Sequential roles of receptor binding and low pH in forming prehairpin and hairpin conformations of a retroviral envelope glycoprotein

A general model has been proposed for the fusion mechanisms of class I viral fusion proteins. According to this model a metastable trimer, anchored in the viral membrane through its transmembrane domain, transits to a trimeric prehairpin intermediate, anchored at its opposite end in the target membrane through its fusion peptide. A subsequent refolding event creates a trimer of hairpins (often termed a six-helix bundle) in which the previously well-separated transmembrane domain and fusion peptide (and their attached membranes) are brought together, thereby driving membrane fusion. While there is ample biochemical and structural information on the trimer-of-hairpins conformation of class I viral fusion proteins, less is known about intermediate states between native metastable trimers and the final trimer of hairpins. In this study we analyzed conformational states of the transmembrane subunit (TM), the fusion subunit, of the Env glycoprotein of the subtype A avian sarcoma and leukosis virus (ASLV-A). By analyzing forms of EnvA TM on mildly denaturing sodium dodecyl sulfate gels we identified five conformational states of EnvA TM. Following interaction of virions with a soluble form of the ASLV-A receptor at 37 degrees C, the metastable form of EnvA TM (which migrates at 37 kDa) transits to a 70-kDa and then to a 150-kDa species. Following subsequent exposure to a low pH (or an elevated temperature or the fusion promoting agent chlorpromazine), an additional set of bands at >150 kDa, and then a final band at 100 kDa, forms. Both an EnvA C-helix peptide (which inhibits virus fusion and infectivity) and the fusion-inhibitory agent lysophosphatidylcholine inhibit the formation of the >150- and 100-kDa bands. Our data are consistent with the 70- and 150-kDa bands representing precursor and fully formed prehairpin conformations of EnvA TM. Our data are also consistent with the >150-kDa bands representing higher-order oligomers of EnvA TM and with the 100-kDa band representing the fully formed six-helix bundle. In addition to resolving fusion-relevant conformational intermediates of EnvA TM, our data are compatible with a model in which the EnvA protein is activated by its receptor (at neutral pH and a temperature greater than or equal to room temperature) to form prehairpin conformations of EnvA TM, and in which subsequent exposure to a low pH is required to stabilize the final six-helix bundle, which drives a later stage of fusion.     

On the molecular weight and subunit composition of calf thymus ribonuclease H1

We have reinvestigated the molecular weight and subunit composition of calf thymus ribonuclease H1. Earlier studies suggested a variety of molecular weights for the enzyme in the range of 64K-84K and reported that the enzyme either was a single polypeptide of 74 kDa or consisted of from two to four subunits in the range of 21-34 kDa. Although we too find bands in this lower molecular weight range in our highly purified preparations following SDS-PAGE, our data suggest that the native structure of RNase H1 is a dimer of 68-kDa subunits. The evidence includes the following: (1) Western blot analysis of fractions taken at various stages of the purification indicates that the predominant antigenic form of the enzyme in crude extracts has a molecular weight of 68K but that during purification in the absence of sufficient protease inhibitors a variety of lower molecular weight forms appear concomitant with the disappearance of the 68-kDa band. (2) Activity gel analysis of the highly purified enzyme prepared in the presence of a battery of protease inhibitors reveals that the 68-kDa band (as well as several bands of lower molecular weight) possesses RNase H activity. (3) The 68-kDa band recognized by Western blotting with anti-RNase H immune sera is not detected by using preimmune sera. Furthermore, when immune sera are used, a trace of a 140-150-kDa antigenic form can sometimes be detected, consistent with the existence of a dimeric form of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of class Pi glutathione transferase activity by sulfhydryl group modification

