Introduction of a Low Molecular Weight Agonist Peptide for Complement C3a Receptor into Soybean Proglycinin A1aB1b Subunit by Site-directed Mutagenesis

LPLPR, a complement C3a agonist peptide, with hypocholesterolemic activity was introduced into the homologous site of soybean proglycinin A_1aB_1b subunit by site-directed mutagenesis. This modified proglycinin was expressed in E. coli and recovered from the insoluble fraction. LPLPR was released by the action of chymotrypsin and trypsin as expected. Furthermore, two peptides (RPSYLPLPR and PSYLPLPR) with extended sequence at the amino-terminus of LPLPR were obtained. Their ileum-contracting activity was 9 ～ 13 times stronger than that of LPLPR. The overall yields of purified LPLPR, RPSYLPLPR and PSYLPLPR were 25%, 12%, and 0.7% respectively.     

Introduction of enterostatin (VPDPR) and a related sequence into soybean proglycinin A1aB1b subunit by site-directed mutagenesis

Enterostatin (VPDPR), having anoretic and hypocholesterolemic activities, and its homologue LPYPR, a hypocholesterolemic peptide found in the glycinin A5A4B3 subunit, were introduced into the corresponding site (TNGPQ) of the proglycinin A1aB1b subunit by site-directed mutagenesis. Modified proglycinins were expressed in E. coli and recovered from the insoluble fraction. VPDPR and LPYPR were released by the action of chymotrypsin and trypsin as expected. The overall yields of purified VPDPR and LPYPR were 40% and 62%, respectively.     

Crystal structure of soybean proglycinin A1aB1b homotrimer

Soybean glycinin is a member of the 11 S globulin family. The crystal structure of proglycinin was determined by X-ray crystallography at 2.8 A resolution with an R-factor of 0.199 and a free R-factor of 0.250. A trimer molecule was found in an asymmetric unit of crystals. The trimer model contains three A1aB1b subunits and comprises 1128 amino acid residues and 34 water molecules. The constituent protomers of the homo-trimeric protein are arranged around a 3-fold symmetry axis with dimensions of 95 Ax95 Ax40 A. The protomer model is composed of five fragments which correspond roughly to conserved regions based on the sequence alignment of various 11 S globulins. The core of the protomer consists of two jelly-roll beta-barrels and two extended helix domains. This structure of proglycinin is similar to those of canavalin and phaseolin belonging to the 7 S globulin family, strongly supporting the hypothesis that both 7 S and 11 S globulins are derived from a common ancestor. The inter and intra-chain disulfide bonds conserved in the 11 S globulin family are clearly observed. It is found that the face with the inter-chain disulfide bond (IE face) contains more hydrophobic residues than that with the intra-chain disulfide bond. This suggests that a mature hexamer is formed by the interaction between the IE faces after processing.     

Amino acid sequence analysis of bitter peptides from a soybean proglycinin subunit synthesized in Escherichia coli

The cDNA encoding A1aB1b proglycinin was expressed in E. coli, for the efficient isolation of a single peptide responsible for the bitterness. The 55-kD proglycinin was highly purified, hydrolyzed, and further purified through a series of chromatographic steps to yield fractions with the major bitter peptides. The most bitter-tasting fractions contained peptides with average molecular weights lower than 1,700 Da. An analysis of the amino acid sequences indicated that many small bitter peptides (< 1,000 Da) are composed of uncharged polar amino acids as well as hydrophobic amino acids, with a charged residue often being present at either end. This suggests the involvement of a certain structural requirement in taste perception.     

