Purification and characterization of renin binding protein (RnBP) from porcine kidney

Renin binding protein (RnBP) was purified from porcine kidney using pepstatin affinity column chromatography, DEAE-Sepharose column chromatography, gel filtration on Ultrogel-AcA 34, aminohexyl-Sepharose 4B column chromatography, and high performance liquid chromatography (HPLC) on TSK-gel G-3000 SW. The purified preparation was homogeneous as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, polyacrylamide disc gel electrophoresis and isoelectric focusing on polyacrylamide gel. The isoelectric point was at pH 4.85, and the apparent molecular weight of RnBP was estimated to be 42,000 by SDS-polyacrylamide gel electrophoresis. The preparation did not show any renin activity and was stable for 30 min at 37 degrees C between pH 5.0 and 9.0 or on storage for 4 weeks at 4 degrees C or -80 degrees C. The activity of renin was greatly inhibited by RnBP. From the kinetic analysis of the inhibition we roughly estimated the dissociation constant between renin and RnBP to be about 0.2 nM, assuming that the stoichiometry in the complex, i.e., high molecular weight (HMW) renin, is one to one, and that the complex is inactive. The inhibitory activity of RnBP was lost by acidification at pH 3.0 and the activity of renin was restored. The purified RnBP formed a single precipitin line with the antiserum prepared with the purified HMW renin as antigen, which is RnBP-renin complex (Takahashi, S., et al. (1983) J. Biochem. 93, 265-274), and this line fused with one of the two precipitin lines formed between HMW renin and anti-HMW renin antiserum. The other of the two lines was between renin and anti-HMW renin antiserum. The purified preparation was thus identified as RnBP. The HMW renin was reconstituted with the purified RnBP and renin, and the apparent molecular weight of the reconstituted specimen was estimated to be 60,000 by gel filtration on Ultrogel AcA 44.     

A study on renin binding protein (RnBP) in the human kidney

We purified, from human kidney, a protein that reacts with rabbit anti-porcine kidney renin binding protein (RnBP) antiserum by trapping with porcine kidney renin. The purified preparation showed a single protein peak on gel filtration by high performance liquid chromatography (HPLC) and two protein bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The latter two kinds of protein were identified as the porcine renin and human kidney protein from their electrophoretic mobilities and reactivity toward rabbit anti-porcine kidney renin and RnBP antisera. The molecular weights of the purified preparation and the human kidney protein were estimated to be 56,000 by HPLC and 43,000 by SDS-PAGE, respectively. The specific activity of porcine renin in the purified preparation was 8.6 mg angiotensin I per mg of protein per h at 37 degrees C and pH 6.5. This specific activity was about one-fifth that of free porcine renin. Therefore, it is suggested from the reactivity toward the anti-porcine RnBP antiserum and inhibitory action toward porcine renin that the human kidney protein is RnBP and that the human RnBP is purified as a complex with porcine renin.     

Purification of a high molecular weight somatomedin binding protein from human plasma

The somatomedins insulin-like growth factor I and II (1,2) are in serum bound to high-molecular weight binding proteins (6,7,8). By use of a four step chromatographic procedure a somatomedin binding protein was isolated from outdated human plasma. Exclusion chromatography on Sephadex G-200 disclosed a molecular weight of 150 kDa. After lyophilization however, the binding activity was found in a lower molecular weight range of 35-45 kDa. A partial amino acid sequence analysis of the lyophilized material revealed a possible N-terminal sequence of Ala-Pro-Trp. This sequence is identical to the N-terminal sequence of the 35 kDa somatomedin binding protein previously isolated from human amniotic fluid (16).     

Effects of nucleotides on the interaction of renin with GlcNAc 2-epimerase (renin binding protein, RnBP)

Renin binding protein (RnBP), a cellular renin inhibitor, was identified as an enzyme, GlcNAc 2-epimerase. Recombinant RnBP inhibited porcine renin activity in a dose dependent manner. However, the inhibition was neutralized by nucleotides, such as ATP, dATP, dGTP, dCTP or dTTP. Moreover, ATP inhibited the formation of hetero-complex of renin with RnBP, called high molecular weight (HMW) renin. On the other hand, N-ethylmaleimide (NEM), a SH-alkylating reagent inhibited the GlcNAc 2-epimerase activity concomitant with the decaying of the dimer to the monomer of the enzyme. The inhibition was modulated in the presence of ATP. These results indicate that nucleotides stabilize the dimeric form RnBP (GlcNAc 2-epimerase) and inhibited the formation of the renin-RnBP hetero complex, HMW renin.     

