Substrate preference of metalloproteinases secreted by ts-110 Moloney murine sarcoma virus-transformed normal rat kidney cells.

In this study, the two forms of affinity-purified transformation-associated proteins (TAPs) (68 and 64 kD) were shown to have different substrate preferences. For the 68-kD TAP, the order of substrate preference was collagen types I, III, and V; fibronectin; gelatin; and collagen IV. For the 64-kD TAP, the order of substrate preference was collagen I, III, and V and gelatin. The 64-kD TAP did not cleave collagen IV and fibronectin. We also found a 71-kD metalloproteinase in the concentrated purified TAPs that reacted only weakly with a TAP monoclonal antibody and showed this substrate preference: collagen I, III, and V; gelatin; and collagen IV. Whether this 71-kD TAP is a new member of the rat metalloproteinase family will be investigated.     

Temperature-sensitive splicing defect of ts110 Moloney murine sarcoma virus is virus encoded.

ts110 Moloney murine sarcoma virus (Mo-MuSV)-nonproductively infected cells (6m2) have a transformed phenotype at 28 to 33 degrees C and a normal phenotype at 39 degrees C. At temperatures permissive for transformation, 6m2 cells contain P58gag produced from the 4.0-kilobase (kb) viral RNA genome and P85gag-mos translated from a 3.5-kb spliced mRNA. At 39 degrees C, only the 4.0-kb RNA and its product P58gag are detected. Two temperature-sensitive defects have been observed in ts110-infected 6m2 cells: (i) the splicing of the 4.0-kb RNA to the 3.5-kb RNA; and (ii) the thermolability of P85gag-mos and its kinase activity relative to the wild-type revertant protein, termed P100gag-mos (R.B. Arlinghaus, J. Gen. Virol. 66:1845-1853, 1985). In the present study, we examined the mos gene products of two cell lines (204-2F6 and 204-2F8) obtained by infection of normal rat kidney cells with ts110 Mo-MuSV as a simian sarcoma-associated virus pseudotype to see whether the temperature-sensitive splicing defect could be transferred by viral infection. Southern blot analysis of these two cell lines showed that viral DNAs containing restriction fragments from cellular DNA are different from those in 6m2 cells, indicating that 204-2F6 and 204-2F8 cells have different ts110 provirus integration sites from those of 6m2 cells. Northern blots, S1 mapping analyses, and immunoprecipitation experiments showed unequivocally that the splicing defect of ts110 Mo-MuSV is virus encoded and is independent of host cell factors.     

Potassium inhibition of transforming protein P85gag-mos and reversal of the transformed phenotype in 6m2 cells

K+ at high concentrations (52-72 mM hypertonic KCl) has been reported to induce reverse transformation in the 6m2 cell, which is a clone of normal rat kidney cells (NRK) infected with a temperature-sensitive transformation virus. When exposed to high K+, 6m2 cells grown at the permissive temperature (33 degrees C) exhibit normal morphology and reduced soft agar growth, characteristics of cells grown at nonpermissive temperature (39 degrees C). In the current study, flattening of cells and rearrangement of surface microvilli were demonstrated by scanning electron microscopy to occur within 6 hr of exposure to high K+, similar to the effect of temperature shift to 39 degrees C. Exposure to K+ resulted in a 90% inhibition of P85gag-mos-associated serine kinase activity within 5 min, with a subsequent reduction of up to 75% of the synthesis of this protein. These alterations in the putative transforming protein were similar to those induced by temperature shift and were considered to be the basis for retrotransformation. The cell microtubular system and F-actin cables were affected more slowly by K+ than by a temperature shift to 39 degrees C. The former did not achieve the fine reticulum network seen in NRK cells until 72 hr later, but the latter remained aberrant. The effect on the enzyme might be mediated by alteration in phosphorylation, but the mechanism by which kinase inactivation induces retrotransformation is not yet known.     

Xeroderma pigmentosum group A correcting protein from calf thymus.

