The sucrose carrier of the plant plasmalemma. III. Partial purification and reconstitution of active sucrose transport in liposomes.

The proteins from plasma membranes from sugar beet leaves were solubilized by 1% CHAPS and separated by size exclusion chromatography and by ion-exchange chromatography. The fractions enriched in sucrose transporter were monitored in three ways: differential labeling, ELISA, and reconstitution in proteoliposomes. When the plasma membranes were differentially labeled by N-ethylamaleimide in the presence of sucrose, a major peak of differential labeling was found at 120 kDa upon gel filtration. When this peak was recovered, denaturated by sodium dodecyl sulfate and reinjected on the gel filtration column, it yielded a peak of differential labeling at 42 kDa. When unlabeled membranes were used, the fractions eluted from the column were monitored by ELISA for their ability to recognize a serum directed against a 42 kDa previously identified as a putative sucrose carrier. The results paralleled those obtained by differential labeling, i.e. a major ELISA-reactive peak was found at 120 kDa upon gel filtration, and this peak yielded a peak most reactive at 40 kDa after denaturation. The 120 kDa peak prepared from unlabeled membranes was further separated on a Mono-Q column. The fractions were monitored by ELISA as described above, and reconstituted into proteoliposomes using asolectin. Active transport of sucrose, but not of valine could be observed with the reconstituted 120 kDa fraction. When the eluates from the Mono-Q column were reconstituted, the fractions exhibiting highest transport activity were enriched with a 42 kDa band. The data provide the first report concerning reconstitution of sucrose transport activity and confirm the involvement of a 42 kDa polypeptide in sucrose transport.     

Polypeptide composition of the 8S form of prolyl-tRNA synthetase from rat liver.

Rat liver Fraction X containing the 24S complex of nine aminoacyl-tRNA synthetases, including prolyl-tRNA synthetase, was centrifuged on a 15-35% sucrose density gradient to obtain the 8S form of prolyl-tRNA synthetase. The enzyme was purified on a prolyldiaminohexyl-Sepharose 4B affinity column, specifically binding prolyl-tRNA synthetase to Sepharose-bound proline. After SDS-polyacrylamide gel electrophoresis, two peptides of 58 and 61 kDa were detected in the peak of prolyl-tRNA synthetase activity eluted from the affinity column. The 58 and 61 kDa peptides were also present in the 24S complex containing prolyl-tRNA synthetase activity isolated on the sucrose density gradient.     

Ribonucleic acid-dependent ribonucleic acid polymerase in the immune response.

Ribonucleic acid (RNA)-dependent RNA polymerase activity was demonstrated in the microsomal and ribosomal fraction from the spleen cells of immunized mice. The enzyme activity was solubilized by Triton X-100 from the fraction and partially purified by Biogel A 1.5 m column chromatography. The RNA-dependent RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RAN preparation (iotaRNA) as template made from the spleens of immunized mice but very low activity was found with an RNA preparation made from the spleens of normal mice. Incorporation of 3H-UTP markedly decreased in the presence of RNase but not in the presence of DNase. DNA preparations made from the spleens of immunized mice were inactive as template for this enzyme. The iotaRNA preparation was fractionated by sucrose density gradient centrifugation. A fraction corresponding to 12-13 S was most active as a template. It was followed by a fraction corresponding to 6-7 S. Sucrose gradient analysis of the 3H-UTP-labeled product was attempted. Some properties of this enzyme are described.     

Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts

The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex.     

Characterization of monoclonal antibodies to protoplast membranes of Nicotiana tabacum identified by an enzyme-linked immunosorbent assay

Murine monoclonal antibodies to protoplast membrne antigens were generated using mouse myelomas and spleen cells from mice immunized with Nicotiana tabacum L. leaf protoplasts. For selecting antibody-secreting clones, a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for monoclonal antibody binding to immobilized cellular membrane preparations or immobilized protoplasts was developed. With intact protoplasts as immobilized antigen, the ELISA is selective for antibodies that bind to plasma-membrane epitopes present on the external surface of protoplasts. Using the membrane ELISA, a total of 24 hybridoma lines were identified that secreted antibodies to plant membrane epitopes. The protoplast ELISA and subsequent immunofluorescence studies identified four hybridoma lines as secreting antibodies which bound to the external surface of protoplasts and cells. The corresponding antigens were not species- or tissue-specific, were periodatesensitive, and were located in membranes which equilibrated broadly throughout a linear sucrose gradient. When protein blots of electrophoretically separated membrane proteins were probed with these antibodies, a band of M_r 14 kilodaltons (kDa) and a smear of bands of M_r 45–120 kDa were labeled. An additional set of three antibodies appeared by immunofluorescence to bind to the plasma membrane of broken but not intact protoplasts and labeled membranes equilibrating at a density of approx. 1.12 kg·l^-1 in a linear sucrose density gradient. These classes of monoclonal antibodies enlarge the library of monoclonal antibodies (Norman et al. 1986, Planta 167, 452–459) available for the study of plant plasma-membrane structure and function.Abbreviations ELISA Enzyme-linked immunosorbent assay - Ig immunoglobulin - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Identification and Purification of Calcium-Independent Phospholipase A2 from Bovine Brain Cytosol

Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KCl. However, when buffer with 1 M KCl was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero-3-phosphoethanolamine, whereas the latter was active with both diacylglycero-3-phosphoethanolamine and 1-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel filtration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high-resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40-kDa molecular mass and two minor bands with 14- and 25-kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high-resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

MULTIPLE FORMS OF PROTEIN KINASES IN MYELIN AND MICROSOMAL FRACTIONS OF BOVINE BRAIN

Abstract— The activity profiles of the solubilized protein kinases from the microsomal and myelin fractions of bovine brain were examined by column chromatography and sucrose density gradient centrifugation. The main peak of adenosine 3',5'-monophosphate (cyclic AMP)-dependent activity with histone as substrate for each membrane enzyme was eluted with about 0.2 m -NaCl on a DEAE-cellulose column. A peak of activity stimulated with cyclic AMP was also eluted with about 0.1 m -NaCl for the microsomal enzyme. A peak with protamine and casein as substrate for the microsomal or myelin enzyme, respectively, was larger than that with histone as substrate for each enzyme. The first peak with histone as substrate on a DEAE–cellulose column appeared as two peaks on the Sepharose 6B column. The second peak with histone as substrate on DEAE–cellulose column was shown to be a holoenzyme consisting of regulatory and catalytic subunits. The holoenzyme and subunits were eluted at similar positions to each other between both membrane enzymes on Sepharose 6B column. The holoenzyme sedimented as two peaks of activity on sucrose density gradient centrifugation, both of which were stimulated with cyclic AMP. The preincubation of the holoenzyme with cyclic AMP resulted in shifting to a position of a smaller molecular size.
The results indicate the occurrence of multiple forms of protein kinases in membrane fractions of brain with respect to substrate specificity and physical property.     

Expressing and purifying membrane transport proteins in high yield

Structural analysis of native or recombinant membrane transport proteins has been hampered by the lack of effective methodologies to purify sufficient quantities of active protein. We addressed this problem by expressing a polyhistidine tagged construct of the cardiac sodium-calcium exchanger (NCX1) in Trichoplusia ni larvae (caterpillars) from which membrane vesicles were prepared. Larvae vesicles containing recombinant NCX1-his protein supported NCX1 transport activity that was mechanistically not different from activity in native cardiac sarcolemmal vesicles although the specific activity was reduced. SDS-PAGE and Western blot analysis demonstrated the presence of both the 120 and 70 kDa forms of the NCX1 protein. Larvae vesicle proteins were solubilized in sodium cholate detergent and fractionated on a chelated Ni(2+) affinity chromatography column. After extensive washing, eluted fractions were mixed with soybean phospholipids and reconstituted. The resulting proteoliposomes contained NCX1 activity suggesting the protein retained native conformation. SDS-PAGE revealed two major bands at 120 and 70 kDa. Purification of large amounts of active NCX1 via this methodology should facilitate biophysical analysis of the protein. The larva expression system has broad-based application for membrane proteins where expression and purification of quantities required for physical analyses is problematic.     

Partial Isolation of Two Classes of Dopamine β-Hydroxylase-Containing Particles Undergoing Rapid Axonal Transport in Rat Sciatic Nerve