Glutathione transferases (GSTs) in Class Pi (rat GST-P (7-7) and human GST-pi) were inactivated by treatment with 0.05-1 mM hydrogen peroxide (H2O2), while GSTs in Class Alpha (1-2) and Class Mu (3-3, 3-4) were not, even with 5 mM H2O2. In the presence of 1 mM reduced glutathione (GSH), the inactivated GST-P (-pi) was effectively reactivated by the action of thioltransferase, which had been partially purified from rat liver by GSH-Sepharose affinity chromatography and gel filtration using Sephadex G-75. Thus, inactivation of GST-P by H2O2 was indicated to involve concomitant formation of disulfide bonds between cysteinyl residues. Single GST-P or GST-pi subunits are known to have four cysteinyl residues at the same positions, which can react with sulfhydryl group modifiers. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, GST-P treated with 1 mM H2O2 showed several extra bands, at least three, with apparent molecular weights of 21.5, 18, 37 kDa in addition to the native GST-P subunit band with a molecular weight of 23.5 kDa. These extra bands were identified as inactive forms since they returned to the native band with accompanying restoration of the activity when treated with dithiothreitol, mercaptoethanol, or thioltransferase. Disulfide bonds were formed mainly within subunits, causing an apparent reduction in molecular weight, only small amounts of binding between subunits being observed.     

Investigation of peroxisomal lipid beta-oxidation enzymes in guinea pig liver peroxisomes by immunoblotting and immunocytochemistry.

We investigated the immunoreactivity of the peroxisomal lipid beta-oxidation enzymes acyl-CoA oxidase, trifunctional protein, and thiolase in guinea pig liver and compared it with that of homologous proteins in rat, using immunoblotting of highly purified peroxisomal fractions and monospecific antibodies to rat proteins. In addition, the immunocytochemical localization of beta-oxidation enzymes in guinea pig liver was compared with that of catalase. All antibodies showed crossreactivity between the two species, indicating that these peroxisomal proteins have been well conserved, although all exhibited some differences with respect to molecular size and, in the case of acyl-CoA oxidase, in frequency of the immunoreactive bands. In the latter case, a distinct second band in the 70 KD range was observed in guinea pig, in addition to the regular band due to subunit A present in rat liver. This novel band could be due either to trihydroxycoprostanoyl-CoA oxidase or to the non-inducible branched chain fatty acid oxidase described recently. All three beta-oxidation enzymes were immunolocalized by light and electron microscopy to the matrix of peroxisomes, in contrast to catalase, which is also found in the cytoplasm and the nucleus of hepatocytes in guinea pig liver.     

The subunit structure of the follitropin (FSH) receptor. Photoaffinity labeling of the membrane-bound receptor follitropin complex in situ

Human follicle-stimulating hormone (hFSH) was acylated with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB) and radioiodinated (55 microCi/micrograms) for use as a photoaffinity probe to investigate the subunit structure of the FSH receptor in calf testis. After incubation with the photoaffinity probe and photolysis with UV light, the cross-linked hormone-receptor complex was solubilized from the membrane and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Autoradiography of the polyacrylamide gels revealed two major bands, 64 kDa and 84 kDa. These were equivalent in molecular mass to those observed in a previous study (Branca, A. A., Sluss, P. M., Smith, A. A., and Reichert, L. E., Jr. (1985) J. Biol. Chem. 260, 9988-9993) in which performed hormone-receptor complexes were solubilized with detergent prior to formation of covalent cross-linkages through the use of homobifunctional cross-linking reagents. Reduction with dithiothreitol resulted in the loss of radioactivity from the 84-kDa band with a concomitant increase in the intensity of the 64-kDa band. Since dithiothreitol increases the dissociation of intact radioiodinated azidobenzoyl-FSH into subunits, it is suggested that the conversion of the 84-kDa band to the 64-kDa band by dithiothreitol is due to the loss of non-cross-linked hFSH subunit from the 84-kDa band and that the two bands observed after photoaffinity labeling arise from covalent bond formation between hFSH and a receptor subunit having a relative molecular weight (Mr) of 48,000. In addition to the predominant photolabeling of the receptor to yield the 64-kDa and 84-kDa bands, several other, less intense bands (54 kDa, 76 kDa, 97 kDa, and 116 kDa) were also consistently observed on autoradiographs. The appearance of all bands, however, was inhibited by the inclusion of unlabeled hFSH in the initial binding incubation mixtures. The results of this study indicate that the calf testis FSH receptor has a multimeric structure containing at least one 48-kDa subunit and suggest the presence of other nonidentical receptor subunit proteins.     

