Detergent-solubilized Preparations Research Articles

Interaction of insecticides with mammalian P-glycoprotein and their effect on its transport function

We studied the effects of four commonly used insecticides (methylparathion, endosulfan, cypermethrin and fenvalerate) on P-glycoprotein isolated from multidrug-resistant cells. All the pesticides stimulated P-glycoprotein ATPase activity, with maximum stimulation of up to 213% in a detergent-solubilized preparation, and up to 227% in reconstituted liposomes. The ATPase stimulation profiles were biphasic, displaying lower stimulation, and in the case of methylparathion, inhibition of activity, at higher insecticide concentrations. Quenching of the intrinsic Trp fluorescence of purified P-glycoprotein was used to quantitate insecticide binding; the estimated Kd values fell in the range 4–6 μM. Transport of the fluorescent substrate tetramethylrosamine (TMR) into proteoliposomes containing P-glycoprotein was monitored in real time. The TMR concentration gradient generated by the transporter was collapsed by the addition of insecticides, and prior addition of these compounds prevented its formation. The rate of TMR transport was inhibited in a saturable fashion by all the compounds, indicating that they compete with the substrate for membrane translocation. Taken together, these data suggest that the insecticides bind to Pgp with high affinity and effectively block drug transport. Inhibition of Pgp by pesticides may compromise its ability to clear xenobiotics from the body, leading to a higher risk of toxicity.

Direct Evidence for Substrate-induced Proton Release in Detergent-solubilized EmrE, a Multidrug Transporter

A novel approach to study coupling of substrate and ion fluxes is presented. EmrE is an H(+)-coupled multidrug transporter from Escherichia coli. Detergent-solubilized EmrE binds substrate with high affinity in a pH-dependent mode. Here we show, for the first time in an ion-coupled transporter, substrate-induced release of protons in a detergent-solubilized preparation. The direct measurements allow for an important quantitation of the phenomenon. Thus, stoichiometry of the release in the wild type and a mutant with a single carboxyl at position 14 is very similar and about 0.8 protons/monomer. The findings demonstrate that the only residue involved in proton release is a highly conserved membrane-embedded glutamate (Glu-14) and that all the Glu-14 residues in the EmrE functional oligomer participate in proton release. Furthermore, from the pH dependence of the release we determined the pK of Glu-14 as 8.5 and for an aspartate replacement at the same position as 6.7. The high pK of the carboxyl at position 14 is essential for coupling of fluxes of protons and substrates.

Packing of the transmembrane helices of Na,K-ATPase: direct contact between beta-subunit and H8 segment of alpha-subunit revealed by oxidative cross-linking.

Spatial relationships among the transmembrane (TM) segments of alpha- and beta-subunits of the Na,K-ATPase molecule have been investigated using oxidative induction of disulfide bonds. The catalytic alpha-subunit contains 10 TM alpha-helices (H1-H10) with 9 Cys residues located within or close to the membrane moiety. There is one Cys residue in the single TM segment of beta-subunit (Hbeta). Previously, the cross-linking products containing the beta-subunit and two fragments of alpha-subunit (the N-terminal containing H1-H2 helices and the C-terminal containing H7-H10 helices) have been identified in experiments with membrane-bound or detergent-solubilized preparations of the membrane moiety of trypsin-digested Na,K-ATPase [Sarvazyan, N. A., Modyanov, N. N., and Askari, A. (1995) J. Biol. Chem. 270, 26528-26532 and Sarvazyan, N. A., Ivanov, A., Modyanov, N. N., and Askari, A. (1997) J. Biol. Chem. 272, 7855-7858]. Here, we have shown that Cu(2+)-phenanthroline treatment of digitonin-solubilized preparation provides the most efficient formation of intersubunit cross-linked product that is predominantly a dimer of beta-subunit and a 22-kDa C-terminal alpha-fragment containing H7-H10 helices. This cross-linked product was isolated and subjected to CNBr cleavage. The resulting fragments were electrophoretically separated and sequenced. A 17-kDa peptide composed of Ile853-Met942 alpha-fragment and Ala5-Met56 beta-fragment was identified as a product of intersubunit disulfide cross-link between Cys44 of Hbeta and either Cys911 or Cys930, located in H8. This provides the first direct experimental evidence of the juxtaposition of Hbeta and H8 within the Na,K-ATPase molecule. The second detected cross-linked product was composed of alpha-fragments Lys947-Met963 and Tyr974-Tyr1016 linked by induced disulfide bridge between Cys964 (H9) and Cys983 (H10). The spatial proximity of these Cys residues defines the mutual orientation of H9 and H10 helices of alpha-subunit.