A proteinous factor was purified from calf thymus and HeLa cells, which specifically corrects the excision repair defect of xeroderma pigmentosum complementation group A (XP-A) cells. Recovery of UV-induced unscheduled DNA synthesis after microinjection of XP-A cells was used as a quantitative assay for the correcting activity of protein preparations. XP-A correcting protein appears to be very stable as it withstands heating to 100 degrees C and treatment with SDS or 6 M urea. A molecular weight of 40-45 kD was found both under native (gel filtration) and denaturing (SDS-PAGE) conditions. Calf XP-A protein binds to single-stranded DNA more strongly than to double-stranded DNA, but shows no clear preference for UV-irradiated DNA. Polyclonal antibodies raised against human recombinant XP-A protein, which strongly inhibit UV-induced unscheduled DNA synthesis of normal human cells, completely abolished XP-A correcting activity when mixed with calf thymus preparations. This indicates a close relationship between human gene product and the calf protein. In the final preparation two main protein bands were present. Only one band at approx. 41 kD showed both DNA binding activity in Southwestern blots and immune reaction with human XP-A antibody, suggesting that this is the active calf XP-A correcting factor.     

The bacteriophage P4 alpha gene is the structural gene for bacteriophage P4-induced RNA polymerase

Two temperature-sensitive mutants of satellite phage P4 which do not synthesize P4 DNA at the nonpermissive temperature have been isolated. One of these phage is mutated in the P4 alpha gene. It complements a P4 delta mutant, but not a P4 alpha amber mutant; both mutants are phenotypically identical to alpha amber mutants in all properties studied. They synthesize P4 early proteins 1 and 2 as well as two additional P4-induced early proteins, 5 and 6, which are described here. P4 late proteins are not synthesized by these mutants and cannot be transactivated by helper phage P2. The mutants are unable to transactivate P2 late proteins from a P2 AB mutant. The P4 RNA polymerase activity which has been suggested to be involved in P4 DNA synthesis is not detected at the nonpermissive temperature. The P4 polymerase activity in partially purified extracts prepared from cells infected with the mutant at the permissive temperature is temperature sensitive. Reduced activity is found in vitro when these extracts are preincubated at 41 degrees C or assayed at temperatures higher than 37 degrees C. Thus, the P4 RNA polymerase is the product of the alpha gene. Temperature shift experiments show that the alpha gene product is required until late in the P4 cycle.     

Characterization of a temperature-sensitive membrane alteration in chick embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus

The intramembrane particles of freeze-fractured chick embryo fibroblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (TS68) are distributed differently at the permissive and non-permissive temperatures if, and only if, the cells are treated with glycerol before fixation. Few aggregates of intramembrane particles are present in glycerol-treated cells grown at the permissive temperature for transformation (36 degrees C), while numerous large aggregates of particles are present at the non-permissive temperature (41 degrees C). Changes in the distribution of particles after cells are shifted from 36 to 41 degrees C are observed after 20 min, while a temperature shift from 41 to 26 degrees C causes changes in glycerol-induced redistributions after 1 h. The changes observed in temperature shifts from 36 to 41 degrees C and from 41 to 36 degrees D do not require protein synthesis or RNA synthesis.     

Characterization and purification of mouse macrophage procoagulant-inducing factor

Macrophage procoagulant-inducing factor (MPIF) is a product of mouse Lyt-1+2- cells that induces macrophage procoagulant activity (MPCA) on mouse peritoneal exudate cells or on the macrophage-like tumor cell line WEHI-265. Supernatants from Sepharose-bound concanavalin A-stimulated cells were fractionated by using DEAE-Sephacel, heparin-Sepharose, and isoelectric focusing. This procedure resolved three different MPIF: MPIF alpha (pI 8.5), MPIF beta (pI 8.8 to 9.2), and MPIF gamma (pI 5 to 5.5). MPIF alpha and beta were small molecules (approximately 14 kD and 20 to 25 kD) as determined by gel filtration on Sephadex G200 and Biogel P100. MPIF beta was sharply resolved as a peak eluting after lysozyme by gel filtration on HPLC columns I-150 and I-125, although SDS-PAGE of the HPLC-enriched material resolved two well-defined bands of 70 and 120 kD and some poorly defined material of 14 kD. Silver staining failed to detect components of MPIF alpha after SDS-PAGE. MPIF gamma activity was associated with material that separated over a broad range (20 to 60 kD and 60 to 200 kD), possibly due to aggregation with other components of the supernatants. Crude supernatants were stable to heating at 56 degrees C for 30 min and pH 2 treatment, although more highly enriched fractions were unstable to these treatments. Heating at 90 degrees C for 5 min totally destroyed MPIF activity. The properties of the two basic MPIF differ from other lymphokines known to affect macrophage function, e.g., colony-stimulating factor, migration-inhibition factor, interferon-gamma, and interleukin 1.     

