Post-translational processing of membrane-associated recombinant human stem cell factor expressed in Chinese hamster ovary cells.

This report describes the structure of soluble human stem cell factor isolated from the conditioned medium of Chinese hamster ovary (CHO) cells transfected with stem cell factor (SCF) cDNA, which encodes a leader sequence plus 248 additional amino acids. The 248 amino acids include a hydrophobic transmembrane region at positions 190-212. The isolated material is glycosylated and three bands (apparent M(r) 28,000, M(r) 35,000, and M(r) 40,000) are evident by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. After complete deglycosylation, the molecular weight by SDS-polyacrylamide gel electrophoresis is 18,000-19,000. Structural analyses of the intact SCF, the deglycosylated SCF, and a deglycosylated C-terminal peptide were performed by laser desorption, fast atom bombardment, or electrospray mass spectrometry. Pulse-labeling of cells with 35S-labeled Met and Cys resulted in cell-associated glycosylated SCF of M(r) 33,000-45,000 which was converted to M(r) 33,000 by in vitro treatment with glycosidases. During a chase with unlabeled Met and Cys, labeled SCF of M(r) 28,000, M(r) 35,000, and M(r) 40,000 appeared in the medium; it was converted to M(r) 18,000-19,000 by glycosidase treatment. SCF at the surface of the transfected CHO cells could be demonstrated by immunofluorescence. The data obtained indicate that the recombinant human stem cell factor, as isolated, represents proteolytically processed forms containing amino acids 1-165, derived from the initially synthesized membrane-bound form of 248 amino acids. Further characterization indicated that the M(r) 28,000 form is glycosylated at Asn120, the M(r) 35,000 form at Asn120 and Asn65, and the M(r) 40,000 form at Asn120, Asn93, and Asn65. Each form also contains O-linked carbohydrate. The N-linked glycosylation, particularly that at Asn93 and at Asn65, adversely affects in vitro biological activity and receptor binding.     

Purification and properties of the aromatic amino acid aminotransferase from gramicidin S-producing Bacillus brevis

The aromatic amino acid aminotransferase was purified to a homogenous state from a gramicidin S-producing strain of Bacillus brevis. The enzyme shows a molecular weight of about 71,000 on gel-filtration. The subunit molecular weight is about 35,000 as determined by sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is a dimer. The enzyme exhibits absorption maxima near 425 and 330 nm at neutral pH. One mole of pyridoxal phosphate is bound per subunit. The enzyme has amino donor specificity for aromatic amino acids, L-phenylalanine, L-tyrosine, and L-tryptophan, and utilizes 2-oxoglutarate as the amino acceptor. This enzyme activity was separated from both the aspartate aminotransferase activity and the branched chain amino acid aminotransferase activity by chromatography on DEAE-Sephadex.     

Some properties of human epidermal growth factor (hEGF)-like immunoreactive material originating from platelets during blood coagulation

During blood coagulation, a considerable amount of human epidermal growth factor (hEGF)-like immunoreactive material (designated as platelet hEGF-LI) was liberated from platelets. The molecular nature of the platelet hEGF-LI was examined. The molecular weight of platelet hEGF-LI estimated by gel filtration was approximately 60,000-70,000. On chromatofocusing chromatography, platelet hEGF-LI was eluted mainly at pH 4.75 as a sharp peak with a minor peak at 4.30, like urine EGF. By treatment with 2-mercaptoethanol, the factor was converted into a material with molecular weight of 35,000-40,000. These results suggest that the majority of hEGF-LI in platelets may exist either in a covalently bound-form with some protein(s) in platelets or as a dimer intermolecularly cross-linked by an S-S linkage. Details of the biological properties and the physiological significance of the platelet hEGF-LI remain to be clarified.     

Subunit structure of Achromobacter collagenase.

