Distinct Protein Bands Research Articles

Protein expression by Streptococcus uberis in co-culture with bovine mammary epithelial cells

Three strains of Streptococcus uberis isolated from dairy cows with mastitis were co-cultured with a bovine mammary epithelial cell line (MAC-T) in Dulbecco's modified Eagle's medium without fetal bovine serum. Protein profiles from culture supernatants and bacterial pellets among different treatments were compared by electrophoresis. There were proteins induced or having increased expression in both supernatant and surface-associated samples from S. uberis co-cultured with MAC-T cells. Some of these proteins were recognized by antibodies in serum obtained from a cow infected by S. uberis. In supernatant samples, there were two distinct protein bands at 35 and 36.8 kDa for all three strains of S. uberis co-cultured with MAC-T cells. These two bands were absent when bacterial protein synthesis was inhibited by chloramphenicol. This study clearly indicates that bacterial protein expression was regulated in response to co-culture with mammary epithelial cells.

Protein expression by Streptococcus uberis in co-culture with bovine mammary epithelial cells.

Three strains of Streptococcus uberis isolated from dairy cows with mastitis were co-cultured with a bovine mammary epithelial cell line (MAC-T) in Dulbecco's modified Eagle's medium without fetal bovine serum. Protein profiles from culture supernatants and bacterial pellets among different treatments were compared by electrophoresis. There were proteins induced or having increased expression in both supernatant and surface-associated samples from S. uberis co-cultured with MAC-T cells. Some of these proteins were recognized by antibodies in serum obtained from a cow infected by S. uberis. In supernatant samples, there were two distinct protein bands at 35 and 36.8 kDa for all three strains of S. uberis co-cultured with MAC-T cells. These two bands were absent when bacterial protein synthesis was inhibited by chloramphenicol. This study clearly indicates that bacterial protein expression was regulated in response to co-culture with mammary epithelial cells.

Ubiquitous Expression of bcl-2 Protein in Mitotic Nuclei and Chromosomes.

We investigated the expression of bcl-2 oncoprotein in human cells by immunocytochemistry using an anti-bcl-2 antibody. Expression of bcl-2 was detected in and around mitotic nuclei and chromosomes from prophase to early telophase in all types of long-term cultured cell lines irrespective of the level of its expression in the cytoplasm. Western blot analysis showed a discrete but distinct protein band, which corresponded to the 26kDa α-form of bcl-2, in the lysate of cells showing only mitotic bcl-2. No other immunoreactions, including those corresponding to the β-form, were detected. RT-PCR combined with nested PCR showed bcl-2 mRNA in the same cell lines and in situ hybridization confirmed its presence in the cytoplasm of almost all interphase cells. The appearance of nuclear bcl-2 was not due to aberrant genetic regulation induced by an artificial in vitro environment. It was also not unique to neoplastic cells as nuclear and chromosomal bcl-2 was detected in freshly prepared specimens from B-cell lymphomas, myelodysplastic syndrome, and primary cultures of normal fibroblasts during mitosis. On the other hand, the level of cytoplasmic bcl-2 varied from one cell line to another; 8 carcinoma lines did not express cytoplasmic bcl-2, whereas many B cell lines expressed high levels of the protein. Bcl-2 appeared to accumulate in mitotic nuclei, because immunocytochemical reaction product was always stronger in mitotic nuclei than in the cytoplasm when the expression of bcl-2 was induced in peripheral blood lymphocytes by ConA stimulation. Our results indicate that bcl-2 protein is ubiquitously expressed in all cells around mitotic nuclei or chromosomes, whereas its distribution in the cytoplasm seems to reflect differences in genetic regulation based on cell lineage, differentiation stage or neoplastic state.

SPECIES SUBSTITUTION OF RESTAURANT FISH ENTREES

ABSTRACTFish species substitution has been a common problem in the retail markets. However, this problem has not been documented in restaurant‐cooked fish entrees. This study investigated fish substitution in restaurant‐cooked snapper and swordfish entrees from four major cities in the U.S. The species of 16 snappers including 13 red snapper and 21 swordfish samples were verified as presented on the menu using isoelectric focusing (IEF). Fish samples were cleaned and desalted by rinsing with distilled water before protein extraction. Protein separation was carried out in a polyacrylamide gel with a wide pH gradient range of 3.5–9.5. Distinctive IEF protein band patterns of unknown samples were compared with the authentic standards. Six out of 13 (46%) red snapper samples and two out of 21 (10%) swordfish samples were misrepresented. The method used in this study adequately and efficiently verified the species of restaurant‐cooked snapper and swordfish samples.