Properties and topography of the major integral plasma membrane protein of a unicellular organism

The cellular distribution, membrane orientation, and biochemical properties of the two major NaOH-insoluble (integral) plasma membrane proteins of Euglena are detailed. We present evidence which suggests that these two polypeptides (Mr 68 and 39 kD) are dimer and monomer of the same protein: (a) Antibodies directed against either the 68- or the 39-kD polypeptide bind to both 68- and 39-kD bands in Western blots. (b) Trypsin digests of the 68- and 39-kD polypeptides yield similar peptide fragments. (c) The 68- and 39-kD polypeptides interconvert during successive electrophoresis runs in the presence of SDS and beta- mercaptoethanol. (d) The 39-kD band is the only major integral membrane protein evident after isoelectric focusing in acrylamide gels. The apparent shift from 68 to 39 kD in focusing gels has been duplicated in denaturing SDS gels by adding ampholyte solutions directly to the protein samples. The membrane orientation of the 39-kD protein and its 68-kD dimer has been assessed by radioiodination in situ using intact cells or purified plasma membranes. Putative monomers and dimers are labeled only when the cytoplasmic side of the membrane is exposed. These results together with trypsin digestion data suggest that the 39- kD protein and its dimer have an asymmetric membrane orientation with a substantial cytoplasmic domain but with no detectable extracellular region. Immunolabeling of sectioned cells indicates that the plasma membrane is the only cellular membrane with significant amounts of 39- kD protein. No major 68- or 39-kD polypeptide bands are evident in SDS acrylamide gels or immunoblots of electrophoresed whole flagella or preparations enriched in flagellar membrane vesicles, nor is there a detectable shift in any flagellar polypeptide in the presence of ampholyte solutions. These findings are considered with respect to the well-known internal crystalline organization of the euglenoid plasma membrane and to the potential for these proteins to serve as anchors for membrane skeletal proteins.     

Purification and some properties of human placental glucose-6-phosphate dehydrogenase

Glucose-6-phosphate dehydrogenase was purified from human placenta using DEAE-Sepharose fast flow, 2',5'-ADP Sepharose 4B column chromatography, and chromatofocusing on PBE 94 with PB 74. The enzyme was purified with 62% yield and had a specific activity of 87 units per milligram protein. The pH optimum was determined to be 7.8, using zero buffer extrapolation method. The purified placental glucose-6-phosphate dehydrogenase gave two activity bands on native PAGE: one band, constituting about 3--5% of total activity, moved slower than the remaining 95%. Among the activity bands only the faster moving band gave a band on protein staining. The slower moving band, which probably corresponded to the higher polymeric form of the G6PD with high specific activity, was not seen on native PAGE due to insufficient protein for Coomassie brilliant blue staining. The observation of one band on SDS--PAGE with an M(r) of 54 kDa and a specific activity lower than expected, suggests the presence of both forms of the G6PD, the high polymeric form at low concentration and the inactive form at high concentration, in our preparation. Measuring the activities of placental glucose-6-phosphate dehydrogenase between 20 and 50 degrees C, the activation energy, activation enthalpy, and Q(10) were calculated to be 8.16 kcal/mol, 7.55 kcal/mol, and 1.57, respectively. It was found that human placental G6PD obeys Michaelis-Menten kinetics. K(m) values were determined using the concentration ranges of 20--300 microM for G6P and 10--200 microM for NADP(+). The K(m) value for G6P was 40 microM; the K(m) value NADP(+) was found to be 20 microM. Double-reciprocal plots of 1/Vm vs 1/G6P (at constant [NADP(+)]) and of 1/Vm vs 1/NADP(+) (at constant [G6P]) intersected at the same point on the 1/V(m) axis to give V(m) = 87 U/mg protein.     