Immunoaffinity isolation of Na+ channels from rat skeletal muscle. Analysis of subunits

Polyclonal antiserum and monoclonal antibodies raised against the sodium channel from rat skeletal muscle sarcolemma have been immobilized on Sepharose and used to immunoaffinity purify this channel directly from skeletal muscle without the intervening purification of surface membranes. These antibodies isolate a approximately 260-kDa protein from whole muscle, although each purifies predominantly a 150-kDa component when isolated sarcolemmal membranes are used as starting material. A 45-kDa band is also found in the material purified from sarcolemma but not that obtained from whole muscle. In addition, these immunoaffinity columns isolate a 38-kDa band from both whole muscle and sarcolemma that copurifies with the 260-kDa protein. In some preparations this component appears as two closely spaced bands of 37 and 39 kDa. These small subunits coelute with the 260-kDa subunit when thiocyanate gradients are used to displace protein bound to the immunoaffinity columns and behave as integral components of the sodium channel. Estimates of stoichiometry were made for the large and small subunits of the muscle channel protein. After correction for labeling efficiency, values consistent with a ratio of one 260-kDa subunit to one 38-kDa subunit were obtained. We conclude that the rat skeletal muscle sodium channel contains a large alpha subunit of approximately 260 kDa that is sensitive to proteolytic nicking during the isolation of sarcolemmal membranes. In addition, at least one 38-kDa beta subunit is associated with each alpha subunit in the native channel.     

Purification of two hexosaminidases from human kidney.

Hexosaminidase forms A and B were isolated from human kidney in a homogeneous state as demonstrated by electrophoretic and enzymic criteria. The enzymes were stable for at least 18 months when stored at -20 degrees C in 0.025 M-phosphate buffer, pH 6.5. The molecular weights of forms A and B were estimated by gel filtration to be 111 000 +/- 1500 and 114 000 +/- 1600 respectively. The molecular weights of hexosamidase A and B subunits were determined by using polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Hexosaminidase A dissociated into one subunit with mol.wt. 68 000. Hexosaminidase B dissociated into three subunits with mol. wts. 100 000, 68 000 and 37000 respectively, and one protein band of mol.wt. 140 000. After treatment of hexosaminidases A and B with iodoacetic acid, the molecular weights of the carboxymethylated polypeptide subunits were also estimated. Carboxymethylated hexosaminidase A dissociated into one major subunit of mol.wt. 18 000 and two other protein bands of mol.wts. 65 000 and 100 000. Carboxymethylated hexosaminidase B dissociated into one major subunit for mol.wt. 19 000 and an additional band of mol.wt. 37 000. The Km of the enzymes for the synthetic substrate p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside was 0.8 mM. Both enzymes were inhibited or activated by various metal ions. Double pH optima for the enzymes were found at pH 4.5 and 4.8.     

Identification of F0 subunits in the rat liver mitochondrial F0F1-ATP synthase.

In order to identify the subunits constituting the rat liver F0F1-ATP synthase, the complex prepared by selective extraction from the mitochondrial membranes with a detergent followed by purification on a sucrose gradient has been compared to that obtained by immunoprecipitation with an anti-F1 serum. The subunits present in both preparations that are assumed to be authentic components of the complex have been identified. The results show that the total rat liver F0F1-ATP synthase contains at least 13 different proteins, seven of which can be attributed to F0. The following F0 subunits have been identified: the subunit b (migrating as a 24 kDa band in SDS-PAGE), the oligomycin-sensitivity-conferring protein (20 kDa), and F6 (9 kDa) that have N-terminal sequences homologous to the beef-heart ones; the mtDNA encoded subunits 6 (20 kDa) and 8 (less than 7 kDa) that can be synthesized in isolated mitochondria; an additional 20 kDa protein that could be equivalent to the beef heart subunit d.     

