Interference with the determination of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and its properties by a microsomal enzymic activity.

Rat liver microsomes and microsomal extracts contain an enzymic activity which competes with 3-hydroxy-3-methylglutaryl coenzyme A reductase for 3-hydroxy-3-methylglutaryl coenzyme A. The presence of this activity in enzyme preparations causes errors in the determination of reductase activity and its properties. This contaminant can be removed by gel filtration using Bio-Gel A 1.5m, by washing the microsomes, or by incubating the microsomal extract at 37 °C. The K_m's of the reductase (free of this competing enzymic activity) for d-3-hydroxy-3-methylglutaryl coenzyme A and NADPH are 1.3 and 26 μm, respectively.     

Production of a monoclonal antibody directed against rat liver glutathione-insulin transhydrogenase

A hybridoma cell line secreting monoclonal antibody specific for glutathione-insulin transhydrogenase has been produced by fusing mouse myeloma cells with spleen cells from mice immunized to purified rat liver glutathione-insulin transhydrogenase. The secreted antibody isotypes were found to be: Ig gamma 1 heavy chains and kappa light chains. This monoclonal antibody has been used to screen glutathione-insulin transhydrogenase in various rat tissue extracts (liver, fat, heart, testis, spleen, lung and kidney) following separation on NaDodSO4/urea polyacrylamide disc-gel electrophoresis and electrophoretic transfer to nitrocellulose. Screening with the monoclonal antibody showed the presence of one immunoreactive protein band equal in molecular weight to that of purified rat liver GIT (Mr 53,000) in extracts of all tissues studied and a second immunoreactive protein band of lower molecular weight (Mr 49,000) in spleen and lung tissue extracts. Separation of these two proteins by HPLC using a TSK-DEAE column demonstrated that both proteins exhibit insulin degrading activity. These data indicate that GIT may occur in multiple forms in some tissues.     

Induction mechanism of 3-hydroxy-3-methylglutaryl-CoA reductase in potato tuber and sweet potato root tissues

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC1.1.1.34), the key enzyme in isoprenoid biosynthesis, was purified from microsomes of potato tuber tissue, and a polyclonal antibody and two monoclonal antibodies against the purified enzyme were prepared. HMGR protein content was measured by immunotitration and radioimmunoassay using these antibodies. HMGR activity was very low in the fresh tissues of both potato tuber and sweet potato root. The activity in potato tuber was increased by cutting and further by additional fungal infection of the cut tissues. In sweet potato root tissue, the activity was scarcely increased after cutting alone, but was markedly increased by additional fungal infection or chemical treatment. The HMGR protein contents in both fresh potato tuber and sweet potato root tissues were also very low, and increased markedly in response to cutting and fungal infection. From these results, we proposed a hypothesis on the induction mechanism of HMGR after cutting and fungal infection in potato tuber and sweet potato root tissues.     

The existence and properties of two dimers of rat liver ecto-5''-nucleotidase.

Immunoaffinity-purified rat liver 5'-nucleotidase contained two subunits of Mr 70 000 (alpha) and 38 000 (beta). Charge-shift electrophoresis and chemical cross-linking revealed that approx. 80% of the solubilized enzyme activity occurred as an alpha alpha-dimer of Mr 140 000. The remaining 20% was an alpha beta-dimer of Mr 108 000. The beta-subunit did not possess enzymic activity. Peptide mapping and immunoblotting with antibodies against the alpha- and beta-subunits showed that the beta-subunit was homologous with a part of the alpha-subunit. Three monoclonal antibodies against rat liver 5'-nucleotidase were characterized as binding to the extracellular domain of the enzyme. All three monoclonal antibodies and concanavalin A bound to the alpha-subunit, but no binding could be detected to the beta-subunit. It was therefore concluded that the beta-subunit was a fragment of an alpha-subunit that had lost an extracellular domain. Both forms of the enzyme occurred in freshly solubilized membrane preparations as well.     

Expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in maize tissues

The activity of the enzyme 3-hydroxy-3-methlglutaryl-coenzyme A reductase (HMGR, EC 1.1.1.34) is highly expressed in 4-day-old etiolated seedlings of normal (cv. DeKalb XL72AA), dwarf ( d ₅) and albino ( lw ₃) maize ( Zea mays L.). HMGR activity of maize seedlings appeared to be exclusively associated with the microsomal rather than the plastidic fraction of maize cells. Maize tissues with high meristematic activity such as germinating seeds, leaf bases, root tips and the site of origin of lateral roots contained high levels of microsomal HMGR activity. The activity of HMGR extracted from leaf tips of normal, dwarf and albino maize seedlings is regulated by light. Microsomal HMGR activity from leaf tips of 4-day-old maize seedlings was inhibited significantly following exposure to strong light (600 μmol m⁻² s⁻¹) for more than 10 h. By comparison, microsomal HMGR activity from leaf bases and root tips of maize was not inhibited by exposure to strong light. These results suggest that the microsomal HMGR which is highly expressed in maize may be related to sterol biosynthesis and membrane biogenesis rather than plastidic-associated isoprenoid synthesis and that light may regulate HMGR activity indirectly by increasing cell differentiation.     

Comparative electron microscopic study on the location of ribosomal proteins S3 and S7 on the surface of the E. coli 30S subunit using monoclonal and conventional antibody

Summary Mice were immunised with 30S subunits from E. coli and their spleen cells were fused with myeloma cells. From this fusion two monoclonal antibodies were obtained, one of which was shown to be specific for ribosomal protein S3, the other for ribosomal protein S7. The two monoclonal antibodies formed stable complexes with intact 30S subunits and were therefore used for the three-dimensional localisation of ribosomal proteins S3 and S7 on the surface of the E. coli small subunit by immuno electron microscopy. The antibody binding sites determined with the two monoclonal antibodies were found to lie in the same area as those obtained with conventional antibodies. Both proteins S3 and S7 are located on the head of the 30S subunit, close to the one-third/two-thirds partition. Protein S3 is located just above the small lobe, whereas protein S7 is located on the side of the large lobe.     

Sex differences in the subunits of glutathione-S-transferase isoenzyme from rat and human kidney

Glutathione-S-transferase (GST) isoenzymes were purified from cytosolic preparations from kidneys of male and female rats and kidney cortical specimens from 2 male and 1 female human subjects. GST isoenzyme expression was analyzed by SDS-PAGE, measurement of catalytic activities with specific substrates and determination of their subunits by ELISA and Western blotting using specific antibodies. GST from female rat kidneys showed a preponderance of subunits 3 and 4; levels of these isoenzymes were 3-4 times greater in females than in males. Levels of subunits 1 and 2 were 1.5-2 times greater in the male rat kidneys. Additional minor bands at 24 and 22 kD were observed in GST preparations from both male and female rat kidneys while a band at 25.3 kD was observed only in the male rat kidney. These bands did not react with antibodies to GST 1-1, GST 2-2 or GST 3-4. Both male and female human kidney samples contained GST isoenzymes comparable to the near-neutral (25-5 kD) and basic forms (25 kD) of GSTs found in human liver. In addition a 28-kD band was present in GST preparations from both male and female human kidneys. Additional bands at 29 and 25.2 kD were present only in male human kidneys. Both the kidney cytosol and the total GSTs prepared from female rats shared 2- to 4-fold greater activity with 1,2-dichloro-4-nitrobenzene, ethacrynic acid and trans-4-phenyl-3-buten-2-one than those from males. The measurement of specific subunit amounts by ELISA were in agreement with these results.(ABSTRACT TRUNCATED AT 250 WORDS)     

牛血清白蛋白单克隆抗体杂交瘤细胞株的建立及鉴定

用牛血清白蛋白(BSA)免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,培养上清经过双抗体夹心法检测初步筛选分泌鼠IgG的杂交瘤细胞,将此种杂交瘤细胞注射小鼠产生的腹水用间接ELISA法筛选,获得4株能稳定分泌抗BSA单克隆抗体的杂交瘤细胞株,分别命名为2A5、3A3、3G6、4A8。鉴定结果显示,2A5细胞分泌IgG2a/κ,其余3株细胞分泌IgG1/κ;纯化后4株腹水单抗的纯度达90%以上,对BSA的ELISA滴度均可达到1∶100000以上;4株单抗均不与人以及马、猪、羊、兔、豚鼠等血清发生交叉反应;W estern B lotting试验证明4株单抗均识别分子量为68000的BSA;用间接ELISA法测定4株单抗相对亲和力及相对敏感度大小依次为3A3>2A5>3G6>4A8;杂交瘤细胞株连续培养3个月以及冻存半年后复苏,细胞生长良好,杂交瘤细胞分泌的抗体效价稳定。     