The effects of steroidal and non-steroidal ovarian hormones on pituitary responsiveness to gonadotrophin surge-attenuating factor.

Primary pituitary cultures from adult female rats were used to investigate the effects of steroidal (oestradiol and progesterone) and non-steroidal (inhibin, follistatin) ovarian hormones on the suppressive actions of the ovarian factor gonadotrophin surge-attenuating factor (GnSAF) in the control of gonadotrophin secretion. The source of GnSAF was a chromatographic preparation from follicular fluid containing four distinct protein bands as resolved on SDS-PAGE. Oestradiol and progesterone added alone had no effect on gonadotrophin secretion but had a wide range of effects on the suppression of both LH and FSH secretion caused by the non-steroidal factors. Oestradiol, progesterone and oestradiol+progesterone enhanced the suppressive actions of GnSAF on GnRH-induced LH secretion (causing 19.3 +/- 5.2% (P < 0.05), 41.9 +/- 3.4% (P < 0.001) and 32.2 +/- 5.3% (P < 0.001) greater suppression than GnSAF alone). Progesterone and oestradiol+progesterone completely abolished the suppression of basal FSH secretion caused by inhibin (causing 157.1 +/- 22.2%, P < 0.001, and 160.9 +/- 11.3%, P < 0.001, stimulation compared with inhibin alone). Separately the steroids had no effect on the suppression of gonadotrophin secretion caused by follistatin. However, in combination, oestradiol+progesterone potentiated the suppressive actions of follistatin on GnRH-induced LH secretion causing 29.9 +/- 5.3% (P < 0.05) greater suppression than follistatin alone. In combination, high-dose follistatin and GnSAF caused 31.1 +/- 6.5% (P < 0.01) greater suppression than GnSAF alone. Thus in combination high-dose follistatin and GnSAF have additive effects on the suppression of GnRH-induced LH secretion. Recombinant human inhibin and GnSAF added in combination had little further effect compared with either alone suggesting that they may have a similar mechanism of action at the pituitary level. These results demonstrate that while FSH secretion in vitro is mainly controlled by inhibin and follistatin, LH secretion is affected by the presence of a whole range of factors. We have demonstrated that oestradiol and progesterone potentiate the suppressive actions of GnSAF in vitro. These data are compatible with the suggestion that in the late follicular phase it is falling levels of GnSAF that allow positive feedback of the steroids on the pituitary to elicit the LH surge, rather than increases in the stimulatory effects of the ovarian steroids overcoming GnSAF. The actions of GnSAF on the pituitary may be modulated by follistatin but it is unlikely that inhibin has any modulatory effects on the GnSAF-induced suppression of LH secretion.

Cultured leptomeningeal cells secrete cerebrospinal fluid proteins.

To extrapolate the function of the leptomeninges, we examined the profile of the proteins secreted from the cultured leptomeningeal cells prepared from 1-2-day-old rats. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the medium conditioned with the cultured cells, 20-25 differentially distinctive protein bands were noted. Through several chromatographic procedures (Sephadex G-75, Mono Q, and 7C8-300), altogether 18 proteins were purified to homogeneity, and the partial amino acid sequence of each protein was determined. Homology search revealed that the major proteins included prostaglandin-D-synthase or beta-trace protein, insulin-like growth factor (IGF)-II, IGF-binding protein-2, apolipoprotein E, beta 2-microglobulin, cystatin C, transferrin, peptidyl-prolyl cis-trans isomerase or cyclophilin C, secreted protein acidic and rich in cysteine, ubiquitin, lysozyme C, extracellular superoxide dismutase, and collagen alpha-1 (III). Most of these proteins are known to be the major brain-derived protein constituents of CSF and are thought to play important roles in certain biological events in the brain. Considering the morphological features, the present findings suggest the importance of the leptomeninges as an origin of such proteins in CSF.

Isolation and characterization of the hyperthermostable serine protease, pyrolysin, and its gene from the hyperthermophilic archaeon Pyrococcus furiosus.