Filamentous fungus Aspergillus oryzae has two types of alpha-1,2-mannosidases, one of which is a microsomal enzyme that removes a single mannose residue from Man9GlcNAc2.

alpha-Mannosidase activities towards high-mannose oligosaccharides were examined with a detergent-solubilized microsomal preparation from a filamentous fungus, Aspergillus oryzae. In the enzymatic reaction, the pyridylaminated substrate Man(9)GlcNAc(2)-PA was trimmed to Man(8)GlcNAc(2)-PA which lacked one alpha-1,2-mannose residue at the nonreducing terminus of the middle branch (Man8B isomer), and this mannooligosaccharide remained predominant through the overall reaction. Trimming was optimal at pH 7.0 in PIPES buffer in the presence of calcium ion and kifunensine was inhibitory with IC(50) below 0.1 microM. These results suggest that the activity is the same type as was previously observed with human and yeast endoplasmic reticulum (ER) alpha-mannosidases. Considering these results together with previous data on a fungal alpha-1,2-mannosidase that trimmed Man(9)GlcNAc(2) to Man(5)GlcNAc(2) (Ichishima, E., et al. (1999) Biochem J, 339: 589-597), the filamentous fungi appear to have two types of alpha-1,2-mannosidases, each of which acts differently on N-linked mannooligosaccharides.

Analysis of the steroid binding domain of rat steroid 5α-reductase (Isozyme-1): The steroid D-ring binding domain of 5α-reductase

We have previously shown that the photoactive 4-azasteroid, [1,2 3H]N-4(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5α-androstan-17β-carboxamide is an effective probe of rat steroid 5α-reductase (isozyme-1) (5αR-1). In the current investigation, PEG-fractionated (6.5%) detergent-solubilized preparations containing 5αR-1 activity were ultraviolet (UV)-photolyzed with [ 3H]-4MABP and subsequently purified by 8.75% preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions corresponding to the radioactive peak following the dye front were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed the presence of a single, labeled, 26 KDa protein band, the apparent molecular weight of 5αR-1. TCA precipitation of the labeled fractions, followed by long-term digestion of the TCA pellet with chymotrypsin and high-performance liquid chromatography analysis, indicated that the majority of the radioactivity eluted with a peak retention time of 55–56 min. Rechromatography of this fraction using a modified gradient (elution 54–55 min), followed by sequence analysis, yielded a single N-terminal tetrapeptide with the sequence, -L-E-G-F-, corresponding to residues 15–18 of the 5αR-1 sequence. Site-directed mutagenesis studies indicated that mutant F18L showed an ∼12-fold increase in the K m for testosterone, whereas the K m for reduced nicotinomide adenine dinucleotide phosphate remained virtually unaltered.

Hexokinase present in human sperm is not tyrosine phosphorylated but its antibodies affect fertilizing capacity.

The present study was conducted to investigate the presence of hexokinase in human sperm cell, its subcellular localization, its modulation and role in capacitation/acrosome reaction, and tyrosine kinase activity, if any. These studies were conducted using antibodies (Ab) raised against rat brain hexokinase (type I isozyme) that have significant cross-reaction with various human tissue hexokinases and neutralize the catalytic activity of the enzyme. Hexokinase Ab reacted with acrosomal, mid-piece and tail regions of methanol-fixed (approximately 70-80%) and live (approximately 35-52%) sperm in the indirect immunofluorescence technique (IFT) and the immunobead binding technique (IBT), respectively. Hexokinase Ab specifically recognized a band of 116 kDa on the Western blot of detergent-solubilized human sperm preparation that was different from the 95-kDa phosphotyrosine protein. Hexokinase Ab caused a significant (P < 0.01 to < 0.001) and concentration-dependent inhibition of human sperm penetration of zona-free hamster oocytes in the sperm penetration assay (SPA). These data indicate that the hexokinase of 116-kDa molecular weight is present in acrosomal, mid-piece, and tail regions of human sperm. The sperm hexokinase is a glycoprotein that is different from the 95-kDa phosphotyrosine protein and is not phosphorylated at tyrosine residues; however, its antibodies cause agglutination and a concentration-dependent inhibition of fertilizing capacity of human sperm.