The highly active form of collagenase (EC 3.4.24.3) from Achromobacter iophagus (specific activity 2 microkat/mg) has a molecular weight of 70,000 and the sedimentation coefficient s20,2 = 4.4 S. It is composed of two subunits of molecular weight 35,000 and s20,w of 2.9 S. The dissociation of the dimer under different conditions resulted in the complete and irreversible loss of enzymic activity. A unique N-terminal sequence Thr-Ala-Ala-Asp-Leu-Glu-Ala-Leu-Val- indicates that the two subunits are identical, at least in the N-terminal part of the polypeptide chain. Reduction and pyridylethylation of the subunit change neither molecular weight nor amino acid composition: therefore each subunit of molecular weight 35,000 consists of a single polypeptide chain. Another active and homogeneous form of Achromobacter collagenase (specific activity 1.64 microkat/mg) gives a value for the apparent molecular weight of 80,000 on sodium dodecyl sulphate-polyacrylamide electrophoresis. It is also a dimer in which each of the two subunits of molecular weight 35,000 binds non-covalently a peptide of molecular weight 5000. The dissociation of this form of collagenase is also accompanied by irreversible loss of enzymic activity. The amino acid composition of the subunits which were isolated from both 70,000 and 80,000 collagenases is the same. The role of dimer-monometer equilibrium in the biological function of collagenase is discussed.     

A new activator protein that activates tryptophan 5-monooxygenase and tyrosine 3-monooxygenase in the presence of Ca2+-, calmodulin-dependent protein kinase. Purification and characterization

Rat brain tryptophan 5-monooxygenase was activated by incubation with ATP, Mg2+, calmodulin, and micromolar concentrations of Ca2+. The activating activity was resolved into two distinct peaks upon gel filtration on Sepharose CL-6B: one, Ca2+-, calmodulin-dependent protein kinase, and the other, a heat-labile activator protein. The activator protein was purified to apparent homogeneity from rat brain by a procedure involving calmodulin-Sepharose 4B, Sephadex G-150, and phenyl-Sepharose CL-4B column chromatography. The molecular weight of the activator protein was determined to be 70,000 by sedimentation equilibrium and by gel filtration on Sephadex G-150. The protein gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of which was estimated to be 35,000, indicating that the protein might be composed of two identical subunits. Analysis of cross-linked activator protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis also suggested that the protein might be a dimer of identical subunits. Some other molecular properties of the activator protein were: sedimentation coefficient, 4.3 S; Stokes radius, 3.6 nm; diffusion coefficient, 6.0 x 10(-7) cm2/s; frictional ratio, 1.32; and partial specific volume, 0.73 cm3/g. The activator protein activated tyrosine 5-monooxygenase as well as tryptophan 5-monooxygenase in the presence of ATP, Mg2+, Ca2+, calmodulin, and Ca2+-, calmodulin-dependent protein kinase.     

The structure and function of acid proteases. VIII. Purification and characterization of cathepsins D from Japanese monkey lung.

Two kinds of cathepsin D were found in Japanese monkey lung and were named cathepsins D-I and D-II. Cathepsin D-I was partially purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography. It had properties common to other ordinary cathepsins D in terms of the elution position from a DEAE-cellulose column at pH 8.0, the pH-dependence of activity toward acid-denatured hemoglobin, and the molecular weight of 35,000 as determined by Sephadex G-100 gel filtration. On the other hand, cathepsin D-II was purified about 1,000-fold by a combination of ammonium sulfate fractionation and column chromatographies on DEAE-cellulose and Sephadex G-100. It was a very acidic protein as judged from its elution position from a DEAE-cellulose column at pH 8.0, and the high mobility toward the anode on disc gel electrophoresis at pH 8.6. Its molecular weight was determined to be 35,000 by Sephadex G-100 gel filtration and 39,000 by SDS-polyacrylamide gel electrophoresis. It was optimally active at pH 2.8 against acid-denatured hemoglobin as a substrate, showing 80% of the optimal activity at pH 1.0, and almost no activity above pH 4.0. This pH-profile of activity was similar to that of monkey pepsin C (gastricsin). It did not hydrolyze N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine, a synthetic substrate for pepsin, but was inhibited by a series of pepsin inhibitors such as pepstatin, 1,2-epoxy-3-(p-nitrophenoxy)propane, p-bromophenacyl bromide, and diazoacetyl-DL-norleucine methyl ester, although the diazo reagent was a rather weak inhibitor of the enzyme. The amino acid composition of cathepsin D-II was found to be fairly different from those of other cathepsins D. However, it showed a striking resemblance to that of Japanese monkey pepsinogen C, suggesting some evolutionary relationship between them.     

Dimeric nature of apo-low density lipoprotein extracted with Triton X-100.