The hyperthermostable serine protease pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus was purified from membrane fractions. Two proteolytically active fractions were obtained, designated high (HMW) and low (LMW) molecular weight pyrolysin, that showed immunological cross-reaction and identical NH2-terminal sequences in which the third residue could be glycosylated. The HMW pyrolysin showed a subunit mass of 150 kDa after acid denaturation. Incubation of HMW pyrolysin at 95 degrees C resulted in the formation of LMW pyrolysin, probably as a consequence of COOH-terminal autoproteolysis. The 4194-base pair pls gene encoding pyrolysin was isolated and characterized, and its transcription initiation site was identified. The deduced pyrolysin sequence indicated a prepro-enzyme organization, with a 1249-residue mature protein composed of an NH2-terminal catalytic domain with considerable homology to subtilisin-like serine proteases and a COOH-terminal domain that contained most of the 32 possible N-glycosylation sites. The archaeal pyrolysin showed highest homology with eucaryal tripeptidyl peptidases II on the amino acid level but a different cleavage specificity as shown by its endopeptidase activity toward caseins, casein fragments including alphaS1-casein and synthetic peptides.

Differential Phosphorylation of the T Lymphocyte Costimulatory Receptor CD28: ACTIVATION-DEPENDENT CHANGES AND REGULATION BY PROTEIN KINASE C

Treatment of T lymphocytes with phorbol ester and anti-CD28 monoclonal antibody (mAb) can induce proliferation and interleukin 2 production by triggering still undefined intracellular signaling pathways. We have developed a deglycosylation procedure that allows the precise identification of a distinct CD28 protein band, facilitating the analysis of activation-dependent changes in the phosphorylation state of CD28. Phorbol 12-myristate 13-acetate (PMA) treatment induced the in vitro phosphorylation of CD28 on threonine as detected in immune complex kinase assays. This effect of PMA was (i) rapid, preceding a PMA-induced increase in CD28 surface expression; (ii) occurred using kinase buffer containing either manganese or magnesium; and (iii) was found in human peripheral T cells, Jurkat T cells, and in a Jurkat subclone, J.Cam1, that is deficient in Lck tyrosine kinase activity. In contrast, anti-CD28 monoclonal antibody stimulation led to in vitro phosphorylation of CD28 on tyrosine that was manganese-dependent and required Lck tyrosine kinase activity, as it was undetectable in J.Cam1 cells. Importantly, CD28 was phosphorylated on tyrosine in vivo as detected with anti-phosphotyrosine antibodies after stimulation with anti-CD28 monoclonal antibody. The in vivo tyrosine phosphorylation of CD28 was inhibited by PMA treatment and was absent in J.Cam1 cells. Thus, the CD28 coreceptor can trigger different intracellular signaling pathways, depending upon the nature of the initial costimulatory signal.

Characterization of a novel co-stimulatory molecule: A 155–160 kD B cell surface protein provides accessory help to CD4 + T cells to proliferate and differentiate

Optimal activation of T cells to clonally expand requires at least two distinct biological signals; one is generated by the interaction of the T cell receptor (TcR) with peptides bound to MHC molecules. The other signal(s) is (are) generated by a functionally defined event called the co-stimulatory pathway. We have characterized the co-stimulatory property of a murine B lymphocyte membrane protein (155–160 kD) on resting CD4 + T cells. The study involved the isolation of a 155–160 kD protein (B1) from the membranes of LPS-stimulated B cells. When reconstituted into lipid vesicles, B1 exerted a dose-dependent proliferative response to CD4 + T cells, resulting in the predominant secretion of IL-4 and IL-5 after cross-linking receptors with anti-CD3 mAb. This protein is a phosphoglycoprotein which gives a single spot on two-dimensional gel electrophoresis under reducing conditions and as a distinct peak on reverse phase-HPLC. The B1 binds to the T cell surface as is demonstrated by electron microscopic autoradiography and scanning electron microscopy, as well as competitive binding assays. It does not cross-react with antibodies directed against ICAM-1, LFA-1 alpha, B7, HSA and VCAM-1, suggesting the novelty of the protein. Activation of CD4 + T cells with B1 in the presence of anti-CD3 resulted in the translocation of protein kinase C (PKC). The B1 is barely detectable on the surface of resting B cells and digestion of this protein with V8 protease and peptide N-glycosidase F resulted in distinct protein bands on an autoradiogram.