Inactive hepatic lipase in rat plasma

Hepatic lipase activity is detectable in liver but also in adrenal glands, ovaries, and plasma. The subunit size of hepatic lipase in liver, adrenal glands, and nonheparin plasma was compared. Hepatic lipase in liver and adrenal glands appeared as a 55 kDa band. In liver, a faint band of lower size was also detected. In nonheparin plasma, hepatic lipase appeared as a doublet of 57 kDa and 59 kDa. When activity/mass ratio was calculated, similar values were obtained for liver and adrenal glands. In plasma this value was much lower. After heparin administration in vivo, hepatic lipase activity in plasma increased nearly 100-fold with appearance of an additional 55 kDa band in postheparin plasma. This band coeluted with activity after preparative polyacrylamide gel electrophoresis. Differences in size persisted after digestion with peptide-N-glycosidase F. A progressive increase in 57 kDa and 59 kDa in postheparin plasma followed disappearance of the 55 kDa band, suggesting that these larger bands originate from the smaller form. In plasma, both smaller and larger forms were associated with HDL, but not with LDL or VLDL. We conclude that rat plasma contains a larger form of hepatic lipase that is inactive in in vitro assay.     

Modulation of the rhythmic patterns of expression of phosphoprotein phosphatases in human leukaemia cells

Temporal variations in the expression of phosphoprotein phosphatase 1 (PP1), phosphoprotein phosphatase 2A (PP2A) and protein tyrosine phosphatase 1B (PTP1B) were monitored in the human acute, promyelocytic leukaemia cell line, HL60. Granulocytic differentiation was induced using all-trans retinoic acid (ATRA) and monocytic differentiation by phorbol-12-myristate-13-acetate (PMA). Expression of the enzyme proteins in cell extracts was determined by SDS-PAGE and Western immunoblotting using specific antibodies. For PP1, a single immunospecific band of molecular mass 38 kDa was detected corresponding to the catalytic subunit; induction of differentiation with either ATRA or PMA showed differences in the patterns of expression and, in the case of the latter, the mean value. Two immunospecific bands, of mass 34 and 37 kDa, possibly corresponding to dephosphorylated and phosphorylated forms, respectively, were detected for PP2A, as well as a minor band of mass 46 kDa; dynamic variations in the expression of all 3 forms were observed and there were differences between the control and treated cells. The catalytic domain of PTP1B was detected as a 46 kDa band. A 42 kDa form of the protein was also seen, which may represent a change in phosphorylation state, or be the result of proteolytic cleavage; usually the 46 kDa band was the major form, but on occasion there was a change to predominance of the 42 kDa band.     

Biochemical and ultrastructural studies of the C-type lectin bovine conglutinin

The aim of this study was to correlate the supramolecular organization of conglutinin (BK) with its primary and tertiary structure and to gain more knowledge of functionally important regions of the molecule. BK analyzed by SDS-PAGE under standard reducing conditions (40 mM DTT) showed a major band at 43 kDa and weaker bands at 86 and 180 kDa. In contrast, reduction with 6-50 mM L-cysteine resulted in 37-kDa subunits indicating the presence of intrachain disulfide bonds within this subunit. Hydroxylamine treatment indicated presence of ester bonds in the 86- and 180-kDa subunits. Collagenase digestion and SDS-PAGE under reducing and nonreducing conditions resulted in bands of 20 and 15 kDa, respectively, indicating the presence of intrachain, rather than interchain, disulfide bonds in the carboxy terminus. Deglycosylation and glycan differentiation analysis of BK revealed the presence of O-linked glycans of GalNAc and alpha (2-3) linked sialic acid type, whereas no N-linked glycans were demonstrated. Binding experiments with GlcNAc-gold suggested that multivalency is required for carbohydrate binding to BK. Electron microscopy showed mostly tetramers, 96 nm in diameter, but also mono-, di-, and trimers were seen. The tetramers consisted of 40-nm strands, each with a peripheral globular head composed of subunits and connected to a common central lobe built from four ring-formed structures. The strands occasionally showed two bends, one close to the central lobe and another 25 nm from the lobe. These bends most likely correspond to the interrupted Gly-Xaa-Yaa repeats at residues 38 and 123.     

Anomalous mobilities of Na,K-ATPase alpha subunit isoforms in SDS-PAGE: identification by N-terminal sequencing.