Improved bile acid-binding ability of soybean glycinin A1a polypeptide by the introduction of a bile acid-binding peptide (VAWWMY)

We have previously identified a potential bile acid-binding peptide sequence (VAWWMY) in acidic polypeptide A1a of the soybean glycinin A1aB1b subunit (Choi, S. K., et al., Biosci. Biotechnol. Biochem., 66, 2395-2401 (2002)). In this study, we introduced the nucleotide sequence encoding this peptide in the coding DNA which corresponds to amino acids between 251 and 256, and 282 and 287 into the A1a polypeptide by replacement to respectively give modified versions A1aM1 and A1aM2. A fluorescence analysis demonstrates that their bile acid-binding ability was improved compared to A1a. Moreover, modified proglycinin A1aB1b with the VAWWMY sequence at the same sites as those of A1aM1 and A1aM2 was judged to assume the correct conformation. These results suggest the possibility of developing transgenic crops to accumulate the modified glycinin.     

Improved Bile Acid-binding Ability of Soybean Glycinin A1a Polypeptide by the Introduction of a Bile Acid-binding Peptide (VAWWMY)

We have previously identified a potential bile acid-binding peptide sequence (VAWWMY) in acidic polypeptide A1a of the soybean glycinin A1aB1b subunit (Choi, S. K., et al., Biosci. Biotechnol. Biochem., 66, 2395–2401 (2002)). In this study, we introduced the nucleotide sequence encoding this peptide in the coding DNA which corresponds to amino acids between 251 and 256, and 282 and 287 into the A1a polypeptide by replacement to respectively give modified versions A1aM1 and A1aM2. A fluorescence analysis demonstrates that their bile acid-binding ability was improved compared to A1a. Moreover, modified proglycinin A1aB1b with the VAWWMY sequence at the same sites as those of A1aM1 and A1aM2 was judged to assume the correct conformation. These results suggest the possibility of developing transgenic crops to accumulate the modified glycinin.     

Improvement of nutritional value and functional properties of soybean glycinin by protein engineering

Glycinin is one of the predominant storage proteins of soybean. To improve its functional properties (heat-induced gelation and emulsification) and/or nutritional value, the A1aB1b proglycinin subunit was modified on the basis of genetically variable domains suggested from the comparison of amino acid sequences of glycinin-type globulins from various legumes and nonlegumes and the relationships between the structure and the functional properties of glycinin. Thus, nucleotide sequences corresponding to each of the variable domains were deleted from the cDNA encoding the A1aB1b proglycinin, and a synthetic DNA encoding four continuous methionines was inserted into the cDNA region corresponding to each of the variable domains. Expression plasmids carrying the modified cDNAs were constructed and expressed in Escherichia coli strain JM105. Some of the modified proteins were accumulated as soluble proteins in the cells at a high level and self-assembled. They exhibited functional properties superior to those of the native glycinin from soybean, which establishes the possibility of creating theoretically designed novel glycinins with high food qualities.     

Protein disulfide isomerase family proteins involved in soybean protein biogenesis

Protein disulfide isomerase family proteins are known to play important roles in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disulfide isomerase family genes from soybean leaf (Glycine max L. Merrill cv. Jack) mRNA by RT-PCR using forward and reverse primers designed from the expressed sequence tag clone sequences. The cDNA encodes a protein of either 364 or 362 amino acids, named GmPDIS-1 or GmPDIS-2, respectively. The nucleotide and amino acid sequence identities of GmPDIS-1 and GmPDIS-2 were 68% and 74%, respectively. Both proteins lack the C-terminal, endoplasmic reticulum-retrieval signal, KDEL. Recombinant proteins of both GmPDIS-1 and GmPDIS-2 were expressed in Escherichia coli as soluble folded proteins that showed both an oxidative refolding activity of denatured ribonuclease A and a chaperone activity. Their domain structures were identified as containing two thioredoxin-like domains, a and a', and an ERp29c domain by peptide mapping with either trypsin or V8 protease. In cotyledon cells, both proteins were shown to distribute to the endoplasmic reticulum and protein storage vacuoles by confocal microscopy. Data from coimmunoprecipitation and crosslinking experiments suggested that GmPDIS-1 associates with proglycinin, a precursor of the seed storage protein glycinin, in the cotyledon. Levels of GmPDIS-1, but not of GmPDIS-2, were increased in cotyledons, where glycinin accumulates during seed development. GmPDIS-1, but not GmPDIS-2, was induced under endoplasmic reticulum-stress conditions.     