The rapid bidirectional transport of dopamine beta-hydroxylase (DBH) in adrenergic axons provides a means of analyzing the life cycle of adrenergic storage vesicles. We compared the physical characteristics of DBH-containing particles traveling to or returning from the terminal varicosities of ligated rat sciatic nerves. Density gradient centrifugation and Sephacryl S1000 gel-permeation chromatography were used to fractionate extracts from nerve segments proximal or distal to the ligatures. A series of experiments indicated the existence of at least two populations of rapidly transported DBH-containing particles, a "light" 85-nm particle and a larger "dense" 120-nm particle. The 85-nm particles were prevalent in unligated nerve, but accounted for only one-third of the total anterogradely transported DBH activity accumulated after 18 h. The 120-nm particles were barely detectable in the unligated nerve, but they accumulated at twice the rate of the 85-nm particles and accounted for the rest of the anterogradely transported particulate DBH activity. These two populations of particles were readily isolated from proximal nerve extracts by sucrose density gradient centrifugation. Similar-appearing dense and light peaks of particulate DBH activity were obtained from distal nerve extracts. Much of the retrogradely transported DBH of the extracts, however, was associated with large particles (greater than 300 nm) not resolved by Sephacryl S1000. Retrogradely transported exogenous NGF was found only in the dense sucrose gradient peak. We propose that the 85-nm DBH-containing particles correspond to "large dense-cored vesicles," and that the 120-nm particles are derived from the dense tubules visualized in adrenergic nerves by the chromaffin reaction.     

Evidence for two distinct adenylate cyclase catalysts in rat brain

The Lubrol-soluble adenylate cyclase activity of brain synaptosomal membranes appeared, upon gel filtration or sucrose gradient centrifugation, as two overlapping peaks. Fractions corresponding to the peak of the largest Stokes radius (Biogel pool 1) or highest s value (gradient pool 1) contained an adenylate cyclase activity which could be detected whatever the enzyme assay conditions. In contrast, in fractions from the second peak (Biogel pool 2 or gradient pool 2), forskolin was needed to reveal adenylate cyclase activity. The enzyme activity of each Biogel pool was retained by forskolin-agarose and eluted by forskolin with a 34-83% yield. A polypeptide of 155 kDa made up 80% of the forskolin-agarose eluate 1, whereas it was almost absent from eluate 2. Since data from various groups point to the 155 kDa polypeptide as a brain adenylate cyclase catalyst, still another distinct catalyst of lower molecular mass is likely to be present in brain.     

Isolation and characterization of oxygen-evolving thylakoid membranes and photosystem II particles from a glaucocystophyte, Cyanophora paradoxa

Conditions for preparing oxygen-evolving thylakoid membranes and PSII complexes, and those for observing the PSII activity were investigated in a glaucocystophyte, Cyanophora paradoxa. The active thylakoid membranes were isolated either with a medium containing glycerol or with that containing high concentrations of sucrose, phosphate, and citrate. Active PSII particles were solubilized by octyl-beta-D-glucoside from thylakoid membranes and were separated by sucrose density gradient centrifugation. The thylakoid membranes and PSII particles showed an oxygen-evolving activity only in high-ionic-strength media. The extrinsic 33 kDa protein (PsbO) and the cytochrome c(550) (PsbV) were found to be present in the PSII particles as in cyanobacteria or red algae, but no 12 kDa protein (PsbU) was detected. The PsbO protein was classified as a land-plant type by its N-terminal amino acid sequence.     

Ultrastructure, fractionation and biochemical analysis of Cryptosporidium parvum sporozoites

Sporozoites of the apicomplexan parasite Cryptosporidium parvum were subjected to cell disruption and subcellular fractionation using a sucrose density step gradient. With this procedure, highly enriched preparations of the parasite membrane, the micronemes, dense granules and amylopectin granules were produced. No separate fraction containing rhoptries was obtained, however this organelle was found in defined fractions of the gradient, still associated with the apical tip of the sporozoites. Using negative staining, the internal structure of the micronemes was revealed by transmission electron microscopy. Micronemes and dense granules showed characteristic protein compositions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The micronemes contained three major proteins of approximately 30, 120 and 200 kDa and the dense granules contain five major proteins in the 120-180 kDa range.     

Evidence for the presence of a very high concentration of arylsulfatase A in the pig thyroid: identification of arylsulfatase A subunits as the two major glycoproteins in purified thyroid lysosomes