Identification of the NADP(H) binding site of rat liver microsomal 5 alpha-reductase (isozyme-1): purification of a photolabeled peptide corresponding to the adenine binding domain.

We have previously shown that [2'-32P]-2-azido-NADP+ is an effective probe of the NADP-(H) binding site of rat liver microsomal 5 alpha-reductase (5 alpha R-1) [Bhattacharyya et al. (1994) Steroids 59, 634-641]. PEG-fractionated (6.5%) detergent-solubilized preparations (40 mg) containing 5 alpha R-1 activity were UV-photolyzed with [32P]-2-azido-NADP+ and subjected to preparative gel electrophoresis on 8% SDS-PAGE. Fractions corresponding to the second major [32P]-labeled peak following the dye-front were analyzed by 10% SDS-PAGE and showed a single [32P]-labeled species with an apparent molecular mass of approximately 26 kDa (5 alpha R-1). TCA precipitation (13.6%) of the labeled fractions resulted in recovery of > 70% of the total radioactivity in the protein pellet. Trypsin digestion of the resuspended pellet followed by immobilized-Al3+ affinity chromatography indicated that > 90% of the radioactivity remained bound to the affinity column. The [32P]-2N3-NADP(+)-labeled peptide was eluted with potassium phosphate, concentrated, and further purified by reverse-phase (C8) HPLC. Sequence analysis of the purified peptide indicated that it consisted of 11 amino acids with the sequence N-L-R-K-P-G-E-T-G-Y-K, corresponding to residues 170-180 of the rat 5 alpha R-1 sequence [Andersson et al. (1989) J. Biol. Chem. 264, 16249-16255].

Activation of a Gi protein in digitonin/cholate-solubilized membrane preparations of mouse sperm by the zona pellucida, an egg-specific extracellular matrix.

Mammalian sperm possess guanine nucleotide-binding regulatory proteins (G proteins) that are involved in signal transduction pathways leading to zona pellucida (ZP)-mediated acrosomal exocytosis. We have previously examined ZP-G protein dynamics in mouse sperm homogenates, as well as cell-free membrane preparations, and our data support the existence of ZP receptor-G protein complexes in sperm membranes. However, the composition of this complex has not been identified due to experimental limitations of the membrane preparations. In the present study, a detergent-solubilized preparation from mouse sperm membranes that retained the signaling properties of cell homogenates and cell-free membrane preparations was developed using buffers containing digitonin and cholate. GTP gamma S, a poorly hydrolyzable analogue of GTP, bound to these solubilized preparations in a specific and concentration-dependent fashion that reached saturation at 100 nM. Incubation of this solubilized membrane preparation with heat-solubilized ZP resulted in an increase in specific GTP gamma S binding in a concentration-dependent manner, with a maximal response at 4-6 ZP/microliters. Mastoparan (50 microM) increased GTP gamma S binding to levels similar to that seen with solubilized ZP. Mastoparan plus ZP stimulated GTP gamma S binding to the same extent as mastoparan or ZP alone. Pertussis toxin completely inhibited ZP-stimulated GTP gamma S binding and decreased mastoparan-stimulated GTP gamma S binding by 50-60%. Purified ZP3, the ZP component that possesses quantitatively all of the sperm binding and acrosomal exocytosis-inducing activities of the intact ZP, stimulated GTP gamma S binding to an extent similar to that of solubilized ZP.(ABSTRACT TRUNCATED AT 250 WORDS)

Mannosyltransferase activities in membranes from various yeast strains.