Purification of a high molecular weight actin filament gelation protein from Acanthamoeba that shares antigenic determinants with vertebrate spectrins

I have purified a high molecular weight actin filament gelation protein (GP-260) from Acanthamoeba castellanii, and found by immunological cross-reactivity that it is related to vertebrate spectrins, but not to two other high molecular weight actin-binding proteins, filamin or the microtubule-associated protein, MAP-2. GP-260 was purified by chromatography on DEAE-cellulose, selective precipitation with actin and myosin-II, chromatography on hydroxylapatite in 0.6 M Kl, and selective precipitation at low ionic strength. The yield was 1-2 micrograms/g cells. GP-260 had the same electrophoretic mobility in SDS as the 260,000-mol-wt alpha-chain of spectrin from pig erythrocytes and brain. Electron micrographs of GP-260 shadowed on mica showed slender rod-shaped particles 80-110 nm long. GP-260 raised the low shear apparent viscosity of solutions of Acanthamoeba actin filaments and, at 100 micrograms/ml, formed a gel with a 8 microM actin. Purified antibodies to GP-260 reacted with both 260,000- and 240,000-mol-wt polypeptides in samples of whole ameba proteins separated by gel electrophoresis in SDS, but only the 260,000-mol-wt polypeptide was extracted from the cell with 0.34 M sucrose and purified in this study. These antibodies to GP-260 also reacted with purified spectrin from pig brain and erythrocytes, and antibodies to human erythrocyte spectrin bound to GP-260 and the 240,000-mol-wt polypeptide present in the whole ameba. The antibodies to GP-260 did not bind to chicken gizzard filamin or pig brain MAP-2, but they did react with high molecular weight polypeptides from man, a marsupial, a fish, a clam, a myxomycete, and two other amebas. Fluorescent antibody staining with purified antibodies to GP-260 showed that it is concentrated near the plasma membrane in the ameba.     

Modulation of active renin secretion by renin-binding protein (RnBP) in mouse pituitary AtT-20 cells transfected with human renin and RnBP cDNAs.

To investigate the role of renin-binding protein (RnBP) in renin metabolism, RnBP expression plasmid, which was constructed to express human RnBP under the control of mouse mammary tumor virus long terminal repeat, was transfected into mouse pituitary AtT-20 cells together with the expression plasmid encoding human renin. The transfectant secreted prorenin and active renin, whereas RnBP was expressed only in the presence of dexamethasone and without secretion into the medium. The secretion of active renin was stimulated by forskolin, and the stimulation was repressed by dexamethasone. The secretion of prorenin, however, was insensitive to forskolin irrespective of the presence or absence of dexamethasone. Moreover, the forskolin-stimulated release of active renin was hardly repressed by dexamethasone in AtT-20 cells transfected with the renin expression plasmid and a selectable plasmid pMAMneo. Coexistence of RnBP and renin mRNAs in human Wilms' tumor G-401 cells was shown by means of polymerase chain reaction of respective cDNAs from the cells. These results suggest that RnBP modulates the release of active renin in renin-producing cells.     

Purification and characterization of a low molecular weight zinc binding protein from human placenta

A low molecular weight, native zinc binding, cytosolic protein (LMZP) has been isolated, purified and characterized from human normal term placenta. Gel filtration of heat treated placental cytosol after sequential acetone precipitation (80% ppt) revealed a major zinc binding protein in the range of low molecular weight. This partially purified zinc binding fraction was further fractionated on DEAE-Sephadex A-25. The zinc was eluted in one of the three peak fractions. Further, the purity of zinc binding protein was confirmed on fast protein liquid chromatography (FPLC). The purified placental LMZP was homogenous on SDS-polyacrylamide gel electrophoresis with a single band. Ultraviolet (UV) spectrum of LMZP showed an absorption maximum at 257 nm which disappeared at pH 2. Molecular weight of LMZP as determined by gel chromatography, SDS-polyacrylamide gel electrophoresis and amino acid analysis was 6 kDa. It was calculated that 1 g atom of zinc was bound to 1 mole of the LMZP. Unlike in classical metallothionein, the amino acid composition of placental LMZP revealed the presence of aromatic amino acids, lower content of cysteine and higher content of histidine, glutamic acid and aspartic acid (10, 9 and 5 residues/mole, respectively).     