Human low density lipoprotein (LDL) was dissolved in 0.3 to 2.0% Triton X-100 at pH 7.5 and apo-LDL (B protein) was extracted from LDL to form B protein-Triton complex. Sedimentation equilibrium study of this complex in a solvent nearly isopycnic to Triton X-100 showed that the molecular weight of the protein in the complex was 570,000. The complex eluted almost at the void volume of a Sepharose 6B column, as would be expected for a complex with a total molecular weight of roughly 900,000, on the assumption that 0.52 g of Triton was bound to 1 g of protein (Helenius, A. and Simons, K. (1972) J. Biol. Chem. 247, 3656-3661). The sedimentation coefficient of the complex gave f/fmin = 2.2, indicating that the complex was either as asymmetric as a fibrinogen molecule or not compact. These results show that B protein exists in its complex with Triton X-100 as an elongated or a loosely expanded dimer based on the molecular weight of monomeric B protein of 270,000. B protein may also exist in LDL as a dimer.     

Isolation and characterization of a high molecular weight protein phosphatase from rabbit skeletal muscle

A high molecular weight protein phosphatase (phosphatase H-II) was isolated from rabbit skeletal muscle. The enzyme had a Mr = 260,000 as determined by gel filtration and possessed two types of subunit, of Mr = 70,000 and 35,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On ethanol treatment, the enzyme was dissociated to an active species of Mr = 35,000. The purified phosphatase dephosphorylated lysine-rich histone, phosphorylase a, glycogen synthase, and phosphorylase kinase. It dephosphorylated both the alpha- and beta-subunit phosphates of phosphorylase kinase, with a preference for the dephosphorylation of the alpha-subunit phosphate over the beta-subunit phosphate of phosphorylase kinase. The enzyme also dephosphorylated p-nitrophenyl phosphate at alkaline pH. Phosphatase H-II is distinct from the major phosphorylase phosphatase activities in the muscle extracts. Its enzymatic properties closely resemble that of a Mr = 33,500 protein phosphatase (protein phosphatase C-II) isolated from the same tissue. However, despite their similarity of enzymatic properties, the Mr = 35,000 subunit of phosphatase H-II is physically different from phosphatase C-II as revealed by their different sizes on sodium dodecyl sulfate-gel electrophoresis. On trypsin treatment of the enzyme, this subunit is converted to a form which is a similar size to phosphatase C-II.     

Purification and characterization of alpha-N-acetylgalactosaminidase from skipjack liver

By means of a simple procedure involving two gel filtrations and an ion-exchange chromatography, alpha-N-acetylgalactosaminidase was purified to an electrophoretically homogeneous form from skipjack liver, in which the enzyme is the dominant glycosidase. The final alpha-N-acetylgalactosaminidase preparation contained practically no other glycosidase activities except alpha-galactosidase activity, which amounted to 0.8% of the alpha-N-acetylgalactosaminidase activity and may be an intrinsic activity of the enzyme. The molecular weight of the enzyme was estimated to be 80,000 at pH 4.2 and 40,000 at pH 7.2 by molecular sieve chromatography, and to be 35,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 4 and was inactive above pH 7. These results suggest that skipjack alpha-N-acetylgalactosaminidase exists as an active dimer at acidic pH and as inactive monomer at neutral or alkaline pH. The enzyme efficiently liberated the N-acetylgalactosamine unit from ovine submaxillary glycoprotein which had been desialylated by neuraminidase. The Km value and maximum velocity were 4.28 mM and 409 mumol/min X mg for p-nitrophenyl alpha-N-acetylgalactosaminide, and 0.0543 mM and 1.19 mumol/min X mg for ovine submaxillary asialoglycoprotein.     

Purification and characterization of a pectin-releasing enzyme produced by Kluyveromyces wickerhamii

A pectin-releasing enzyme produced by Kluyveromyces wickerhamii IFO 1675 (PPase-W) was purified to homogeneity from a culture filtrate by cation-exchange and size-exclusion chromatographies. This enzyme had a molecular weight of 35,000 determined by both size exclusion chromatography and ultracentrifugal analysis, and of 40,000 by SDS-PAGE. It contained 2.4% sugar, and its isoelectric point was at pH 5.2. PPase-W catalyzed the release of highly polymerized pectin from various protopectins, and also showed endopolygalacturonase (endo-PGase) activity. The purified enzyme had optimum PGase activity at about pH 5.2 and 50°C and was stable in the range of pH from 4.0 to 7.0 and up to 50°C. The properties of PPase-W were compared with those of PPase-F from Kluyveromyces fragilis IFO 0288, and some differences were found. Also, some preliminary data dealing with the relationship between enzyme activities (PPase and endo-PGase) and protein structure are discussed.     