Purification and characterization of a DnaK-like and a GroEL-like protein from Porphyromonas gingivalis

Heat-shock proteins of Porphyromonas gingivalis were demonstrated and two of them were purified and further characterized. The amplified de novo synthesis of two different proteins, with apparent molecular weights of 75 kDa and 68 kDa, was observed by autofluorography when a P. gingivalis culture incubated in a 14C-labeled amino acid mixture was shifted from 37 degrees C to 44 degrees C. Both proteins possessed ATP-binding abilities and were purified to almost homogeneity employing affinity chromatography on ATP-agarose followed by preparative SDS-PAGE. Purified 75 kDa and 68 kDa proteins had isoelectric points of 4.4 and 4.6, respectively. They were shown to be immunoreactive with commercial anti-DnaK and anti-GroEL polyclonal antibodies, respectively. Immunoblotting analysis of whole cells using antiserum raised against each purified protein from P. gingivalis, confirmed elevated synthesis of both proteins during thermal shock. A GroEL protein reacted strongly with antiserum against the 68 kDa protein. However, a DnaK protein reacted weakly with antiserum to the 75 kDa protein. Analysis of the N-terminal amino acid sequence of the DnaK-like protein (75 kDa) showed a high degree of homology with those of the HSP70 family including both prokaryotic and eukaryotic cells. The N-terminal amino acid analysis of the GroEL-like protein (68 kDa) indicated that it was identical to those of cloned GroEL homologues from P. gingivalis.     

Evidence that the Rous sarcoma virus transforming gene product is associated with glycerol kinase activity

This communication provides biochemical, immunological, and genetic evidence that pp60src, the Rous sarcoma virus transforming gene product, is associated with glycerol kinase activity. Our investigations demonstrated that the compound phosphorylated by pp60src or by glycerol kinase (EC 2.7.1.30) from Candida mycoderma share the same electrophoretic and chromatographic mobilities. The glycerol kinase and protein kinase activities of pp60src were inhibited similarly by preincubation with immune IgG. Both activities were reduced 6-9-fold in pp60src preparations derived by immunoaffinity chromatography from cells which were infected with NY68, a temperature-sensitive transformation mutant of Rous sarcoma virus. The thermolability at 41 degrees C of the glycerol kinase activity of pp60src from the mutant virus-infected cells was greater (t/2 = 1.3 min) than the same activity in pp60src preparations from wild type virus-infected cells (t/2 = 4.8 min).     

优质蛋白玉米胚乳贮存蛋白积累的电泳分析

玉米胚乳的２２ ｋＤ和２０ ｋＤ醇溶蛋白在授粉后１５ 天开始积累,编码２２ ｋＤ 和２０ ｋＤ醇溶蛋白的结构基因在胚乳发育过程中同时表达。优质蛋白玉米和ｏ２ 玉米的胚乳中,２２ ｋＤ和２０ ｋＤ醇溶蛋白的合成受到抑制,即ｏ２ 基因对２２ ｋＤ和２０ ｋＤ醇溶蛋白的合成有负的调节作用。Ｍｏ１７／ｏ２ 和Ｍｏ１７ 胚乳醇溶蛋白的双向电泳结果表明,Ｍｏ１７／ｏ２ 的２７ ｋＤ、２２ ｋＤ、２０ ＫＤ和１５ ｋＤ醇溶蛋白的合成均受到强烈的抑制。遗系０４１／ｏ２ 和遗系０４０／ｏ２ 胚乳醇溶蛋白的双向电泳结果表明,二者只在高分子量的蛋白质斑点区域有一些细微的差别。可溶性蛋白的ＳＤＳ－ＰＡＧＥ分析表明,Ｍｏ１７／ｏ２ 胚乳的可溶性蛋白比其同型系Ｍｏ１７ 少３８．７ ｋＤ 和２６．７ ｋＤ两条谱带,多２７．２ ｋＤ和２６．１ ｋＤ两条谱带。二者出现的可溶性蛋白的差异是ｏ２ 基因调控的结果。遗系０４１／ｏ２ 胚乳的可溶性蛋白比其同型系０４０／ｏ２ 多１８．６ ｋＤ和１７．６ ｋＤ两条谱带,少４０．２ ｋＤ 一条谱带,这与ｏ２ 基因修饰因子的作用密切关联     

Acetylcholinesterase in Mouse Neuroblastoma NB2A Cells: Analysis of Production, Secretion, and Molecular Forms