Epstein-Barr virus induction by a serum factor. Purification of a high molecular weight protein that is responsible for induction

Serum contains a factor that induces Epstein-Barr virus antigens in latently infected human lymphoblastoid cell lines and that cooperates with chemical inducers. Here we report the purification of a protein that is responsible for the effect. Using ammonium sulphate precipitation, chromatography on DEAE-agarose and cellulose, molecular sieve chromatography on Bio-Gel A-5, and glycerol gradient centrifugation, the factor was enriched about 300,000-fold compared to calf serum. Under neutral conditions, the protein chromatographed as a high molecular weight protein of about 5.5 X 10(5) both in its active and inactive form. Both forms of the molecule sedimented at about 7 s. The factor is an acidic protein with a pI of 5. It seems to be composed of high molecular and low molecular weight subunits. Nanogram amounts of purified factor are sufficient for measurable inducing effects.     

Claudin-2 forms homodimers and is a component of a high molecular weight protein complex

Tight junctions are multiprotein complexes that form the fundamental physiologic and anatomic barrier between epithelial and endothelial cells, yet little information is available about their molecular organization. To begin to understand how the transmembrane proteins of the tight junction are organized into multiprotein complexes, we used blue native-PAGE (BN-PAGE) and cross-linking techniques to identify complexes extracted from MDCK II cells and mouse liver. In nonionic detergent extracts from MDCK II cells, the tight junction integral membrane protein claudin-2 was preferentially isolated as a homodimer, whereas claudin-4 was monomeric. Analysis of the interactions between chimeras of claudin-2 and -4 are consistent with the transmembrane domains of claudin-2 being responsible for dimerization, and mutational analysis followed by cross-linking indicated that the second transmembrane domains were arranged in close proximity in homodimers. BN-PAGE of mouse liver membrane identified a relatively discrete high molecular weight complex containing at least claudin-1, claudin-2, and occludin; the difference in the protein complex sizes between cultured cells and tissues may reflect differences in tight junction protein or lipid composition or post-translational modifications. Our results suggest that BN-PAGE may be a useful tool in understanding tight junction structure.     

Purification and characterization of a high molecular weight proteinase (macropain) from human erythrocytes

An alkaline proteinase, previously identified in rat liver and heart, has been purified from the soluble fraction of human erythrocytes. The proteinase has an apparent molecular weight of 600 000 and is composed of eight subunits with molecular weights ranging from 32 000 to 21 000. The proteinase degrades both protein and synthetic peptide substrates with a broad pH optimum of 7.5-11.0. Among the synthetic peptides tested, tripeptides with arginine at the P1 position (e.g. Z-Val-Leu-Arg-4-methoxy-2-napthylamine and Boc-Leu-Gly-Arg-4-methylcoumarin-7-amide) are particularly good substrates. The proteinase appears to be sulfhydryl-dependent and is inhibited completely by mersalyl acid and by hemin; inhibitors of serine and metallo-type proteinases have no effect on proteinase activity. Interestingly, a variety of other proteinase inhibitors such as leupeptin, chymostatin and N-ethylmaleimide failed to completely inhibit protein-hydrolyzing activities of the enzyme. These results indicate that these activities may be accounted for by at least two different catalytic sites. Proteinase activity is stable in the presence of 1 M urea, 0.5% Triton X-100 or 0.03% SDS and is not affected by ATP. Based on the high molecular weight and sulfhydryl-dependence, we have named this proteinase macropain.     

Purification and chemical composition of a low molecular weight follicle-stimulating hormone binding inhibitor from porcine follicular fluid