In addition to their general function in cellular homeostasis, thyroid lysosomes play an essential role in the biosynthesis of thyroid hormones by cleaving the macromolecular prohormone, thyroglobulin. In the present work, we have attempted to determine whether the enzyme composition of thyroid lysosomes differs from that of lysosomes from other tissues. Lysosomal enzymes, cathepsin D, beta-D-galactosidase, beta-D-glucosidase, alpha-D-mannosidase, alpha-L-fucosidase, hexosaminidase, and arylsulfatase A and B, were assayed in crude fractions from various pig tissues, heart, brain, liver, kidney, thyroid, adrenals, ovary, and spleen. It appeared that the specific activity of arylsulfatase A was at least 20 times higher in the thyroid than in most other tissues. Thyroid lysosomes purified by isopycnic centrifugation on Percoll gradients contained two major polypeptides with apparent molecular weights of 58,000 and 54,000 representing about 30% of the total protein. These polypeptides were glycosylated and were exclusively found in the intralysosomal soluble fraction obtained by osmotic pressure-dependent lysis. By fractionating intralysosomal soluble proteins by velocity sedimentation on sucrose gradients or gel permeation chromatography we identified a thyroid arylsulfatase A holoenzyme which corresponds to a 120,000 Mr species. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of the gradient or column fractions showed that the 120-kDa protein peak with arylsulfatase A activity essentially contained the 58- and 54-kDa polypeptides in equivalent amounts. In conclusion, arylsulfatase A, a heterodimer of 120 kDa composed of two nonidentical subunits, is the major protein component of thyroid lysosomes. The superabundance of this protein in purified thyroid lysosomes is related to the very high specific activity of the enzyme in the thyroid as compared to other tissues.     

Subunit composition of vacuolar membrane H(+)-ATPase from mung bean

The vacuolar H(+)-ATPase from mung bean hypocotyls was solubilized from the membrane with lysophosphatidycholine and purified by QAE-Toyopearl column chromatography. The purified ATPase was active only in the presence of exogenous phospholipid and was inhibited by nitrate, dicyclohexyl carbodiimide and Triton X-100, but not by vanadate or azide. Dodecyl sulfate/polyacrylamide gel electrophoresis of the purified ATPase yielded ten polypeptides of molecular masses of 68 kDa, 57 kDa, 44 kDa, 43 kDa, 38 kDa, 37 kDa 32 kDa, 16 kDa, 13 kDa and 12 kDa. All polypeptides remained in the peak activity fraction after glycerol density gradient centrifugation. Nine of them, excluding the 43-kDa polypeptide, comigrated in a polyacrylamide gradient gel in the presence of 0.1% Triton X-100. The 16-kDa polypeptide could be labeled with [14C]dicyclohexylcarbodiimide. The amino-terminal amino acid sequence of the isolated 68-kDa polypeptide generally agreed with that deduced from the cDNA for the carrot 69-kDa subunit [Zimniak, L., Dittrich, P., Gogarten, J. P., Kibak, H. & Taiz, L. (1988) J. Biol. Chem. 263, 9102-9112]. Thus, mung bean vacuolar H(+)-ATPase seems to consist of nine distinct subunits.     

Analysis of rat uterine estrogen receptors by high-pressure liquid chromatography methods

Cytosolic (ERc) and nuclear (ERn) estrogen receptors prepared from rat uteri were characterized by size-exclusion and ion-exchange HPLC. The oligomeric ERc eluted as a single, sharp peak near the exclusion volume of the gel column; ERn eluted as a broad peak. When salt-extracted ERn was partially purified sequentially by Sephadex G-200, DEAE-cellulose chromatography and polyacrylamide gel electrophoresis, the partially purified receptor moieties were not distinguishable by the sucrose gradient method, but showed characteristic retention times in the size-exclusion HPLC column. Further distinction in net surface charges was observed between ERc and ERn moieties by ion-exchange high-pressure liquid chromatography (HPLC). Molybdate-stabilized ERc was eluted as sharp peak at 0.27 M salt gradient. In contrast, fresh extracts of ERn emerged as a broad peak in the region of 0.1-0.2 M salt gradient. In the absence of molybdate, ERc dissociated into several 4-5 S molecules, which were well resolved in the DEAE column. This report, therefore, demonstrates the usefulness of size-exclusion and ion-exchange HPLC for steroid receptor analysis.     

Monitoring Phloem Unloading and Post-Phloem Transport by Microperfusion of Attached Wheat Grains