In the yeast Golgi compartments, at least five, and potentially several additional mannosyltransferases are involved in elongating to 'mannan' the core Man8GlcNAc2 oligosaccharide trimmed from Glc3Man9GlcNAc2 in the endoplasmic reticulum. Structural studies on oligosaccharides from alg3 mutant yeast, which lack the four upper arm mannoses donated by Man-P-Dol (where Dol is dolichol), verified that the new alpha 1,6-branch in endo H-resistant mannan in this strain is efficiently initiated in vivo on the alpha 1,3-linked core residue of the lipid-oligosaccharide form of Man5GlcNAc2 (Verostek et al., J. Biol. Chem., 266, 5547-5551, 1991). This Man5GlcNAcGlcNAc[3H]ol isomer (where GlcNAc[3H]ol is N-acetylglucosamin [1-3H] itol) was found to be an excellent acceptor for a number of GDP-Man-dependent Golgi mannosyltransferases in detergent-solubilized yeast membrane preparations: an alpha 1,3-mannosyltransferase (Mnn1p), an alpha 1,6-mannosyltransferase (Och1p) and two alpha 1,2-mannosyltransferases (Mnt1p/Kre2p,?) whose products were readily identified by 1H NMR spectroscopy. The Man6GlcNAcGlcNAc[3H]ol isomers formed were easily defined by alpha 1,2-mannosidase sensitivity and either Bio-Gel P-4 gel filtration or AX-5 high-performance liquid chromatography. In general, mannosyltransferases present in detergent-solubilized microsomes from most yeast strains mimicked the array of sugar linkages observed on their respective glycoproteins. However, in the case of the Saccharomyces pmr1 mutant, an alpha 1,3-mannosyltransferase was active in microsomal extracts, but the alpha 1,3-Man epitope could not be identified on Western blots of cellular glycoproteins using sugar linkage-specific antibodies or lectins. The in vitro transferase assay is simple, rapid and accurate, and in the case of pmr1 suggests that in vivo either invertase is misrouted during secretion or the alpha 1,3-mannosyltransferase is mistargeted after its synthesis in this mutant.

Photoaffinity labeling of rat liver microsomal steroid 5α-reductase by 2-azido-NADP

Preincubation of female rat liver microsomal preparations with [2′- 32P]2N 3-NADP + followed by photolysis with UV lighjt (254 nm) and analysis by SDS-PAGE/autoradiography showed incorporation of 32P into at least 3 major protein bands in the molecular weight range of 14–197 Kd. Labeling of a 26 Kd band, the apparent molecular weight of 5α-reductase in liver microsomes, was accompanied by a loss of enzyme activity, consistent with its covalent modification. The inclusion of 20-fold excess NADP + (100μM) completely inhibited the incorporation of [2′- 32P]2N 3-NADP + and preserved the enzyme activity, whereas excess NAD + (100μM) failed to protect 5α-reductase (5αR) activity. Similar results were obtained with the detergent-solubilized form of 5αR. Polyethylene glycol (PEG) fractionation of detergent-solubilized with the detergent-solubilized preparations of 5αR showed that all the 5αR activity could be recovered in the 6.5%, pellet with a 3—4-fold increase in the specific activity. Photolysis of this fraction with [2′- 32P]2N 3-NADP + resulted in ∼ 2-fold increase in 32P labeling of the 5αR band. Increasing photolysis time and concentration of the [2′- 32P]2N 3-NADP + indicated that the half-life for photoincorporation and the apparent K d were 1.0 min and 2 μM, respectively. Theser results suggest that 2N 3-NADP + is an effective probe of the NADP(H) binding site of 5αR, and is a useful marker during purification of the enzyme.

High-level Expression of Functional Glutamate Receptor Channels in Insect Cells

We have expressed glutamate-gated ion channels in Spodoptera frugiperda Sf21 insect cells using a recombinant baculovirus system. Cells infected with recombinant baculoviruses encoding the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-selective glutamate receptor channel subunits GluR-B and GluR-D displayed specific high-affinity [3H]AMPA binding (apparent dissociation constant Kd of 15 nM for GluR-B and 40 nM for GluR-D) with pharmacological profiles typical of AMPA receptors. The binding reached maximal levels (Bmax of 15-30 pmol per mg of membrane protein) by 3-4 days postinfection. AMPA, glutamate and kainate triggered inward currents in GluR expressing cells, indicating assembly of functional homomeric channels. Formation of heteromeric GluR-B/D channels in doubly-infected cells was evident from the diagnostic current-voltage relations of AMPA-activated whole-cell currents. For the solubilization of the receptor, nonionic detergents Triton X-100, n-octyl-D-glucoside and n-dodecylmaltoside proved most effective. Detergent-solubilized receptor preparations were stable, retained their characteristic ligand-binding properties and bound to immobilized wheat germ lectin, demonstrating the glycosylation of insect cell-expressed GluR subunits. The expression level of 300-400 micrograms of receptor protein per liter of suspension culture should facilitate production of glutamate receptors for biochemical and structural studies.