DENN, a novel human gene differentially expressed in normal and neoplastic cells.

A novel 5843 bp human cDNA sequence was isolated from fetal liver cDNA using the RACE procedure. Within this cDNA was characterised an uninterrupted ORF of 3861 nt encoding a predicted hydrophilic protein of 1287 aa with a calculated molecular mass of 141790 Da and an isoelectric point of 5.41. An RGD cellular adhesion motif was identified within the putative serine- and leucine-rich protein. Northern blot hybridisation with a specific cDNA probe revealed differential levels of expression of 6.5 kb transcripts in 35 human tissues and cancer cell lines, with strongest signals observed in fetal brain and kidney; adult testis, ovary, brain and heart; and in the SW480, K-562, HeLa S3 and HL-60 cell lines. To reflect this feature, the novel gene was designated DENN for differentially expressed in normal and neoplastic cells. Furthermore, an alternative splicing event within DENN involving a 129 nt alternative exon encoding 43 aa was found in fetal liver and in several human cancer cell lines. Based on the predicted aa sequence, two peptides were designed and synthesised to raise rabbit polyclonal antisera which detected a distinct protein band of 140-145 kDa in Western blots of human cell lines. Immunofluorescent labelling of human cells with the same antibodies indicated predominant cell membrane localisation with some cytoplasmic staining.

Photoaffinity Labeling of a Cell Surface Polyamine Binding Protein

Intracellular polyamine pools are partially maintained by an active transport apparatus that is specific for and regulated by polyamines. Although mammalian transport activity has been characterized by kinetic studies, the actual protein itself has yet to be identified, purified, or cloned. As one approach to this problem, we attempted photoaffinity labeling of plasma membrane proteins using two specifically designed and synthesized polyamine conjugates as photoprobes. The first is a spermidine conjugate bearing the photoreactive moiety 4-azidosalicylic acid at the N4 position via an alkyl linkage, and the second is a norspermine conjugate with 4-azidosalicylic acid at the N4 position via an acyl linkage. Labeling of murine L1210 lymphocytic leukemia cells was carried out at 4 degrees C to promote selective alkylation of cell surface proteins. Separation of plasma membrane proteins from cells cross-linked with the N4-spermidine conjugate by SDS-polyacrylamide gel electrophoresis revealed two heavily labeled proteins at approximately 118 and approximately 50 kDa (designated p118 and p50, respectively). Band p118 was more well defined and much more intensely labeled. Analogous proteins were also observed in human U937 lymphoma cells. Specificity of labeling was strongly suggested by competition with polyamines and analogs during labeling and further indicated by the nearly identical labeling of the same protein by the N1-norspermine photoprobe but not by the unconjugated photoreagent. Neuraminidase pretreatment of L1210 cells increased mobility of the p118, suggesting that it was glycosylated and, thus, of plasma membrane origin. In transport-deficient L1210 cells, p118 and p50 were found to have a slightly higher molecular mass and were accompanied by a less distinct protein band (approximately 100 kDa). These findings indicate the presence of a polyamine binding protein at the surface of murine and human leukemia cells, which could be directly or indirectly related to the polyamine transport apparatus.

Cytokines and growth factors modulate cell growth and insulin‐like growth factor binding protein secretion by the human salivary cell line (HSG)

The human salivary cell line (HSG) was investigated for the effect of various growth factors and cytokines on cellular proliferation and on the production of insulin-like growth factor binding proteins (IGFBPs). IGF-I increased cell growth by approximately 25%, and induced the appearances of three distinct protein bands on ligand blot of the cell culture. Two bands with molecular weights of 43 and 45 Kda, respectively, proved to be IGFBP-3 using a specific antibody, and the third was a 24 Kda species (probably, IGFBP-4). Similar IGFBPs were released by the cells following stimulation by EGF and insulin as well as following incubation with the IGF-I receptor antibody alpha IR3. Retinoic acid had an inhibitory effect (50%) on IGF-I-induced cellular proliferation and an attenuative effect on the 24 Kda band when it was combined with IGF-I, and to a lesser effect EGF; however, it enhanced IGFBP-3 production when incubated with IGF-I. The IGF-I receptor antibody had an agonistic effect on IGFBPs production when applied alone or together with IGF-I. TNF-alpha and INF-gamma had minimal effects on cell growth when added alone but when applied in combination, a marked inhibition of cellular proliferation was noted. These cytokines caused increased accumulation of IGFBP-3, -4, and -5. Addition of IGF-I to these cytokines enhanced the expression of these bands. These data demonstrate that growth factors and cytokines which modulate HSG cell growth, induce specific IGFBPs which may play a role in their effects on cell growth.