Role of posttranslational cleavage in glycinin assembly.

Glycinin, like other 11S seed storage proteins, undergoes a complex series of posttranslational events between the time proglycinin precursors are synthesized in endoplasmic reticulum and the mature glycinin subunits are deposited in vacuolar protein bodies. According to the current understanding of this process, proglycinin subunits aggregate into trimers in endoplasmic reticulum, and then the trimers move to the vacuolar protein bodies where a protease cleaves them into acidic and basic polypeptide chains. Stable glycinin hexamers, rather than trimers, are isolated from mature seeds. We used a re-assembly assay in this study to demonstrate that proteolytic cleavage of the proglycinin subunits is required for in vitro assembly of glycinin oligomers beyond the trimer stage. The possibility that the cleavage is a regulatory step and that it triggers the deposition of 11S seed storage proteins as insoluble aggregates in vivo is considered.     

Cosuppression of the alpha subunits of beta-conglycinin in transgenic soybean seeds induces the formation of endoplasmic reticulum-derived protein bodies

The expression of the alpha and alpha' subunits of beta-conglycinin was suppressed by sequence-mediated gene silencing in transgenic soybean seed. The resulting seeds had similar total oil and protein content and ratio compared with the parent line. The decrease in beta-conglycinin protein was apparently compensated by an increased accumulation of glycinin. In addition, proglycinin, the precursor of glycinin, was detected as a prominent polypeptide band in the protein profile of the transgenic seed extract. Electron microscopic analysis and immunocytochemistry of maturing transgenic soybean seeds indicated that the process of storage protein accumulation was altered in the transgenic line. In normal soybeans, the storage proteins are deposited in pre-existing vacuoles by Golgi-derived vesicles. In contrast, in transgenic seed with reduced beta-conglycinin levels, endoplasmic reticulum (ER)-derived vesicles were observed that resembled precursor accumulating-vesicles of pumpkin seeds and the protein bodies accumulated by cereal seeds. Their ER-derived membrane of the novel vesicles did not contain the protein storage vacuole tonoplast-specific protein alpha-TIP, and the sequestered polypeptides did not contain complex glycans, indicating a preGolgi and nonvacuolar nature. Glycinin was identified as a major component of these novel protein bodies and its diversion from normal storage protein trafficking appears to be related to the proglycinin buildup in the transgenic seed. The stable accumulation of proteins in a protein body compartment instead of vacuolar accumulation of proteins may provide an alternative intracellular site to sequester proteins when soybeans are used as protein factories.     

Determination of Optimal Protein Quantity Required to Identify Abundant and Less Abundant Soybean Seed Proteins by 2D-PAGE and MS

Optimizing the amounts of proteins required to separate and characterize both abundant and less abundant proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is critical for conducting proteomic research. In this study, we tested five different levels of soybean seed proteins (75, 100, 125, 150, and 200 μg) by 2D-PAGE. Following 2D-PAGE and spot excision, proteins were identified by mass spectrometry analysis. The number of visible protein spots was increased with an increase in the amount of protein loaded. The intensity of highly abundant proteins [β-conglycinin β-homotrimer and glycinin G4 (A5A4B3) precursors] increased linearly between 75 and 125 μg, whereas the proglycinin G3 (A1ab1b) homotrimer showed linearity between 75 and 150 μg. The spot intensity of less abundant proteins, glycinin G2 (A2b1a) precursor and proglycinin G3 (A1ab1b) homotrimer, increased linearly with an increase in the amount of protein through 200 μg, whereas spot intensity of β-conglycinin β-homotrimer and the allergen Gly m bd 28K increased linearly until 150 μg and did not increase further at 200 μg. These results suggest that 150 μg protein was a suitable amount for the separation of abundant proteins, and 200 μg protein was suitable for the separation of less abundant proteins prepared from soybean seeds. Mention of trade name, proprietary product or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or imply its approval to the exclusion of other products or vendors that also may be suitable.     