Three isoforms of the alpha subunit of Na,K-ATPase, alpha 1, alpha 2, and alpha 3 have been characterized at the DNA, mRNA and protein levels. In admixtures, isoforms migrate as doublets (i.e. alpha 1 and another band originally designated alpha +, comprising alpha 2 + alpha 3) when analyzed by SDS-PAGE. As deduced from cDNA sequences their masses range from 111.7 to 112.6 kDa. With conventional protein standards, however, SDS-PAGE yields nominal masses of 85-105 kDa. In this system, the presence of a doublet that reacted with a polyclonal anti-Na,K-ATPase antibody in the kidney was interpreted as indicating two molecular or conformational species of the kidney alpha sub-unit (Siegel, G.J. and Desmond, T.J. (1989) J. Biol. Chem. 264, 4751-4754). We report that Na,K-ATPase purified from dog, guinea pig and rat kidney medulla or from rat brain, can yield two distinct bands when analyzed by SDS-PAGE or STS-PAGE, migrating between 85 and 105 kDa. An additional band migrating at 117 and 120 kDa appears often in enzyme purified from rat and guinea pig kidney medulla. The apparent molecular weights and relative intensities of these bands vary with temperature and duration of incubation during sample preparation. N-terminal sequencing and monospecific antibody probes revealed that the two distinct bands obtained from the kidney enzyme consist only of the alpha 1 isoform. The band appearing at 117-120 kDa also contains only the alpha 1 N-terminal sequence. In contrast, as reported earlier (Sweadner, K.J. (1979) J. Biol. Chem. 254, 6060-6067), the doublet seen in brain preparations consists of alpha 1 and alpha 2 or (alpha 2 + alpha 3). We conclude that monospecific antibody probes or N-terminal sequencing must be used to identify Na,K-ATPase isoforms by SDS- or STS-PAGE. In addition, gel conditions that may affect the mobilities of the isoforms are discussed.     

The magnetic properties of the nickel cofactor F430 in the enzyme methyl-coenzyme M reductase of Methanobacterium thermoautotrophicum.

Preparations of gamma-aminobutyrate (GABA)/benzodiazepine receptor from pig cerebral cortex are composed of three major bands of polypeptides (51, 55 and 57 kDa) which are purified in a ratio of approx. 2:1:1 respectively. Treatment of purified receptor preparations with cyclic AMP-dependent protein kinase resulted in major incorporation of 32P into the 55 kDa band only. The maximum incorporation achieved was 0.6 mol of 32P/mol of 55 kDa polypeptide. The phosphorylated receptor subunit (beta-subunit) displays the same apparent Mr as a band labelled irreversibly with the GABA receptor agonist [3H]muscimol. The two nonphosphorylated subunit polypeptides (51 and 57 kDa) are each labelled irreversibly with [3H]flunitrazepam and are recognized by anti-peptide antibodies specific for alpha-subunits.     

Antigenic relatedness between phospholipases A2 from Naja naja venom and from mammalian cells

Polyclonal antiserum was prepared against phospholipase A2 from Naja naja and used to prepare a purified antibody. It cross-reacted with the antigen, and with intracellular mammalian PLA2. This antibody was immunoreactive and inhibited the PLA2 activity of Naja naja and of guinea pig alveolar macrophages or rat lymphocytes. By immunoblotting, this antiserum revealed one band of PLA2 from Naja naja (14 kDa) and 3 bands for guinea pig alveolar macrophages and rat lymphocytes (30, 45 kDa and a minor band of 14 kDa). These results show an antigenic relatedness between an extracellular PLA2 and membrane-bound PLA2 from two different mammalian species and cell types.     

The flavin-containing monooxygenase in mouse lung: Evidence for expression of multiple forms

The flavin-containing monooxygenase (FMO) was purified from mouse lung microsomes. On SDS-PAGE, the purified enzyme separated as two bands, a major band of 58,000 daltons and a minor band of 59,000 daltons. Antibodies to mouse liver FMO cross-reacted with both bands in the purified preparations, whereas antibodies to rabbit lung FMO cross-reacted only with the major band. In microsomal preparations the major band was recognized by both antibodies, but neither antibody detected the minor band in microsomes. A cDNA encoding the pig liver FMO hybridized with mRNA isolated from mouse liver, kidney, and lung, whereas cDNA encoding the rabbit lung FMO hybridized only with mouse lung and kidney mRNA. Thermal stability studies showed that the FMO preparation purified from mouse lung consisted of a heat-stable and a heat-labile component. The heat-labile component of lung FMO was inhibited competitively by imipramine, whereas the heat-stable component was insensitive to the presence of imipramine. Immunoprecipitation of purified mouse lung FMO with anti-rabbit lung FMO completely removed the protein band reactive to anti-rabbit lung FMO while leaving reactivity to anti-liver FMO. The catalytic and immunochemical differences seen between FMO from rabbit lung and mouse lung appear to result from the expression of at least two forms of FMO in the mouse lung, one similar to the rabbit pulmonary form and one similar to the major mouse liver form of FMO.     