Guanosine-5'-(3-O-thio)triphosphate-mediated stimulation of phosphoinositidase C in solubilized rat peripheral nerve myelin and its alteration in streptozotocin-induced diabetes.

The regulation of phosphoinositidase C (PIC) activity by guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) was characterized in a cholate-solubilized peripheral myelin-enriched fraction from rat sciatic nerve. The GTP analog maximally enhanced PIC-catalyzed hydrolysis of exogenous phosphatidylinositol-4,5-bisphosphate (PIP2) in a dose-dependent manner only within a narrow range of cholate concentrations. Maximal stimulation was attained at 0.6 microM GTP gamma S and could be completely prevented by 1 microM guanosine-5'-(2-O-thio)diphosphate. Neither adenylyl-imidodiphosphate nor adenosine triphosphate (ATP) enhanced PIC activity. Carbamoylcholine (1 mM) added together with GTP gamma S increased the extent of PIP2 hydrolysis over that elicited by GTP gamma S alone and this stimulation was blocked by the muscarinic receptor antagonist, atropine (50 microM). In detergent-solubilized myelin preparations from streptozotocin-induced diabetic rats, a higher concentration of the guanine nucleotide analog was required to achieve stimulation comparable to that obtained with corresponding preparations from normal animals. These results suggest that sciatic nerve myelin possesses muscarinic receptors coupled via a GTP-binding protein to PIC and that this system can be reconstituted in detergent-solubilized extracts. It is possible that the function of G proteins in cell signaling is impaired in experimental diabetic neuropathy.

Anti-idiotypes against an anti-haloperidol antibody bind to sigma receptors

Anti-idiotypic monoclonal antibodies that interact with the binding site of sigma receptors were generated. First, BALB/c mice were immunized with a haloperidol-bovine serum albumin conjugate, and monoclonal anti-haloperidol antibodies that recognize the piperidinyl moiety of haloperidol molecule were obtained. Second, for generation of anti-idiotypic antibodies, BALB/c mice were immunized with the anti-haloperidol monoclonal antibodies coupled to keyhole limpet hemocyanin. Anti-idiotypic antisera and three hybridomas secreting anti-idiotypic monoclonal antibodies were obtained. All of them were shown to inhibit [ 3H]haloperidol binding to the anti-haloperidol antibodies. The anti-idiotypes were potent in displacing the binding of [ 3H]haloperidol to rat brain sigma receptors. Furthermore, they significantly immunoprecipitated the sigma receptors from a detergent-solubilized preparation. These findings demonstrate the generation of anti-idiotypic monoclonal antibodies specifically interacting with membrane-bound and solubilized sigma receptors.

Reconstitution of channels from preparations enriched in lens gap junction protein MP70.

Detergent-solubilized ovine lens membrane proteins, enriched in the 70-kDa gap junction component (MP70), were reconstituted into planar lipid bilayers and analyzed for channel activities. Three distinct activities were found. Those showing conductance steps of 290 pS (symmetrical 150-mM KCl solutions) had properties similar to those reported earlier for MIP26 (Ehring, G.R., Zampighi, G., Horwitz, J., Bok, D., Hall, J.E. 1990. J. Gen. Physiol. 96:631-664.) of which minor amounts were normally present in the detergent-solubilized preparations. Two novel channel activities had unitary conductances of 90 and 45 pS, were halothane sensitive and did not discriminate between sodium and potassium ions. The 90-pS channel was asymmetrically voltage dependent, and its properties would be consistent with the expected properties of junctional hemichannels.

Specific antibody to the thyrotropin receptor identifies multiple receptor forms in membranes of cells transfected with wild-type receptor complementary deoxyribonucleic acid: characterization of their relevance to receptor synthesis, processing, structure, and function.