Improvement of protein quality in transgenic soybean plants

Glycinin is one of the abundant storage proteins in soybean seeds. A modified Gy1 (A1aB1b) proglycinin gene with a synthetic DNA encoding four continuous methionines (V3-1) was connected between the hpt gene and the modified green fluorescent protein sGFP(S65T) gene, and a resultant plasmid was introduced into soybean by particle bombardment in order to improve nutritional value of its seeds. After the selection with hygromycin, the efficiency of gene introduction was evaluated. More than 60 % of the regenerated plants tolerant to hygromycin yielded the hpt and V3-1 fragment by polymerase chain reaction (PCR) analysis, and the expression of sGFP was detected in about 50 % of putative transgenic soybeans. Southern hybridization confirmed the presence of transgenes in T0 plants and the transgenic soybeans hybridized with the hpt and V3-1 genes were analyzed showed different banding patterns. Most of the transgenic plants were growing, flowering normally and produced seeds. Analysis of seed obtained from transgenic soybean plants expressing hpt and V3-1 genes showed higher accumulation of glycinin compared with non-transgenic plants. In addition, protein expression in transgenic soybean plants was observed by using 2D-electrophoresis.     

Anti-HIV-1 Activity of Flavonoid Myricetin on HIV-1 Infection in a Dual-Chamber In Vitro Model

HIV infection by sexual transmission remains an enormous global health concern. More than 1 million new infections among women occur annually. Microbicides represent a promising prevention strategy that women can easily control. Among emerging therapies, natural small molecules such as flavonoids are an important source of new active substances. In this study we report the in vitro cytotoxicity and anti-HIV-1 and microbicide activity of the following flavonoids: Myricetin, Quercetin and Pinocembrin. Cytotoxicity tests were conducted on TZM-bl, HeLa, PBMC, and H9 cell cultures using 0.01–100 µM concentrations. Myricetin presented the lowest toxic effect, with Quercetin and Pinocembrin relatively more toxic. The anti-HIV-1 activity was tested with TZM-bl cell plus HIV-1 BaL (R5 tropic), H9 and PBMC cells plus HIV-1 MN (X4 tropic), and the dual tropic (X4R5) HIV-1 89.6. All flavonoids showed anti-HIV activity, although Myricetin was more effective than Quercetin or Pinocembrin. In TZM-bl cells, Myricetin inhibited ≥90% of HIV-1 BaL infection. The results were confirmed by quantification of HIV-1 p24 antigen in supernatant from H9 and PBMC cells following flavonoid treatment. In H9 and PBMC cells infected by HIV-1 MN and HIV-1 89.6, Myricetin showed more than 80% anti-HIV activity. Quercetin and Pinocembrin presented modest anti-HIV activity in all experiments. Myricetin activity was tested against HIV-RT and inhibited the enzyme by 49%. Microbicide activities were evaluated using a dual-chamber female genital tract model. In the in vitro microbicide activity model, Myricetin showed promising results against different strains of HIV-1 while also showing insignificant cytotoxic effects. Further studies of Myricetin should be performed to identify its molecular targets in order to provide a solid biological foundation for translational research.     

Preferential assignment of allotype a1 globulin for the production of early IgM anti-para-azobenzenearsonate antibodies in heterozygous a1a3 rabbits.