The mouse neuroblastoma cell line NB2A produces cellular and secreted acetylcholinesterase (AChE). After incubation of the cells for 4 days the ratio between AChE secreted into the medium and AChE in the cells was 1:1. The cell-associated enzyme could be subdivided into soluble AChE (25%) and detergent-soluble AChE (75%). Both extracts contained predominantly monomeric AChE (4.6S) and minor amounts of tetrameric AChE (10.6S), whereas the secreted AChE in the culture supernatant contained only the tetrameric form. All forms were partially purified by affinity chromatography. It could be demonstrated that the secretory and the intracellular soluble tetramers were hydrophilic, whereas the detergent-soluble tetramer was an amphiphilic protein. On the other hand the soluble and the detergent-soluble monomeric forms were amphiphilic and their activity depended on the presence of detergent. By digestion with proteinase K amphiphilic monomeric and tetrameric AChE could be converted to a hydrophilic form that no longer required detergent for catalytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]diisopropylfluorophosphate-labelled AChE gave one band at 64 kilodaltons (kD) under reducing conditions and two additional bands at 120 kD and 140 kD under nonreducing conditions.     

Changes in the synthesis and phosphorylation of cellular proteins in chick fibroblasts transformed by two avian sarcoma viruses

35S- and 32P-labeled proteins from control chick embryo fibroblasts and from fibroblasts transformed by UR2 sarcoma virus, or by a temperature-sensitive mutant (tsLA29) of Rous sarcoma virus, were separated by two-dimensional electrophoresis on giant gels to detect transformation-specific changes in protein synthesis and total phosphorylation. A nontransforming avian retrovirus, UR2-associated virus (UR2AV), was also studied. Virus-coded proteins appear in whole cell lysates of all infected cells. The structural proteins can be identified by comparison with proteins immunoprecipitated with antivirus serum. The transforming proteins pp60src and p68ros, present in cells transformed with Rous sarcoma virus and UR2, respectively, are phosphorylated in vivo. Eighteen increases and eight decreases in cellular phosphoproteins are associated with transformation, and revert toward normal levels when cells infected with tsLA29 are incubated at 42 degrees C. These changes are more extensive than previously reported, but none represent new phosphorylations, since all phosphoproteins seen in transformed cells also appear to be phosphorylated to a certain extent in control cells. Fifteen cellular proteins show increased relative rates of synthesis apparently related either to transformation or to growth at 42 degrees C. Four other proteins are increased exclusively in cells incubated at 42 degrees C, but not at 37 degrees C, whether transformed or not. Eleven additional increases in the synthesis of cellular proteins, many quite large, and one seemingly a de novo induction, appear to be specific for transformation. These changes occur in cells transformed by either UR2 or Rous sarcoma virus at 37 degrees C, do not occur with UR2AV infection, and tend to revert in cells infected with tsLA29 incubated at 42 degrees C. These 11 changes may represent increases in cellular gene expression that are related specifically to the maintenance of the transformed state.     

Membrane association of the transforming protein of avian sarcoma virus UR2 and mutants temperature sensitive for cellular transformation and protein kinase activity.

The localization of the transforming protein P68gag-ros of avian sarcoma virus UR2, which has a hydrophobic region at the N terminus of its ros-specific tyrosine kinase-encoding sequence, was examined by subcellular fractionation. P68 behaved as an integral membrane protein associated with the plasma membrane of transformed cells. P68 became membrane associated very rapidly in its biogenesis. Three temperature-sensitive mutants of UR2 were isolated and characterized. Cells infected with the mutants were temperature sensitive for morphological alteration and colony formation. The mutant P68 proteins were membrane associated in mutant-infected cells regardless of the temperature but were active as protein kinases only at the permissive temperature. The results suggest that P68 is a membrane-associated protein whose kinase activity plays a crucial role in UR2-mediated cell transformation.     

Adenovirus VAI RNA complexes with the 68 000 Mr protein kinase to regulate its autophosphorylation and activity.