An antibody to a peptide of the TSH receptor, residues 352-366 which are not present in gonadotropin receptors, specifically identifies three major forms of the receptor on Western blots of detergent-solubilized membrane preparations from Cos-7 cells transfected with full-length rat and human TSH receptor cDNA: 230, 180, and 95-100 kilodaltons (kDa), based on simultaneously run protein standards. The 95- to 100-kDa protein is absent in cells transfected with a mutant receptor with no signal peptide and is sensitive to endoglycosidase-F. Its size is consistent with the sum of amino acids predicted from its cDNA sequence (84 kDa after subtracting the signal peptide) plus its carbohydrate content (14 kDa estimated from glycosylation mutants). It alone is absent in two deletion mutants that have lost TSH binding and activity after transfection: M1 missing residues 37-121 and M2 missing residues 110-307. It, thus, appears to be the processed glycosylated functional receptor on the cell surface. The 230-kDa protein is a nonprocessed form of the receptor, as evidenced by its insensitivity to endoglycosidase-F and its continued presence in cells transfected with a mutant receptor with no signal peptide. It is the primary form identified in rat FRTL-5 thyroid cells that have a functioning TSH receptor; it is not present in rat FRT thyroid cells with no functioning TSH receptor or receptor RNA. It appears, therefore, to be a early synthetic form of the functional TSH receptor. The 180-kDa protein is endoglycosidase-F sensitive and appears to be a processed intermediate between the 230-kDa early synthetic form and the 95- to 100-kDa functional receptor, rather than a dimer of the latter. Thus, with decreases in size appropriate to a receptor monomer, it remains present in membranes from the M1 and M2 deletion mutants that contain the 230-kDa protein but are missing the 95- to 100-kDa receptor form in association with lost TSH binding and activity after transfection. Minor receptor forms (54 kDa in rat receptor transfectants, 54 and 48 kDa in human receptor transfectants) appear to be degraded forms of the processed and glycosylated 95- to 100-kDa receptor. The presence or absence of reducing agents in the detergent solubilization mixture does not change the pattern or amount of the receptor forms recognized by the antibody, including the 54-kDa form; however, boiling does.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies to a C-terminal peptide of the rat brain glutamate receptor subunit, GluR-A, recognize a subpopulation of AMPA binding sites but not kainate sites

Antibodies were made to a thirteen amino acid synthetic peptide corresponding to the C-terminal portion of the glutamate (glu) receptor, GluR-A. The immunoprecipitation of kainic acid (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) binding sites by the anti-peptide antibodies was studied using a detergent-solubilized preparation of rat brain membranes. Under these conditions a subpopulation of AMPA binding sites was recognized by the antibodies, but no KA binding sites were recognized. Scatchard analysis of this subpopulation of AMPA binding sites yields a curvilinear plot which fits a two-site model with dissociation constants of 4.6 and 323 nM. These studies show that the glu receptor complex, GluR-A, binds AMPA but not KA and suggest that (i) the binding sites for these two ligands reside on different proteins, and (ii) the KA receptor identified physiologically is not equivalent to the KA binding sites identified with 3H-labelled KA.

Measurement of β-galactosyltransferase activity in cell extracts with an ELISA-based assay

An enzyme-linked immunosorbent assay (ELISA)-based glycosyltransferase assay has been used to measure UDP-Gal: N-acetylglucosamine β-1,4-galactosyl-transferase (EC 2.4.1.38) activity in detergent extracts of chinese hamster ovary (CHO) cells. LEC 11 cells (a mutant of the CHO cell line, Pro −5), which are known to express a complex array of carbohydrate structures, were used to develop the assay for use with whole cell extracts. A detergent-solubilized preparation of the enzyme from whole cells was used to convert the substrate, lactotriglycosylceramide, to the product, neolactotetraglycosylceramide. The monoclonal antibody, 1B2, which specifically binds to the Galβ1-4GlcNAc epitope, was used in an ELISA to identify and quantify the product. The enzyme activity in the preparations was found to be similar to that obtained by conventional radioactive assay methods. The β-galactosyltransferase found in LEC11 cell detergent extracts exhibited an absolute requirement for the nucleotide sugar and MnCl 2. The activity of the enzyme was also strictly dependent on the presence of exogenous glycolipid acceptor. When Triton X-114 was used to solubilize the LEC11 β-galactosyltransferase, activity was found in both the hydrophilic and the hydrophobic phases, suggesting the presence of two forms of the enzyme. The ELISA-based assay was used to compare β-1,4-galactosyltransferase activity in detergent extracts of four CHO cell lines: Pro −5, Lec1, LEC11, and LEC12 and in detergent-solubilized microsomes from human leukemia cells. The results from this study demonstrate the utility of the ELISA-based assay for measuring glycosyltransferase activity in detergent-solubilized whole cells and microsome preparations.