Purification, characterization, and immunological properties of fumarase from Euglena gracilis var. bacillaris.

A rapid three-step procedure utilizing heat treatment, ammonium sulfate fractionation, and affinity chromatography on Matrex gel Orange A purified fumarase (EC 4.2.1.2) 632-fold with an 18% yield from crude extracts of Euglena gracilis var. bacillaris. The apparent molecular weight of the native enzyme was 120,000 as determined by gel filtration on Sephacryl S-300. The preparation was over 95% pure, and the subunit molecular weight was 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme is a dimer composed of two identical subunits. The pH optimum for E. gracilis fumarase was 8.4. The Km values for malate and fumarate were 1.4 and 0.031 mM, respectively. Preparative two-dimensional gel electrophoresis was used to further purify the enzyme for antibody production. On Ouchterlony double-immunodiffusion gels, the antifumarase serum gave a sharp precipitin line against total E. gracilis protein and purified E. gracilis fumarase. It did not cross-react with purified pig heart fumarase. On immunoblots of purified E. gracilis fumarase and crude cell extracts of E. gracilis, the antibody recognized a single polypeptide with a molecular weight of approximately 60,000, indicating that the antibody is monospecific. This polypeptide was found in E. gracilis mitochondria. The antibody cross-reacted with an Escherichia coli protein whose molecular weight was approximately 60,000, the reported molecular weight of the fumA gene product of E. coli, but it failed to cross-react with proteins found in crude mouse cell extracts, Bacillus subtilis extracts, or purified pig heart fumarase.     

The HIV-1 gp120 envelope protein has the intrinsic capacity to stimulate monokine secretion

Results and conclusions concerning the ability of HIV glycoprotein (gp) 120 to stimulate monokine secretion have been equivocal, based on observations using natural gp120 derived from infected human cells and a Chinese hamster ovary (CHO) cell-derived recombinant fusion protein. Current studies were designed to determine whether differences in recombinant gp120 proteins could result in failure to trigger monokine production. We found that natural gp120 could stimulate monocytes to release TNF-alpha, IL-1 beta, IL-6, and granulocyte-macrophage-CSF, and this effect could be blocked with soluble CD4. Full-length rgp120 either expressed from an adenovirus vector and purified from infected human cells, or derived from CHO cells, could function similarly. In contrast, full-length recombinant envelope protein expressed in a baculovirus system and a CHO cell-derived recombinant fusion protein tested previously, consistently failed to stimulate monokine production. The stimulatory capacity of both natural and full-length CHO cell-derived gp120 was eliminated by heating at 100 degrees C, and could be blocked with excess CHO cell-derived gp120 fusion protein. Inasmuch as the baculovirus-expressed gp120 and the CHO cell-derived recombinant fusion protein can bind to CD4, these results suggest that HIV gp120 binding to CD4 on the monocyte surface may of itself be insufficient for stimulation of monokine secretion. Therefore, primary protein structure, as well as posttranslational protein modifications, may determine this activity.     

Human interferon omega 1: isolation of the gene, expression in Chinese hamster ovary cells and characterization of the recombinant protein.

A gene encoding human interferon omega-1 (IFN-omega 1) was isolated from a cosmid library, sequenced and expressed in Chinese hamster ovary (CHO) cells under the control of an SV40-derived promoter/enhancer sequence. Culture supernatants of stably transfected cell clones contained biologically active IFN-omega 1 at concentrations up to 10 micrograms/l. Amplification of the expression vector containing a dhfr gene under methotrexate selection pressure resulted in yields up to 200 micrograms/l. Production of IFN-omega 1 was further enhanced 2- to 3-fold by propagation of the cells in the presence of n-butyrate. IFN-omega 1 was purified from culture supernatants by monoclonal antibody affinity chromatography. The resulting protein was at least 95% pure as determined by reverse-phase HPLC and size-exclusion HPLC. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed two bands of about the same intensity with apparent molecular masses of 24.5 and 22.5 kDa. Upon treatment with peptide:N-glycosidase F, both bands were shifted to lower molecular masses (20.5 and 18.5 kDa), indicating that CHO cell-derived IFN-omega 1 is glycosylated; Asn-78 was identified as the glycosylation site. Analysis of the carbohydrate moiety using glycosidases and lectins revealed the presence of biantennary complex oligosaccharides containing neuraminic acid. Amino acid sequencing showed that only about 40% of the molecules have the expected N-terminus, whereas the others carry two additional amino acids derived from the signal sequence. C-terminal amino acid sequencing using carboxypeptidase P demonstrated that the smaller form of the protein lacks nine amino acids. Disulfide bridges were shown to connect Cys residues 1 and 99 as well as 29 and 139, respectively, as in IFN-alpha. The specific antiviral activity of recombinant, glycosylated human IFN-omega 1 on human cells was 2.6 x 10(8) IU/mg, not significantly different from that of the authentic, human leukocyte-derived protein.     