Characterization of a novel bovine leukocyte protein involved in cell-cell adhesion.

Preliminary characterization of an apparently novel bovine leukocyte adhesion protein is described. Two IgG1 monoclonal antibodies, UC-C1 and UC-H5, raised against established cultures of IL-2-dependent bovine peripheral blood lymphocytes (PBL) were found to react with an antigen expressed by the majority of bovine peripheral blood leukocytes. Immunoprecipitation and polyacrylamide gel electrophoresis of the antigen produced a distinct protein band of molecular weight 160,000, and additional diffuse protein bands of approximate molecular weight 180,000, 175,000, and 150,000. Two-color flow cytometric analyses showed that the antigen was expressed at low density on a small proportion of circulating B lymphocytes, but was highly expressed on all circulating T lymphocytes. The majority of monocytes and all granulocytes expressed the antigen at a density lower than that of T lymphocytes. Peripheral blood lymphocytes stimulated with concanavalin A had an approximately 3-fold increased expression of the antigen, which was apparent within 18 h and remained stable in long-term cultures. Expression of the antigen in thymus, analyzed by the immunoperoxidase technique, was predominantly restricted to thymocytes in the immediate subcapsular cortex and medulla; expression in lymph nodes and spleen was predominantly confined to lymphocytes in T-cell areas. Flow-cytometric analysis demonstrated that thymocytes and the majority of peripheral and mesenteric lymph node-derived T cells had relatively low surface density of antigen compared to circulating T cells. Binding of UC-C1 or UC-H5 to the antigen on lymphocytes induced homotypic aggregation. UC-C1 completely blocked binding of FITC-conjugated UC-H5 to blood mononuclear cells, suggesting that the antibodies recognize the same epitope or proximal epitopes.

Insulin-induced translocation of the glucose transporter GLUT4 in cardiac muscle: studies on the role of small-molecular-mass GTP-binding proteins.

Subcellular fractions obtained from rat cardiac ventricular tissue were used to elucidate a possible functional relationship between small-molecular-mass G-proteins and the insulin-responsive glucose transporter GLUT4. Proteins were separated by SDS/PAGE and transferred to nitrocellulose membranes. Incubation with [alpha-32P]GTP revealed the presence of two major distinct GTP-binding protein bands of 24 and 26 kDa in both plasma and microsomal membranes. Immunoadsorption of microsomal membranes to anti-GLUT4 antibodies was used to isolate GLUT4-enriched membrane vesicles. This material was found to contain a much decreased amount of small G-proteins, with the exclusive presence of the 24 kDa species. Insulin treatment in vivo had no effect on the microsomal membrane content of small GTP-binding proteins, but significantly decreased the 24 kDa species in GLUT4-enriched vesicles by 36 +/- 5% (n = 3). This correlated with a decreased (30-40%) recovery of GLUT4-enriched vesicles from insulin-treated animals. Western-blot analysis of microsomal membranes with a panel of antisera against rab GTP-binding proteins indicated the presence of rab4A, with a molecular mass of 24 kDa, whereas rab1A, rab2 and rab6 were not observed. rab4A was barely detectable in GLUT4-enriched vesicles; however, insulin produced an extensive shift of rab4A from the cytosol and the microsomal fraction to the plasma membrane with a parallel increase in GLUT4. These data show that a small GTP-binding protein is co-localized with GLUT4 in an insulin-responsive intracellular compartment, and strongly suggest that this protein is involved in the exocytosis of GLUT4 in cardiac muscle. Furthermore, the observed translocation of rab4A is compatible with insulin-induced endosome recycling processes, possibly including the glucose transporters.