Porcine follicular fluid contains several factors capable of inhibiting the binding, in vitro, of follicle-stimulating hormone (FSH) to receptor, including an agonist and an antagonist of FSH biological activity in vitro. FSH receptor-binding inhibitory activity (FSH-BI) was determined with assays using radioligand (125iodide-human FSH) receptor (calf-testes membrane); in vitro biological assays (cultured immature rat Sertoli cells) were used to determine antagonist/agonist activity. FSH antagonist activity is due to a low (less than 5000) molecular weight FSH-BI that is soluble in acidic acetone and insoluble in diethyl ether allowing preparative scale isolation. Additional purification was achieved by anion-exchange and reverse-phase high-performance liquid chromatography. Highly purified, biologically active FSH-BI contained the amino acids Ser, Gly, Arg, Thr, Ala, Pro, Val, and Lys; hexoses (phenol-sulfuric acid-positive reaction); and ethanolamine. Thus, this FSH antagonist appears to be a complex glycopeptide--possibly derived from membrane components, as suggested by the presence of ethanolamine and carbohydrate residues. Porcine follicular fluid, therefore, contains a low molecular weight FSH antagonist that, along with the high molecular weight FSH agonist previously identified, may regulate gonadal responsiveness to FSH through interactions with the FSH receptor.     

Purification of normal cellular prion protein from human platelets and the formation of a high molecular weight prion protein complex following platelet activation

A method for the extraction and purification of PrP(C), in its native monomeric form, from outdated human platelet concentrates is described. Both calcium ionophore platelet activation and lysis in Triton X-100 were evaluated as methods for the extraction of soluble platelet PrP(C) in its monomeric form. Following platelet activation, the majority of released PrP(C) was detected as a disulphide linked high molecular weight complex, which under reducing conditions could be separated into what appear to be stable non-disulphide linked PrP dimers or PrP covalently linked to another as yet unidentified protein. This phenomenon appears to be unique to activation since only monomeric PrP(C) was detected following lysis of resting platelets. Subsequently, PrP(C) was purified from the Triton X-100 lysate by sequential cation ion exchange and Cu2+ affinity chromatography. From 10 L of outdated platelet concentrate, we were able to recover 1.29 mg PrP(C) at a purity of 92%.     

New molecular evidence that Teladorsagia circumcincta (Nematoda: Trichostrongylidea) is a species complex

In the polymorphic Teladorsagia circumcincta (morphs circumcincta and trifurcata), a sheep and goat line (SGL) and a goat line (GL) have been previously described on the basis of the malate dehydrogenase allozyme polymorphism (MDH-2) and of the morphology of the dorsal ray. The GL were never found alone in 1 host, so the status of species was not given to these 2 lines. To investigate further whether there are other genetic markers that will delineate them, we collected T. circumcincta worms from goat and sheep at 8 farms in Touraine (west-central France). The worms were identified individually as being SGL or GL on the basis of MDH-2 polymorphism. This distinctiveness was corroborated by sequences of the beta-tubulin isotype I gene, the second internal transcribed spacer (ITS2) of their rDNA, and the nicotinamide dehydrogenase (ND4) gene of their mDNA. The extent of the divergence in the 3 additional genetic markers was such that SGL and GL may be considered as 2 species. A third putative species was found in the SGL line based exclusively on the ND4 gene. These findings suggest that T. circumcincta is a species complex and that further investigation is required on a wider geographic scale.     

Purification of cofilin, a 21,000 molecular weight actin-binding protein, from porcine kidney and identification of the cofilin-binding site in the actin sequence

Cofilin, a 21,000 molecular weight protein originally purified from porcine brain that is capable of binding to actin filaments in a molar ratio of the protein to actin monomer of 1:1 in the filament (Nishida et al. (1984) Biochemistry 23, 5307-5313), was purified from porcine kidney in the present study. The two cofilins from brain and kidney were indistinguishable from each other with respect to the mobility on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the one-dimensional peptide map, and the mode of interaction with actin. Treatment of the actin-cofilin complex with a zero-length cross-linker, 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide (EDC), generated a cross-linked product with an apparent molecular weight of 63,000. Analysis of this product by peptide mapping (Sutoh (1982) Biochemistry 21, 3654-3661) showed that cofilin was cross-linked with the N-terminal segment of actin containing residues 1-12.     