Phloem unloading and post-phloem transport in developing wheat (Triticum aestivum L.) grains were investigated by perfusing the endosperm cavities of attached grains. Relative unloading ratio (RUR) and the rate of sucrose release into the endosperm cavity (SRR) were calculated, respectively, from 14C import and from sucrose washout from the cavity. RUR and SRR continued at or near in vivo rates over a wide range of cavity sap osmolality (90 to approximately 500 milliosmolal) and sucrose concentration (14-430 mM) and for long times (29 h). These are much greater ranges than have been observed for the endosperm cavity in vivo (230-300 milliosmolal, and 40-120 mM, respectively), indicating that neither the cavity sap osmolality nor sucrose concentration are controlling factors for the rate of assimilate import into the cavity. The maintenance of in vivo transport rates over a wide range of conditions strongly implicates the role of transport processes within the maternal tissues of the wheat grain, rather than activities of the embryo or endosperm, in determining the rate of assimilate import into the grain. RUR was decreased by high concentrations of sucrose and sorbitol, but not of mannitol. By plasmolyzing some chalazal cells, sorbitol appeared to block symplastic transport across the crease tissues, but neither sucrose nor mannitol caused plasmolysis in maternal tissues of attached grains. The inhibition of RUR by KCN and carbonyl cyanide m-chlorophenyl (CCCP) and the continued import of sucrose into grains against its concentration gradient suggest that solute movement into the endosperm cavity might occur by active membrane transport. However, the evidence is weak, since KCN and CCCP appeared to act primarily on some aspect of symplastic (i.e. nonmembrane) transport. Also, sucrose could move from the endosperm cavity into the maternal tissues (i.e. opposite to the normal direction of sucrose movement), suggesting that transmembrane movement in the nucellus may be a reversible process. Pressure-driven flow into the grain could account for movement against a concentration gradient.     

Fluorescence monitoring of flavins during preparation of respiratory membranes

Azotobacter vinelandii large and small membrane particles were examined by fluorescence spectroscopy through purification to qualitatively monitor contamination by non-respiratory flavin. Flavin was analyzed by observing the effects of reduction by dithionite or NAD(P)H and subsequent oxidation. Flavin of the large particles did not change significantly with purification on a sucrose gradient. The small particle or R3 fraction contained relatively large amounts of non-respiratory flavin. Small particles eluted from a Sepharose CL-6B column with a fluorescence peak but still contained contaminating flavin. After centrifugation on a sucrose gradient, the flavin of these particles was essentially the same as the large particles. This method is an improvement over just observation of fluorescence intensity for monitoring flavoprotein purity of membrane particle preparations.     

Proton-Coupled Sucrose Transport in Plasmalemma Vesicles Isolated from Sugar Beet (Beta vulgaris L. cv Great Western) Leaves

Sucrose is the predominant form of photosynthetically reduced carbon transported in most plant species. In the experiments reported here, an active, proton-coupled sucrose transport system has been identified and partially characterized in plasmalemma vesicles isolated from mature sugar beet (Beta vulgaris L. cv Great Western) leaves. The isolated vesicles concentrated sucrose fivefold in the presence of an imposed pH gradient (basic interior). The presence of carbonyl cyanide m-chlorophenylhydrazone, a protonophore, prevented sucrose accumulation within the vesicles. ΔpH-dependent sucrose transport exhibited saturation kinetics with an apparent K_m of 1.20 ± 0.40 millimolar, suggesting translocation was carrier-mediated. In support of that conclusion, two protein modifiers, diethyl pyrocarbonate and p-chloromercuribenzenesulfonic acid, were found to be potent inhibitors with 50% inactivation achieved at 750 and 30 micromolar, respectively. ΔpH-Dependent sucrose transport was not inhibited by glucose, fructose, raffinose, or maltose suggesting the transport system was specific for sucrose. Transport activity was associated with the plasmalemma because ΔpH-dependent sucrose transport equilibrated on a linear sucrose gradient at 1.17 grams per cubic centimeter and comigrated with a plasmalemma enzyme marker, vanadate-sensitive K⁺, Mg²⁺-ATPase. Taken together, these results provide the first In vitro evidence in support of a sucrose-proton symport in the plasmalemma of mature leaf tissue.     

On the homogeneity of 11-S acetylcholinesterase

11-S acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) purified by affinity chromatography of trypsin-digested homogenates was shown to be contaminated with three other active forms of enzyme. The initial purification used an affinity column of the inhibitor, N-methylacridinium ion. Chromatography of the "affinity-pure" sample on hydroxyapatite resulted in two peaks of acetylcholinesterase activity. One peak contained only a form sedimenting at 11-S (approx. 85% of the recovered activity). The other peak consisted of a 9.5-S form, in addition to 14-S and 18-S forms. The 9.5-S form (approx. 7% of the activity) co-electrophoresed with 11-S in 6% polyacrylamide gels and co-sedimented with the same form in sucrose density gradients containing 0.1 M NaCl. The purified 11-S enzyme was shown to be homogeneous by sucrose density gradient centrifugation and electrophoresis. These results indicate that 11-S acetylcholinesterase may be unsuitable for some characterization studies due to undetected contamination by the 9.5-S form.