MRNA and protein expression of p53 mutations in human bladder cancer cell lines

We investigated mRNA and protein expression in p53 gene mutations in four human bladder cancer cell lines using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and Northern blot and Western blot analyses. The following mutations were identified in three of the four cell lines: a missense transversion at codon 110, a missense transition at codon 250 and a non-sense transversion at codon 126. These mutations were located outside previously identified hot spot codons and have rarely been reported in bladder cancer tissues or other neoplasms. Positive intranuclear p53 immunostaining in neoplastic cells in the two missense mutations and the premature stop codon in the non-sense mutation suggested the presence of structural and functional alterations in the p53 protein. Northern and Western blot analyses revealed either an intense or a weak p53 mRNA band together with an intense p53 protein band in the missense mutations, but no p53 mRNA or protein band in the non-sense mutation. A weak p53 mRNA band, but no distinct p53 protein band was observed in the cell line without a mutation and in normal control bladder cells. Our findings suggest that regulation of p53 expression in these cell lines differs at the posttranscriptional and/or post-translational level between the wildtype and the mutant p53 genes and also among different mutant p53 genes. The three cell lines with mutations were derived from high-grade carcinomas; the cell line without mutation was derived from a low-grade carcinoma.

Isolation and partial polypeptide characterization of bovine neutrophil plasma membranes

Summary The neutrophil plasma membrane has a major role in migration, phagocytosis, and destruction of microorganisms. Neutrophils isolated from blood and mammary secretions were homogenized, and the plasma membrane fraction was isolated on discontinuous sucrose gradient (20, 32, and 50%). Purity of plasma membrane preparation was determined by use of marker enzyme analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) of the membrane proteins was performed under reducing conditions for polypeptide characterization. The membrane proteins were also labeled with 125I externally, using 1,3,4,6-tetrachloro-3α-6α-diphenylgly-couril, and proteins were separated by sds-page and autoradiographed. Compared with whole cell homogenate, the plasma membrane fraction obtained at the 20/32% interface was enriched for the marker enzymes, 5′-nucleotidase (16-fold), alkaline phosphatase (5.5-fold), and total phosphatase (26-fold). The membrane fraction had minimal specific activity for β-glucuronidase (0.4-fold), compared with whole cell homogenate. Plasma membrane protein yield was about 500 μg/ 109 bovine blood neutrophils. The sds-page of plasma membrane proteins revealed 25 protein bands, of which 13 were major bands. There were 3 distinct bands (18, 36, and 65 kd) in the plasma membrane-enriched fraction (20/32 interface) that were not seen in other fractions (30/50% and pellet). Further, 125I-labeling identified 5 distinct protein bands (205, 140, 65, 35, and 30 kd). Blood and mammary neutrophils had similar polypeptide patterns, except that 36- and 65-kd bands were more prominent for blood neutrophils than for mammary neutrophils.

Characterization of in-vitro-translated human regulatory and catalytic subunits of cAMP-dependent protein kinases.

Full-length human cDNAs for all the different regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinases (PKA) were transcribed and translated in a cell-free in vitro system. The resulting proteins were characterized with respect to molecular size, isoelectric focusing, immunoreactivity, cAMP binding, and to what extent the RII protein subunits revealed mobility shifts upon phosphorylation by catalytic subunit of PKA. We were able to express cDNAs for all the human R (RI alpha, RI beta, RII alpha and RII beta) and C (C alpha, C beta and C gamma) subunits in a wheat-germ extract. [35S]Methionine-labelled in-vitro-translated products were analyzed by SDS/PAGE and revealed distinct protein bands with apparent molecular masses of 49 (RI alpha), 54-55 (RI beta), 51 (RII alpha) and 53 kDa (RII beta) for the R subunits. In vitro transcription/translation of the cDNAs for the C subunits of PKA gave proteins with molecular masses of approximately 40 kDa for all the different C subunits. Phosphorylation of RII alpha and RII beta by the C subunit of PKA, revealed a distinct mobility shift of the RII alpha subunit on one-dimensional SDS/PAGE (51-54 kDa), but not of RII beta (53 kDa). Further characterization of the R subunits by two-dimensional SDS/PAGE revealed that RI alpha was more acidic than RI beta, with pIs of 6.1-6.0 and 6.4-6.2, respectively. Furthermore, the RII alpha protein was more basic than RII beta, with pIs of approximately 5.4-5.3 and 5.3-5.1, respectively. All the in-vitro-translated R subunits could be photoaffinity labelled by the cAMP-analog 8-azido-[32P]cAMP and were also detected by immunoprecipitation with subunit-specific antibodies.