High molecular weight insulin-like growth factor binding protein complex. Purification and properties of the acid-labile subunit from human serum

In the human circulation, the insulin-like growth factors (IGFs) circulate as part of a growth hormone-dependent 125- to 150-kDa complex. This complex has been postulated to contain, in addition to IGFs and one or more IGF-binding proteins, an acid-labile subunit (ALS) which does not itself bind IGFs. In this study, the ALS has been purified 1600-fold from human serum, and its binding properties have been examined. Fresh serum was fractionated on DEAE-Sephadex, and active fractions (determined by radioimmunoassay) were purified by affinity chromatography on an IGF-agarose column saturated with the plasma IGF-binding protein BP-53. After further high performance anion exchange chromatography, an ALS preparation was obtained which contained only an 84-86-kDa protein doublet, converting to a single 70-kDa band on N-glycanase treatment, and having an amino-terminal sequence unrelated to IGF-binding proteins or receptors. Pure ALS formed a complex with BP-53 (Ka approximately 5 x 10(8) M-1), immunoprecipitable by anti-BP-53 antiserum, only in the presence of IGF-I or IGF-II. This complex appeared at approximately 150 kDa on high performance gel chromatography. Pure ALS had no intrinsic IGF-binding activity and no effect on the binding of IGF-I or IGF-II to BP-53. These studies suggest that formation of the high molecular weight IGF-binding protein complex requires ALS, BP-53, and IGF.     

Purification of a high molecular weight membrane protein by fast protein liquid chromatography: the ATPase complex of a thermophilic cyanobacterium

Fast protein liquid chromatography (FPLC) with a strong anion-exchange (Mono Q) column is applied to the purification of a high molecular weight membrane protein. The ATPase complex of the thermophilic cyanobacterium Synechococcus 6716, partially purified by ammonium sulfate precipitation, was fractionated in the presence of the detergent octylglucoside. The ATPase complex containing fractions were eluted by a linear NaCl gradient at about 0.4 M and within 10 min. The FPLC fractions were analyzed for protein and pigment contents and by polypeptide composition. The purest fraction is essentially free of pigments and has a high specific ATP hydrolysis activity (about 1.6 mumol ATP X min-1 X mg protein-1) which is sensitive to N,N'-dicyclohexylcarbodiimide.     

Purification and characterization of an alpha-actinin-like protein from porcine kidney

An alpha-actinin-like protein was partially purified from the Triton-insoluble cytoskeleton of porcine kidney by 0.6 M MgCl2 treatment, ammonium sulfate fractionation, DEAE-cellulose chromatography and hydroxyapatite chromatography. Apparent purity of the kidney protein was approximately 90% by quantitative densitometry of Coomassie-stained polyacrylamide gels. The kidney alpha-actinin-like protein is very similar to muscle alpha-actinins by the following criteria: (1) both kidney protein and muscle alpha-actinins bind to F-actin at a similar ratio; (2) both proteins demonstrate no difference in the actomyosin turbidity assay and the ATPase assay for alpha-actinin activity; (3) both native proteins contain a large core of identical molecular weight resistant to trypsin; (4) on two-dimensional gels, both kidney protein and muscle alpha-actinins have similar isoelectric points of 5.9-6.1. However, kidney alpha-actinin-like protein is not identical in every respect with muscle alpha-actinins. Electrophoretic mobility of the kidney protein is slightly greater than that of chicken gizzard alpha-actinin and is identical to that of a component of chicken skeletal muscle alpha-actinin. One-dimensional peptide mappings of the kidney protein and muscle alpha-actinins were significantly different from each other. The interaction between kidney alpha-actinin-like protein and F-actin is sensitive to Ca2+. Similar Ca2+-sensitivity was observed with other non-muscle cell alpha-actinins.     

A high molecular weight diapause-associated protein from the stem-borer Busseola fusca: Purification and properties

The hemolymph of diapausing larvae of the stem borer, Busseola fusca Fuller (Lepidoptera: Noctuidae), contains an electrophoretically distinct protein band on nondenaturing polyacrylamide gels. The protein, called the Busseola diapause protein (BDP), was purified by a combination of density gradient ultracentrifugation, gel permeation, and affinity chromatography. It is a high molecular weight protein (M_r ～5 × 10⁵; pl = 6.1) that is composed of two subunits, I (M_r ～88,000 ± 4,000) and II (M_r ～79,000 ± 1,000), which are not linked by disulfide bridges. The protein contains both lipids (2%) as well as covalently bound carbohydrates (1%). The inability to stain the fluorescein isothiocyanate-conjugated concanavalin A (FITC-Con A) suggests that the carbohydrate moiety of BDP is not of the high mannose type. Amino acid analysis showed a high tyrosine plus phenylalanine content (16 mol%). Labeling studies using [³⁵S]-methionine showed that de novo synthesis by the fat body tissue occurs only in diapausing larval insects. It is proposed that the BDP could serve a storage function by providing the amino acids needed for the synthesis of pupal and adult structures.