Purification and characterization of rat liver glycosylasparaginase.

1. Rat liver glycosylasparaginase [N4-(beta-N-acetylglucosaminyl)-L-asparaginase, EC 3.5.1.26] was purified to homogeneity by using salt fractionation, CM-cellulose and DEAE-cellulose chromatography, gel filtration on Ultrogel AcA-54, concanavalin A-Sepharose affinity chromatography, heat treatment at 70 degrees C and preparative SDS/polyacrylamide-gel electrophoresis. The purified enzyme had a specific activity of 3.8 mumol of N-acetylglucosamine/min per mg with N4-(beta-N-acetylglucosaminyl)-L-asparagine as substrate. 2. The native enzyme had a molecular mass of 49 kDa and was composed of two non-identical subunits joined by strong non-covalent forces and having molecular masses of 24 and 20 kDa as determined by SDS/polyacrylamide-gel electrophoresis. 3. The 20 kDa subunit contained one high-mannose-type oligosaccharide chain, and the 24 kDa subunit had one high-mannose-type and one complex-type oligosaccharide chain. 4. N-Terminal sequence analysis of each subunit revealed a frayed N-terminus of the 24 kDa subunit and an apparent N-glycosylation of Asn-15 in the same subunit. 5. The enzyme exhibited a broad pH maximum above 7. Two major isoelectric forms were found at pH 6.4 and 6.6. 6. Glycosylasparaginase was stable at 75 degrees C and in 5% (w/v) SDS at pH 7.0.     

Cloning, expression and characterization of the pig liver GDP-mannose pyrophosphorylase. Evidence that GDP-mannose and GDP-Glc pyrophosphorylases are different proteins.

GDP-Man, the mannosyl donor for most Man-containing polymers is formed by the transfer of Man-1-P to GTP to form GDP-Man and PPi. This reaction is catalyzed by the widespread and essential enzyme, GDP-Man pyrophosphorylase (GMPP). The pig liver GMPP consists of an alpha subunit (43 kDa) and a beta subunit (37 kDa). Purified pig GMPP catalyzes the synthesis of GDP-Glc (from Glc-1-P and GTP) and GDP-Man (from Man-1-P and GTP), but has higher activity for the formation of GDP-Glc than for synthesis of GDP-Man. In the present study, we report the cloning of the cDNA for the beta subunit of GMPP, and its expression in a bacterial system resulting in the formation of active enzyme. The full length cDNA encoding the beta subunit was isolated from a porcine cDNA library, and its predicted gene product showed high amino-acid sequence homology to GMPPs from other species. The gene was expressed in Escherichia coli cells, and a 37-kDa protein was over-produced in these cells. This gene product reacted strongly with antibody reactive to the native beta subunit of pig GMPP. Most interestingly, this recombinant protein had high activity for synthesizing GDP-Man (from Man-1-P and GTP), but very low activity for the formation of GDP-Glc (from Glc-1-P and GTP). Other properties of the recombinant protein were also analyzed. This study suggests that the beta subunit is the GMPP, whereas the alpha subunit, or a combination of both subunits, may have the GDP-Glc pyrophosphorylase activity.     

Identification of a high molecular weight macrophage colony-stimulating factor as a glycosaminoglycan-containing species.