Rabbits of allotype a1a3 were injected on days 0, 2, and 4 with mixtures containing equal amounts of pigeon erythrocytes (Prbc) coupled to para-azobenzenearsonate (AA) and to para-azobenzene-N-trimethylammonium (TMA). On day 6, the allotypes of antibody from plaque-forming cells (PFC) of the blood were determined by observing the inhibition of plaque formation by anti-allotype sera. Anti-AA PFC appeared to consist for the most part of cells making antibody of allotype a1 since 65% of them were inhibited by anti-a1 serum and only 8% by anti-a3. Anti-TMA PFC, on the other hand, appeared to consist mostly of cells making antibody of allotype a3, since less than 1% of them were inhibited by anti-a1 but 47% by anti-a3. Antibody allotype for spleen PFC was also determined on day 6 and was similar to that found for blood PFC. Anti-AA PFC were inhibited 74% by anti-a1 serum and 15% by anti-a3 whereas anti-TMA PFC were inhibited 19% by anti-a1 and 43% by anti-a3. Serum hemolysin specific for AA hapten from a1a3 animals was also strongly inhibited by anti-a1 serum but not by anti-a3 whereas the converse was true for hemolysin against TMA hapten. The a1a3 rabbits, in whcih the anti-AA was restricted to allotype a1, were mated to produced homozygous a3a3 animals. When the PFC and serum antibodies of these a3a3 offspring were examined by specific inhibition, the anti-AA activity was found to be of allotype a3 rather than being a-negative. The number of anti-AA PFC in the blood of a3a3 rabbits was lower than that in blood of a1a3 or a1a1 animals. In addition, the TMA hapten appeared to inhibit the response to the AA hapten. Thus a1a3 rabbits immunized with AA-Prbc alone had 14-fold more anti-AA PFC or 18-fold higher anti-AA hemolysin titer than a3a3 animals immunized with both AA-Prbc and TMA-Prbc. Our results are discussed in relation to various explanations which have been offered for an imbalance of allotypes in a given antibody.     

High-level heterologous expression and properties of a novel lipase from Ralstonia sp. M1

The mature lipase LipA and its 56aa-truncated chaperone DeltaLipBhis (with 6xhis-tag) from Ralstonia sp. M1 were over-expressed in Escherichia coli BL21 under the control of T7 promoter with a high level of 70 and 12mg protein per gram of wet cells, respectively. The simply purified lipase LipA was effectively refolded by Ni-NTA purified chaperone DeltaLipBhis in molar ratio 1:1 at 4 degrees C for 24 hours in H2O. The in vitro refolded lipase LipA had an optimal activity in the temperature range of 50-55 degrees C and was stable up to 45 degrees C with more than 84% activity retention. The maximal activity was observed at pH 10.75 for hydrolysis of olive oil and found to be stable over alkaline pH range 8.0-10.5 with more than 52% activity retention. The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine (remaining activity 137-334%), but inhibited by 1-butanol and acetonitrile (40-86%). Metal ions Cu2+, Sn2+, Mn2+, Mg2+, and Ca2+ stimulated the lipase slightly with increase in activity by up to 22%, whereas Zn2+ significantly inhibited the enzyme with the residual activity of 30-65% and Fe3+ to a lesser degree (activity retention of 77-86%). Tween 80, Tween 60, and Tween 40 induced the activation of the lipase LipA (222-330%) and 0.2-1% (w/v) of Triton X-100, X-45, and SDS increased the lipase activity by up to 52%. However, 5% (w/v) of Triton X-100, X-45, and SDS inhibited strongly the activity by 31-89%. The inhibitors including DEPC, EDTA, PMSF, and 2-mercaptoethanol (0.1-10mM) inhibited moderately the lipase with remaining activity of 57-105%. The lipase LipA hydrolyzed a wide range of triglycerides, but preferentially short length acyl chains (C4 and C6). In contrast to the triglycerides, medium length acyl chains (C8 and C14) of p-nitrophenyl (p-NP) esters were preferential substrates of this lipase. The enzyme preferentially catalyzed the hydrolysis of cottonseed oil (317%), cornoil (227%), palm oil (222%), and wheatgerm oil (210%) in comparison to olive oil (100%).     