Bacterial phosphoenolpyruvate-dependent phosphotransferase system: association state of membrane-bound mannitol-specific enzyme II demonstrated by inactivation

The quaternary structure of the membrane-bound mannitol permease (EIIMtl) of the bacterial phosphotransferase system in Escherichia coli has been investigated in the membrane by using the radiation inactivation method. The experiments reveal two distinct but interconvertible forms of the permease. The first state is a dimer, and the second state consists of a less active higher molecular weight complex involving the dimer. The equilibrium between these two forms in the membrane can be shifted by changing the pH. At pH 8.1 the dimer is the dominant form. Decreasing the pH results in increased binding of a regulatory protein to the dimer, thus increasing the amount of the higher molecular weight form involving the dimer. Cross-linking EIIMtl in situ, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, resulted in the formation of two cross-linked forms. One is the dimer, and the other has a higher molecular weight. Two-dimensional electrophoresis using a reversible cross-linker revealed no other protein except EIIMtl in these complexes.     

Purification and characterization of a thermostable-cellulase free xylanase from Syncephalastrum racemosum Cohn

Syncephalastrum racemosum Cohn. produces an extracellular xylanase that was shown to potentially bleach pulp at pH 10 and 50 degrees C. The enzyme was found to be a dimer with an apparent molecular weight of 29 kDa as determined by SDS-PAGE. The optimum activity was found at two pH values 8.5 and 10.5; however the activity sharply decreased below pH 6 and above pH 10.5. The enzyme was stable for 72 h at pH 10.5 and at 50 degrees C. Kinetic experiments at 50 degrees C gave V(max) and K(m) of 1,400 U/ml min(-1) mg(-1) protein and 0.05 mg/ml respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by group II b metal ions like Zn2+, Hg2+, etc. Xylan completely protected the enzyme from being inactivated by N-bromosuccinimide.     

Isolation and Partial Properties of a Porin-Like Protein from Vibrio parahaemolyticus Cell Envelope

The cell envelope of Vibrio parahaemolyticus pilot strain K-11 contains a major protein with an apparent molecular weight of 35,000 which was not solubilized with 2% sodium dodecyl sulfate (SDS) at 50 C for 30 min and was resistant to trypsin. The protein was extracted from the SDS-insoluble envelope with SDS containing 0.4 m NaCl and purified by acetone precipitation and gel filtration. The purified protein was completely dissociated into a monomer with a molecular weight of 35,000 in SDS at 60 C. The amino acid composition of the protein was nearly the same as that of porins from Escherichia coli and Salmonella typhimurium. Thus the protein seems to be porin-like.     

Isolation and partial characterization of monophosphoglycerate mutase from human erythrocytes.

Monophosphoglycerate mutase has been purified to homogeneity from outdated human erythrocytes as indicated by exclusion chromatography, polyacrylamide gel electrophoresis, and equilibrium centrifugation. Occasionally, the recommended purification procedure yields a small amount (3% or less) of a single extraneous protein which can be deleted from the enzyme preparation by employing an additional purification step. The native enzyme has a molecular weight of 54,000 to 56,000 as determined by equilibrium centrifugation and exclusion chromatography. Disc gel electrophoresis in the presence of sodium dodecyl sulfate yields a single protein band with a molecular weight of 28,600, indicating that the native macromolecule is a dimer composed of subunits of similar mass. Homogeneous monophosphoglycerate mutase is free of diphosphoglycerate mutase, enolase, and nonspecific phosphatase activities; however, the enzyme manifests intrinsic 2,3-diphospho-D-glycerate phosphatase activity as shown by thermal denaturation studies. The diphosphatase activity is stimulated by PPi and glycolate-2-P, but is inhibited by Cl-, HSO3-, and Pi. The pH optimum for both the diphosphatase and the mutase is 6.8. The Km for 2,3-diphospho-D-glycerate in the phosphatase reaction is 82 muM at 37 degrees and pH 7.2. The amino acid composition of homogeneous monophosphoglycerate mutase is given.     

