)E. According to negative stain electron microscopy, VLDLNR-1 is slightly larger than VLDLNR-2. The third fraction, VLDLR, is composed of smaller particles that are retained by the heparin-Sepharose and contain apoE. The C apolipoproteins of the respective VLDL subfractions transfer to 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) single bilayer vesicles giving three subfractions designated VLDLNR-1-C, VLDLNR-2-C, and VLDLR-C. The protein, phospholipid, and cholesterol (free + esterified) contents decrease in the order VLDLR > VLDLNR-2 > VLDLNR-1. Triglyceride content decreases in the opposite order. POPC treatment of each VLDL subfraction increases the phospholipid and decreases the protein, triglyceride, and cholesteryl ester contents, while free cholesterol remains unchanged. According to immunological analysis of each subfraction with well-characterized monoclonal antibodies, the accessibility of some epitopes of apoB-100 on VLDL is changed by POPC treatment. Electron-microscopic analysis of POPC-treated VLDL subfraction reveals vacancies on the surfaces of each particle. VLDLNR-1, VLDLNR-2, and VLDLR are resistant to thrombin cleavage, whereas the lipoproteins lacking C apolipoproteins are not. Thrombin cleavage (8 h) of apoB-100 of VLDLNR-2-C and VLDLR-C gives two fragments, T1 and T2, that are converted to smaller fragments only after prolonged treatment. In contrast, apoB-100 of VLDLNR-1-C is converted into small fragments after 8 h thrombin treatment. These results suggest that removal of apoCs affects the accessibility and conformation of apoB-100 in the individual VLDL subfractions in the region near residue 3249, which is the primary thrombin cleavage site and the epitope of monoclonal antibody 4C11.

The 110-kDa polypeptide of spinach plastid DNA-dependent RNA polymerase: single-subunit enzyme or catalytic core of multimeric enzyme complexes?

Highly purified RNA polymerase preparations from spinach chloroplasts contain seven major polypeptides of 150, 145, 110, 102, 80, 75, and 38 kDa. I find that RNA polymerase activity can be separated under defined conditions into three different fractions by heparin-Sepharose chromatography. Immunological analysis has shown that the first fraction contains RNA polymerase activity associated with all seven major polypeptides, and other studies have shown that some of these polypeptides (150, 145, 80, and 38 kDa) are associated with an RNA polymerase similar to the Escherichia coli enzyme. However, similar analyses of the remaining fractions show activity associated only with the 110-kDa polypeptide, suggesting the existence of a second kind of chloroplast RNA polymerase. Samples of this 110-kDa polypeptide purified by SDS/PAGE actively synthesize RNA in a reaction dependent on a supercoiled DNA template and the four ribonucleoside triphosphates. Hence, this polypeptide has all of the properties expected of a single-subunit RNA polymerase of the T7 bacteriophage type.

Isolation and characterization of the chains of type V/type XI collagen present in bovine vitreous

Previous studies show that the collagen fibrils of the mammalian vitreous humor are assembled largely from type II collagen with smaller amounts of type IX collagen and either type V or type XI collagen. In this paper, we report the separation of two chains of type V/type XI collagen from type II collagen by heparin-Sepharose chromatography. These chains were characterized by sequencing of selected cyanogen bromide or tryptic peptides with subsequent comparison of these sequences with cDNA-derived amino acid sequences of the alpha 1(V), alpha 1(XI), alpha 2(V), and alpha 2(XI) chains. The results show that vitreous fibrils are assembled from molecules containing the alpha 1(XI) and alpha 2(V) chains. These results, together with recent results from other laboratories, indicate that type V and type XI collagens are not separate collagen types but are part of a larger collagen family in which chains of both type V and type XI collagens participate in the formation of a variety of native molecules.

Different hydrolytic efficiencies of adipose tissue lipoprotein lipase on very-low-density lipoprotein subfractions separated by heparin-Sepharose chromatography

Human very-low-density lipoproteins (VLDL) were subfractionated by heparin-Sepharose chromatography into an unbound (A) and three bound (B, C and D) populations at increasing ionic strengths. Subfractions were characterized regarding their chemical composition and efficiency of triacylglycerol hydrolysis by rat adipose tissue LPL. The triacylglycerol content decreased, whereas the cholesterol and protein contents increased from subfractions A and B to subfraction D. VLDL-D showed the highest apo E/apo C ratio, though all the subfractions contained appreciable apo E. Appearance of VLDL-A resulted from exceeding the binding capacity of the column, since practically all its particles eluted at positions of bound VLDL under re-chromatography. Subfractions B, C and D stimulated LPL activity on emulsified tri[ 14C]oleoylglycerol to a similar extent, indicating that their apo C-II content was equally effective activating LPL. Incubation of tri[ 14C]oleoylglycerol labeled VLDL subfractions with fat pad pieces in the presence or absence of heparin resulted in greater hydrolysis and fatty acid uptake for VLDL-B and -C than for VLDL-D, a pattern observed over a wide range of LPL activities in the media. We conclude: (1) any VLDL particle can interact with heparin, which is consistent with the presence of apo E in all the subfractions, and (2) triacylglycerols in apo E-rich VLDL are less efficiently hydrolyzed by LPL than those in apo E-poor particles. We propose that richness in apo E impairs LPL action upon VLDL and decreases the rate of delivery of fatty acids to peripheral tissues.