Isolation, purification, and characterization of a thermostable xylanase from a novel strain, Paenibacillus campinasensis G1-1

High levels of xylanase activity (143.98 IU/ml) produced by the newly isolated Paenibacillus campinasensis G1-1 were detected when it was cultivated in a synthetic medium. A thermostable xylanase, designated XynG1-1, from P. campinasensis G1-1 was purified to homogeneity by Octyl-Sepharose hydrophobic-interaction chromatography, Sephadex G75 gel-filter chromatography, and Q-Sepharose ion-exchange chromatography, consecutively. By multistep purification, the specific activity of XynG1-1 was up to 1,865.5 IU/mg with a 9.1-fold purification. The molecular mass of purified XynG1-1 was about 41.3 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sequence analysis revealed that XynG1-1 containing 377 amino acids encoded by 1,134 bp genomic sequences of P. campinasensis G1-1 shared 96% homology with XylX from Paenibacillus campinasensis BL11 and 77%~78% homology with xylanases from Bacillus sp. YA- 335 and Bacillus sp. 41M-1, respectively. The activity of XynG1-1 was stimulated by Ca2+, Ba2+, DTT, and beta- mercaptoethanol, but was inhibited by Ni2+, Fe2+, Fe3+, Zn2+, SDS, and EDTA. The purified XynG1-1 displayed a greater affinity for birchwood xylan, with an optimal temperature of 60 degrees C and an optimal pH of 7.5. The fact that XynG1-1 is cellulose-free, thermostable (stability at high temperature of 70 degrees C~80 degrees C), and active over a wide pH range (pH 5.0~9.0) suggests that the enzyme is potentially valuable for various industrial applications, especially for pulp bleaching pretreatment.     

A moderate increase in carnitine palmitoyltransferase 1a activity is sufficient to substantially reduce hepatic triglyceride levels

Nonalcoholic fatty liver disease (NAFLD), hypertriglyceridemia, and elevated free fatty acids are present in the majority of patients with metabolic syndrome and type 2 diabetes mellitus and are strongly associated with hepatic insulin resistance. In the current study, we tested the hypothesis that an increased rate of fatty acid oxidation in liver would prevent the potentially harmful effects of fatty acid elevation, including hepatic triglyceride (TG) accumulation and elevated TG secretion. Primary rat hepatocytes were transduced with adenovirus encoding carnitine palmitoyltransferase 1a (Adv-CPT-1a) or control adenoviruses encoding either beta-galactosidase (Adv-beta-gal) or carnitine palmitoyltransferase 2 (Adv-CPT-2). Overexpression of CPT-1a increased the rate of beta-oxidation and ketogenesis by approximately 70%, whereas esterification of exogenous fatty acids and de novo lipogenesis were unchanged. Importantly, CPT-1a overexpression was accompanied by a 35% reduction in TG accumulation and a 60% decrease in TG secretion by hepatocytes. There were no changes in secretion of apolipoprotein B (apoB), suggesting the synthesis of smaller, less atherogenic VLDL particles. To evaluate the effect of increasing hepatic CPT-1a activity in vivo, we injected lean or obese male rats with Adv-CPT-1a, Adv-beta-gal, or Adv-CPT-2. Hepatic CPT-1a activity was increased by approximately 46%, and the rate of fatty acid oxidation was increased by approximately 44% in lean and approximately 36% in obese CPT-1a-overexpressing animals compared with Adv-CPT-2- or Adv-beta-gal-treated rats. Similar to observations in vitro, liver TG content was reduced by approximately 37% (lean) and approximately 69% (obese) by this in vivo intervention. We conclude that a moderate stimulation of fatty acid oxidation achieved by an increase in CPT-1a activity is sufficient to substantially reduce hepatic TG accumulation both in vitro and in vivo. Therefore, interventions that increase CPT-1a activity could have potential benefits in the treatment of NAFLD.     