We have investigated the interaction of VAI RNA with the interferon-induced, double-stranded (ds) RNA-activated protein kinase, P68, both of which regulate protein synthesis in adenovirus-infected cells. Previous work has shown that during infection by the VAI RNA-negative mutant, dl331, both viral and cellular protein synthesis are inhibited due to phosphorylation of the alpha-subunit of the eukaryotic initiation factor, eIF-2, by the P68 protein kinase. Utilizing monoclonal antibodies specific for P68, we demonstrated that the physical levels of P68 in dl331-infected, wild-type Ad2-infected and uninfected cells were all comparable suggesting that the elevated kinase activity detected during mutant infection was not due to increased P68 synthesis. To examine the basis of the increased activity of P68, the protein kinase was purified from infected-cell extracts using the monoclonal antibody. We found that P68 was heavily autophosphorylated during dl331 infection but not during wild-type or mock infection. The extent of autophosphorylation correlated with elevated P68 activity and the loss of the dsRNA requirements to phosphorylate the exogenous substrates, eIF-1 alpha and histones. We also analyzed VAI RNA function in vitro and present evidence that purified VAI RNA can block the autophosphorylation of P68 in the ribosomal salt wash fraction of interferon-treated cells. Finally we suggest VAI RNA functions through a direct interaction with the P68 protein kinase, since we demonstrated that VAI RNA forms a complex with P68 both in vitro and in vivo.     

Fetuin: a serum component associated with rat Sertoli and spermatogenic cells in coculture

Cocultures of rat Sertoli-spermatogenic cells plated in a culture medium supplemented with 10% fetal bovine serum for 6-12 h and then maintained in serum free, hormone/growth factor-supplemented medium accumulated an acidic glycoprotein of molecular weight of 68,000 dalton (68 kD) and isoelectric point range of about 4.2-3.5. Anion exchange chromatography has allowed the partial purification of this protein, which consists of a major protein band of 68 kD and two minor, low molecular weight components. A rabbit antiserum raised against the 68 kD component also crossreacts with the two low molecular weight components, thus suggesting that these two minor components are antigenically related to the 68 kD protein. The 68 kD protein has been identified as fetuin, the major component of fetal bovine serum, based on similar molecular weight, isoelectric point, immunoreactivity and trypsin inhibitory activity. Labeling experiments with [14C]amino acid mixture show that 68 kD protein is not synthesized by cocultured rat Sertoli and spermatogenic cells. Immunocytochemistry and Western blot approaches carried out under various experimental conditions support the view that the fetuin-68 kD protein is taken up from serum by both Sertoli cells and pachytene spermatocytes. Because fetuin 1) behaves as a carrier protein for growth factors, 2) has protease inhibitory activity, 3) is preferentially internalized by Sertoli cells and pachytene spermatocytes and 4) fetal bovine serum-supplemented medium impairs spermatogenic cell viability, there is a need to further define appropriate conditions for optimizing long-term viability and differentiation of spermatogenic cells in vitro.     

u-PA/t-PA嵌合型纤溶酶原激活剂(ut-PA)的构建、表达、纯化和性质研究

将尿激酶原（ｐｒｏ－ＵＫ）ｃＤＮＡ和组织型纤溶酶原激活剂（ｔ－ＰＡ）Ａ链ｃＤＮＡ克隆到Ｍ１３ｍｐ１８中,经过二次寡核甘酸诱导的大片段定点删除和一次寡核苷酸诱导的多位点突变,得到ｕ－ＰＡ（Ｌｅｕ１４４－Ｇｌｙ４０８）／ｔ－ＰＡ（Ｓｅｒ１－Ｔｈｒ２６３）（ｕｔ－ＰＡ）融合基因．将ｕｔ－ＰＡ融合基因克隆到表达载体ｐＣＭ－βｎｅｏ中,与ｐＣＭ－ｄｈｆｒ共转染ＣＨＯ／ＤＨＦＲ－细胞,筛选稳定表达株．收集无血清表达上清,经苯甲脒柱纯化得到ｕｔ－ＰＡ纯品,ＳＤＳ－ＰＡＧＥ和纤维蛋白自显影显示ｕｔ－ＰＡ有两种分子量形式,分子量分别为６８ｋＤ和６１ｋＤ．纤维蛋白亲和性试验表明,ＬＵＫ（低分子量尿激酶）对纤维蛋白没有亲和性,而含有ＬＵＫ的ｕｔ－ＰＡ则对纤维蛋白表现出很强的亲和性,但ｕｔ－ＰＡ的亲和性略低于亲本ｔ－ＰＡ．