Cloning and characterization of a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Salvia miltiorrhiza involved in diterpenoid tanshinone accumulation

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is a rate-limiting step in the isoprenoid biosynthesis via the MVA pathway. In this study, the full-length cDNA encoding HMGR (designated as SmHMGR2, GenBank accession no. FJ747636) was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE). The cloned gene was then transformed into the hairy root of S. miltiorrhiza, and the enzyme activity and production of diterpenoid tanshinones and squalene were monitored. The full-length cDNA of SmHMGR2 comprises 1959 bp, with a 1653-bp open reading frame encoding a 550-amino-acid protein. Molecular modeling showed that SmHMGR2 is a new HMGR with a spatial structure similar to other plant HMGRs. SmHMGR2 contains two HMG-CoA-binding motifs and two NADP(H)-binding motifs. The SmHMGR2 catalytic domain can form a homodimer. The deduced protein has an isoelectric point of 6.28 and a calculated molecular weight of approximately 58.67 kDa. Sequence comparison analysis showed that SmHMGR2 had the highest homology to HMGR from Atractylodes lancea. As expected, a phylogenetic tree analysis indicates that SmHMGR2 belongs to plant HMGR group. Tissue expression pattern analysis shows that SmHMGR2 is strongly expressed in the leaves, stem, and roots. Functional complementation of SmHMGR2 in HMGR-deficient mutant yeast JRY2394 demonstrates that SmHMGR2 mediates the MVA biosynthesis in yeasts. Overexpression of SmHMGR2 increased enzyme activity and enhanced the production of tanshinones and squalene in cultured hairy roots of S. miltiorrhiza. Our DNA gel blot analysis has confirmed the presence and integration of the associated SmHMGR2 gene. SmHMGR2 is a novel and important enzyme involved in the biosynthesis of diterpenoid tanshinones in S. miltiorrhiza.     

Characterization of ferritin and ferritin-binding proteins in canine serum

Ferritin and ferritin-binding proteins in canine serum were characterized. A certain percentage of ferritin in canine serum, but no tissue ferritin, was precipitated by centrifugation at 16,000×g for 30 min. The precipitated ferritin was found to contain two subunits corresponding to the H and L subunits of canine liver ferritin by immunoblotting, the H subunit being predominant. More ferritin was precipitated from canine sera which had been incubated with anti-rat liver ferritin antibody than from untreated sera, and the H chain also predominated. To evaluate the possibility that the autoantibody was responsible for the precipitation of canine serum ferritin, the ferritin-binding activities of canine antibodies were examined using liver ferritin-coated microtiter plates and alkaline phosphatase-labeled antibodies specific for canine IgM, IgA, and IgG heavy chains. The results showed that IgM and IgA, but not IgG, had considerable ferritin-binding activities. Given these results, we suggest that there is H-chain-rich isoferritin in canine serum, and that ferritin exists as an immune complex.     

The Preparation of a Cellulose-like Polymer from 2,3,6-Tri-O (N-pheny1carbamyl)-D-glucopyranose by the Action of Phosphorus Pentoxide in Dimethyl Sulfoxide

Starch phosphorylase was purified from either freshly harvested or stored roots of sweet potato (.Ipomoea batatas (L.) Lam. cv Tain on 65). Both enzyme preparations in their native state showed on polyacrylamide gel electrophoresis a cluster of about six closely located activity bands, which had common antigenic determinants as they were simultaneously probed by monoclonal antibodies. The molecules of enzymes from stored roots were smaller than those from fresh roots. However, the two enzyme preparations had completely fused precipitin lines in double diffusion assays with an antiserum raised against the fresh root preparation. One large subunit and several small ones were found for both enzyme preparations. The small subunits appeared to be the degradation products of the large ones as revealed by peptide mapping and immunoblotting. Immunofluorescence microscopy showed that the enzyme was present in the amyloplasts and cell walls of root storage parenchyma.     