Chinese hamster ovary cells transfected with a 4.0-kilobase macrophage colony-stimulating factor (M-CSF) cDNA express two different M-CSF species; one has an apparent molecular weight of 85,000 and is identified as a homodimer of a 43-kDa subunit, and the other has an indeterminate structure greater than 200 kDa. In this study, we investigated the structure of the high molecular weight M-CSF by immunochemical procedures. The high molecular weight M-CSF was easily purified, since it bound tightly to DEAE-Sephacel and eluted at a characteristically high salt concentration. The high molecular weight M-CSF migrated as a diffuse band of over than 200,000 on nonreducing sodium dodecyl sulfate-polyacrylamide gels. Analysis of the same samples under reducing conditions revealed that the larger species consisted of a heteromer of the 43- and 150-200-kDa M-CSF subunits. Digestion of the 150-200-kDa M-CSF subunit with chondroitinase, which degrades the chondroitin sulfate glycosaminoglycan chain, yielded a 100 kDa band. This species was secreted instead of 150-200-kDa species when the cells were cultured in the presence of beta-D-xyloside, which inhibits the elongation of the chondroitin sulfate glycosaminoglycan chain in proteoglycans, providing additional evidence for the existence of a chondroitin sulfate chain in the 150-200-kDa M-CSF subunit. Removal of O- and N-linked carbohydrate from the 150-200-kDa subunit yielded a polypeptide chain with a larger molecular mass (approximately 45 kDa) than that of the 43-kDa subunit (approximately 25 kDa). Collectively, these results indicate that the 150-200-kDa M-CSF subunit is a proteoglycan with a core protein that may be an alternatively processed form of M-CSF.     

Intersubunit disulfides of the follitropin receptor

The electrophoretic mobility of radioiodinated follitropin (FSH) alpha and beta subunits as well as the alpha beta dimer changed markedly depending on the concentration of reducing agents such as dithiothreitol. The changes were more dramatic in the beta subunit than in the alpha subunit. 125I-FSH, complexed to the receptor on porcine granulosa cells or in Triton X-100 extracts, was cross-linked with a cleavable (nondisulfide) homobifunctional reagent, solubilized in sodium dodecyl sulfate without reducing agents, and electrophoresed. The cross-linked sample revealed three bands of high molecular mass, in addition to the hormone subunit and dimer bands. The band of lightest mass, 110 kDa, was the major band and the other two of 76 and 62 kDa were barely noticeable. Upon reduction with dithiothreitol, the 110-kDa band decreased while the 76- and 62-kDa bands increased, indicating the existence of disulfides between components of the 110-kDa complex. Formation of the disulfide-linked complexes requires 125I-FSH, specifically bound to the hormone receptor and cross-linking, and can be prevented with an excess of native FSH but not human choriogonadotropin. Complex formation was independent of blocking free sulfhydryl groups with N-ethylmaleimide. When the cross-linked complexes were reduced in the gel matrix and analyzed on fresh gels, the 76- and 62-kDa complexes were generated from the 110-kDa band, indicating the loss of two components. The lost components were estimated to be at 14 and 34 kDa. The rate of formation and cleavage of the cross-linked complexes indicated a sequential and incremental addition of 22-, 14-, and 34-kDa components to the FSH alpha beta dimer. The results of reduction of the cross-linked complexes demonstrate the existence of disulfide linkage between the three components.     

Purification and characterization of methylenetetrahydrofolate reductase from human cadaver liver

Methylenetetrahydrofolate reductase from human cadaver liver was purified to homogeneity. The purified enzyme had a molecular mass of 150 kDa. On SDS-polyacrylamide gel electrophoresis it was dissociated into a single fragment with a molecular mass of 39 kDa. In contrast, fresh lymphocyte enzyme extract showed a major band with a molecular mass of 75 kDa and a minor band of 39 kDa. Fresh liver enzyme was inhibited by S-adenosylmethionine while the purified enzyme from human cadaver liver was not inhibited. These observations suggest that human methylenetetrahydrofolate reductase is composed of two identical subunits of 75 kDa each but is cleaved into a major single band due to autolysis in cadaver liver. The purified cadaver enzyme was a FAD-specific protein. The pH optimum was 6.6 for methylenetetrahydrofolate-NADPH oxidoreductase, 6.5 for methyltetrahydrofolate-menadione oxidoreductase, and 7.2 for NADP-menadione oxidoreductase. The Km values of human liver methylenetetrahydrofolate reductase were 17 microns for NADPH and 38 microns for methyltetrahydrofolate in the reduction of menadione, and 12 microns for NADPH in the reduction of methylenetetrahydrofolate.