Immunologic studies with LFA-1- and Mo1-deficient lymphocytes from a patient with recurrent bacterial infections

A patient and his parents, deficient for lymphocyte function associated antigen-1 (LFA-1) and Mo1 (OKM1), were studied with respect to leukocyte surface marker expression and functional properties. The patient had a history of severe recurrent bacterial infections. Two siblings had already died of bacterial infections. The patient's granulocytes, monocytes, and lymphocytes expressed low but detectable amounts (less than or equal to 10%) of LFA-1 and Mo1. Intracellularly, LFA-1 and Mo1 (OKM1) were detectable and LFA-1 expression was enhanced on patient T cells stimulated with phytohemagglutinin. Granulocytes and monocytes of both the patient's parents expressed markedly decreased amounts of LFA-1 and Mo1. Lymphocytes of the mother expressed 40 to 60% of the amount of LFA-1 expressed on control lymphocytes, but his father's lymphocytes showed a normal LFA-1 expression. Granulocytes of the patient and of his deceased sister showed normal phagocytosis, but they had a dysfunction in the activation of the oxidative metabolism. Functional activities mediated by patient T cells were all normal. Moreover, all lymphocyte functions, including killer (K), natural killer (NK), cytotoxic T cell activity, helper activity for in vitro immunoglobulin (Ig) production by normal B cells, and PHA-induced proliferation were inhibitable by anti-LFA-1 monoclonal antibodies. K and NK activity mediated by patient leukocytes was 100-fold more sensitive to the inhibiting effect of anti-LFA-1 antibody than K and NK activity of normal donor leukocytes. Thus, although the amount of LFA-1 expressed was strongly reduced, it was still sufficient and required for the functional activity exhibited by patient T cells. The major functional defect observed with leukocytes of the patient and his father was an apparent B cell defect. B cells of the father and of the patient failed to produce Ig in the pokeweed mitogen (PWM)-driven system. The B cells of patient and of his father only produced Ig when cultured with T cells of the father, and not with normal donor T cells or T cells of the mother, in the presence of exogenous interleukin 2 (IL 2). In addition, the father's B cells produced Ig when cocultivated with patient T cells in the IL 2-driven system. This restriction of helper T cell activity is noteworthy because PWM- and IL 2-driven Ig synthesis by normal lymphocytes show no histocompatibility requirements between cooperating T and non-T cell populations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of a new acidic glutathione S-transferase, GST-Yn1Yn1, with a high leukotriene-C4 synthase activity from rat brain

A new acidic form of glutathione S-transferase (GST, pI 6.2) was purified from rat brain by S-hexylglutathione affinity chromatography followed by chromatofocusing. This form occupied 20-25% of the total activity bound to the affinity column. It had a molecular mass (subunit 26 kDa) similar to that of a major GST form of rat testis (MT or 6-6) on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. However, it differed from the MT in isoelectric point, activity towards 1,2-dichloro-4-nitrobenzene and immunological properties. On two-dimensional gel electrophoresis the brain form gave a spot which was identical in molecular mass, isoelectric point and immunological properties to a less acidic one (Yn1) of two spots (Yn1 and Yn2) of the testis GST-MT. Therefore, the brain acidic form is a homodimer, and named GST-Yn1Yn1. The activity was inhibited by sulfasalazine, an inhibitor of leukotriene-C4 synthase. This form (GST-Yn1Yn1) showed the highest leukotriene-C4 synthase activity, 496 nmol/mg protein in 5 min, among nine cytosolic GST isoenzymes from the rat. The Km values for leukotriene A4 and glutathione were 26 microM and 3.5 mM respectively. A major GST form of rat brain, occupying about 40% of the total activity, was identical with GST-P (7-7) purified from rat liver bearing preneoplastic hyperplastic nodules and localized at astroglias. GST-P also showed the significant leukotriene-C4 synthase activity, 67.2 nmol/mg protein in 5 min, but the Km for leukotriene A4 was 100 microM, fourfold higher than that of GST-Yn1 Yn1. These results suggest that mainly GST-Yn1 Yn1 may be involved in leukotriene-C4 synthesis in rat brain.