Purification of human liver cytochrome P-450 and comparison to the enzyme isolated from rat liver

Human liver cytochrome P-450 was isolated from autopsy samples using cholate extraction and chromatography on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE-cellulose gels. Purified preparations contained as much as 14 nmol cytochrome P-450 mg^?1 protein, were free of other hemoproteins, and were active in the mixed-function oxidation of d-benzphetamine and 7-ethoxycoumarin when coupled with either rat or human liver NADPH-cytochrome P-450 reductase. Some of the preparations were apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; apparent subunit M_rs estimated for several preparations were 53,000 or 55,500. The amino acid composition of one preparation was determined and found to resemble those of rat liver cytochromes P-450, although some variations were noted. Rabbit antibodies raised to phenobarbital-treated rat liver cytochrome P-450 were more effective in inhibiting d-benzphetamine N-demethylase activity in human liver microsomes than were antibodies raised to 3-methylcholanthrene-treated rat liver cytochrome P-450. These antibodies also inhibited benzo(a)pyrene hydroxylation in human liver microsomes, although the inhibition patterns did not follow a general pattern as in the case of benzphetamine demethylase activity. Microsomes prepared from three different human liver samples were more effective in eliciting complement fixation with antibodies raised to phenobarbitalthan to 3-methylcholanthrene-treated rat liver cytochrome P-450. Complement fixation in such systems appears to result from similarity of certain rat and human liver cytochrome P-450 antigenic determinants, as fixation could be inhibited by removal of cytochrome P-450-directed antibodies from the total immunoglobulin population and purified human cytochrome P-450 was more effective (on a protein basis) than liver microsomes in producing fixation. Human liver microsomes prepared from five different individuals all produced ≥ 90% complement fixation, but variations were observed in the fixation curves plotted either versus microsomal protein or versus spectrally detectable microsomal cytochrome P-450.These results indicate that human liver microsomal cytochromes P-450 can be isolated using modifications of techniques developed for laboratory animals and that human and rat liver cytochromes P-450 share certain features of structural, functional, and immunological similarity. The available data suggest the existence of multiple forms of human liver microsomal cytochrome P-450, but possible artifacts associated with the use of autopsy samples suggest caution in advancing such a conclusion.     

Insig Proteins Mediate Feedback Inhibition of Cholesterol Synthesis in the Intestine

Enterocytes are the only cell type that must balance the de novo synthesis and absorption of cholesterol, although the coordinate regulation of these processes is not well understood. Our previous studies demonstrated that enterocytes respond to the pharmacological blockade of cholesterol absorption by ramping up de novo sterol synthesis through activation of sterol regulatory element-binding protein-2 (SREBP-2). Here, we genetically disrupt both Insig1 and Insig2 in the intestine, two closely related proteins that are required for the feedback inhibition of SREBP and HMG-CoA reductase (HMGR). This double knock-out was achieved by generating mice with an intestine-specific deletion of Insig1 using Villin-Cre in combination with a germ line deletion of Insig2. Deficiency of both Insigs in enterocytes resulted in constitutive activation of SREBP and HMGR, leading to an 11-fold increase in sterol synthesis in the small intestine and producing lipidosis of the intestinal crypts. The intestine-derived cholesterol accumulated in plasma and liver, leading to secondary feedback inhibition of hepatic SREBP2 activity. Pharmacological blockade of cholesterol absorption was unable to further induce the already elevated activities of SREBP-2 or HMGR in Insig-deficient enterocytes. These studies confirm the essential role of Insig proteins in the sterol homeostasis of enterocytes.     

Monoclonal antibodies to 13-deoxy-gibberellins

The production and characterization of two high affinity rat monoclonal antibodies to 13-deoxy-gibberellins is described. Hybrid myelomas were derived from rats immunized with an immunogenic keyhole limpet hemocyanin-gibberellin conjugate, linked at carbon-3 to gibberellin A₄ via a hemisuccinate bridge. The selected monoclonal antibodies were characterized by a competitive radioimmunoassay.