Composition of major outer membrane proteins of Vibrio vulnificus isolates: effect of different growth media and iron deficiency

The outer membrane proteins of Vibrio vulnificus including isolates from humans, seawater and an asari clam were examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. A major outer membrane protein with an apparent molecular weight of 48,000 (48K protein) was common to all the strains grown in 3% NaCl-nutrient broth; however this 48K protein was not produced in any of the strains grown in chemically defined medium. Other major outer membrane proteins with molecular weights ranging from 33,000 to 40,000 varied in number, relative amount and molecular weight depending on the strain. One to three new outer membrane proteins with molecular weights ranging from 74,000 to 85,000 were produced in the cells grown in iron-deficient medium. The 48K protein and one or two major proteins with molecular weights ranging from 35,000 to 37,000 in the cells grown in 3% NaCl-nutrient broth were not solubilized by 2% SDS at 60 C for 30 min and were resistant to trypsin, indicating that they are porins. On the other hand, in cells grown in chemically defined medium, one or two major outer membrane proteins with molecular weights ranging from 33,000 to 40,000 might be porins.     

Sedimentation equilibrium measurements of recombinant DNA derived human interferon gamma

Recombinant DNA derived human interferon gamma (IFN-gamma) from Escherichia coli was examined by equilibrium ultracentrifugation. Short-column equilibrium experiments at pH 6.9 in 0.1 M ammonium acetate buffer gave a z-average molecular weight of 33,500 +/- 1400 at infinite dilution, corresponding to 1.98 +/- 0.08 times the formula weight. Long- (2.6 mm) column experiments at pH 7.5 in 0.04 M imidazole buffer gave a molecular weight of 33,400 +/- 500. Under the latter conditions IFN-gamma behaves somewhat nonideally, with the departure from ideality accounted for by an effective (Donnan) charge of about 6+. No association of this dimer to form tetramer or higher polymers was observed, with the association constant for formation of tetramer from dimer K24 found to be less than 34 L mol-1. Similarly, no dissociation to monomers was observable, with the dissociation constant to monomer K21 being less than 5 X 10(-8) mol L-1. At pH 3.55 in 0.02 M buffer (acetate plus acetic acid), there was virtually complete dissociation of the dimer to monomer. Extreme nonideality was seen in this low ionic strength system, and the effective charge on the protein was estimated to be about 11+. The reduced molecular weight M(1 -upsilon rho) of the monomer was found to be about 4.09 +/- 0.20 kg mol-1; this corresponds to a molecular weight of 16,410 +/- 820, with the Scatchard definition of components. A small amount of a polymer with a molecular weight of about 0.5 X 10(6) was detected under these conditions.     

SOLUTION PROPERTIES OF β NERVE GROWTH FACTOR PROTEIN AND SOME OF ITS DERIVATIVES

Abstract— The molecular weight of β nerve growth factor protein determined by sedimentation equilibrium in sodium acetate buffer, pH 40, and at protein concentrations around 0-5 mg/ml agrees with the value obtained from the amino acid sequence and confirms the dimeric character of the protein under these conditions. At pH values of 5.0 or greater, β nerve growth factor protein shows either partial dissociation into monomers or aggregation to higher polymers or both phenomena. The extent of dissociation or aggregation depends on buffer type and pH and is most pronounced at alkaline pH. The variation of molecular weight of β nerve growth factor with solvent conditions is similar to that of insulin or proinsulin. Removal of either the two COOH-terminal arginine residues or the two NH₂-terminal octapeptide sequences from the protein has no effect on its solution properties at acid pH, the protein remaining a dimer. Species such as 2-5 S nerve growth factor or cyanogen bromide cleaved nerve growth factor which are partically deficient in COOH-terminal arginine residues and/or NH₂-octapeptide or nonapeptide sequences are also dimers at pH40. The protein derivative which lacks the two NH₂-terminal octapeptide sequence does not, like β-nerve growth factor, display dissociation or aggregation behavior at neutral pH, indicating that these sequences are involved in monomer-monomer interactions.