Binding and cross-linking of iodinated rat prolactin to rat hepatic prolactin receptor

Basal parameters for binding and cross-linking of 125I-rat prolactin (rPRL) to lactogenic (PRL) binding species present in crude membrane fraction (CMF) or detergent-solubilized preparations of rat liver have been investigated. (1) The highest specific binding to CMF was obtained with an incubation time of 50 h at 20 °C and with a 50 mM potassium phosphate buffer adjusted to pH 8.5. (2) Cross-linking of 125I-rPRL to binding sites in CMF with disuccinimidyl suberate (DSS) showed the autoradiographic appearance of an M r 40,000 binding species. (3) No specific binding or cross-Unking of rPRL was seen in Triton X-100-solubilized CMF. This is probably due to Triton X-100-induced changes in the physical properties of rPRL. (4) Specific binding of 125I-rPRL was detected in CHAPS-solubilized CMF. Following cross-linking the autoradiographic appearance of a binding species with an M r value of 40,000 was shown. 125I-hGH was cross-linked to three PRL binding species with M r 82,000, 40,000 and 35,000 in CHAPS-solubilized preparations. (5) In Golgi-enriched low-density membrane preparation 125I-rPRL was cross-linked to M r 82,000, 40,000 and 35,000 species. It is proposed that the inability of rPRL to be cross-linked to M r 82,000 and 35,000 species present in CHAPS-solubilized preparation is the result of CHAPS-induced changes of rPRL binding properties and low solubilizing capacity of CHAPS. (6) In conclusion, this study shows that also the iodinated endogenous hormone, rat prolactin, and not only hGH identifies high and low molecular forms of the rat liver prolactin receptor.

Biogenesis of cytochrome c1: Role of cytochrome c1 heme lyase and of the two proteolytic processing steps during import into mitochondria

The biogenesis of cytochrome c1 involves a number of steps including: synthesis as a precursor with a bipartite signal sequence, transfer across the outer and inner mitochondrial membranes, removal of the first part of the presequence in the matrix, reexport to the outer surface of the inner membrane, covalent addition of heme, and removal of the remainder of the presequence. In this report we have focused on the steps of heme addition, catalyzed by cytochrome c1 heme lyase, and of proteolytic processing during cytochrome c1 import into mitochondria. Following translocation from the matrix side to the intermembrane-space side of the inner membrane, apocytochrome c1 forms a complex with cytochrome c1 heme lyase, and then holocytochrome c1 formation occurs. Holocytochrome c1 formation can also be observed in detergent-solubilized preparations of mitochondria, but only after apocytochrome c1 has first interacted with cytochrome c1 heme lyase to produce this complex. Heme linkage takes place on the intermembrane-space side of the inner mitochondrial membrane and is dependent on NADH plus a cytosolic cofactor that can be replaced by flavin nucleotides. NADH and FMN appear to be necessary for reduction of heme prior to its linkage to apocytochrome c1. The second proteolytic processing of cytochrome c1 does not take place unless the covalent linkage of heme to apocytochrome c1 precedes it. On the other hand, the cytochrome c1 heme lyase reaction itself does not require that processing of the cytochrome c1 precursor to intermediate size cytochrome c1 takes place first. In conclusion, cytochrome c1 heme lyase catalyzes an essential step in the import pathway of cytochrome c1, but it is not involved in the transmembrane movement of the precursor polypeptide. This is in contrast to the case for cytochrome c in which heme addition is coupled to its transport directly across the outer membrane into the intermembrane space.

Function of the delipidated beta-adrenergic receptor appears to require a fatty acid or a neutral lipid in addition to phospholipids.

Detergent-solubilized preparations of the beta-adrenergic receptor (R) and of the guanyl nucleotide binding proteins (Gs) were extensively treated to remove phospholipids and cholesterol. Reconstitution of an R-Gs system was subsequently performed in the presence of a mixture of natural phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine or the synthetic dioleoyl derivatives of the same phospholipids. In both cases, an additional lipid was required for the agonist-dependent activation of Gs. The requirement could be fulfilled by alpha-tocopherol, or by unsaturated fatty acids such as oleic acid. Inclusion of this non-phosphorylated lipid in the reconstituted system enhanced the isoproterenol-dependent activation of Gs by guanosine 5'-O-[gamma-thio]triphosphate 16-33-fold. The rate of activation was largely dependent on the addition of the agonist. Efficient functional reconstitution of R-Gs was thus achieved in a totally defined lipid system. Additional studies of the reconstituted system and of the native membrane led to the notion that the non-phosphorylated lipid plays a role in the function of the hormone-R complex.