Detection and purification of modified leghemoglobins from soybean root nodules

A new procedure was developed to purify leghemoglobins (Lbs) from Glycine max L. (Merr). root nodules. This procedure resulted in the purification of a hemoprotein fraction, Lbam as detected by ion-exchange-HPLC was found in nodule extracts prepared in the presence of peptidase inhibitors, and was unstable in acidic solutions. Acidic conditions also emhanced proteolysis of Lba by a nodule enriched in peptidases as observed using a denaturing peptide gel developed for this purpose. In contrast, there was little degradation of Lba by endogenous peptidases at pH 8.0. We incorporated these observations into the purification protocol as follows: (1) nodule extracts were first chromatographed on hydroxylapatite at pH 6.8. Greater than 98% of the peptidases bound to the column and the Lbs were recovered in the wash fraction; (2) subsequent ion-exchange chromatography of the wash fraction at pH 8.0 yielded several fractions related to Lba, including Lbam; and (3) The crude Lbam fraction was resolved into four distinct protein bands by isoelectric focusing using a new buffer system and gel formulation. One of these bands exhibited a p I value identical to that of Lba, whereas the other three proteins displayed more acidic p I values of 4.91, 4.87 and 4.85, respectively. All of these purified Lbam proteins had molecular weights indistinguishable from Lba, similar amino acid compositions and possessed a sequence identical to that of Lba in the first 15 residues from the amino-terminal end.

Partial purification and characterization of extracellular metalloproteases with caseinolytic, aminopeptidolytic and collagenolytic activities from Vibrio anguillarum

Abstract. Proteases produced by Vibrio anguillarum were isolated from culture supernatant by ultrafiltration, gel chromatography and ion exchange chromatography. The enzyme(s) were shown to be collagenolytic when assayed with native collagen substrates. In addition, the enzyme(s) hydrolysed azocasein, azocollagen, the collagenase substrate 4‐phenylazobenzyloxycarbonyl‐Pro‐Leu‐Gly‐Pro‐D‐Arg and the aminopeptidase substrate L‐Leu‐pNA effectively. Separation of the proteases by Mono Q ion exchange chromatography and native polyacrylamide gel electrophoresis revealed four distinct protein bands containing caseinase activity. However, only two of the bands showed aminopeptidase activity. The aminopeptidase activities could be separated from the caseinase activities by isoelectric focusing. Secreted proteases of different serotypcs of V. anguillarum showed a heterogeneous caseinolytic pattern. The molecular mass of the major enzyme was estimated at 35kDa as determined by its mobility on SDS‐polyacrylamide gels. Serine protease inhibitors like PMSF, TPCK, TLCK and benzamidine had no inhibitory effects on the proteolytic activity when tested with azocasein as substrate. However, the enzyme was strongly inhibited by metal chelators like EDTA and 1, 10‐phenanthroline. Also, normal salmon scrum and purified α2‐macroglobulin from salmon serum strongly inhibited the caseinolytic activity of the enzyme.

Isolation and Characterization of the Outer Membrane Proteins of Serratia marcescens W225

The study addressed the general problem of fractionating cell envelopes in order to isolate the outer membranes of gram-negative bacteria. Whereas the cells are normally transformed into spheroplasts prior to disintegration and membrane separation, Serratia marcescens was found to be resistant to spheroplast formation using the procedures available, which were originally developed for Escherichia coli. An efficient technique for spheroplasting S. marcescens was therefore developed; this comprised combining osmotic shock and lysozyme-EDTA treatment of sucrose-conditioned cells. Spheroplasting efficiency and the amount of outer membrane protein recovered were highly dependent on the spheroplasting technique used. Separation of the outer and inner membranes was performed by two methods, isopycnic centrifugation and selective detergent solubilization with Sarkosyl. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the analysis of specific inner membrane marker enzymes revealed that the protein obtained by detergent solubilization was much purer than that obtained by isopycnic centrifugation. The outer membrane isolated accounted for 60% of the envelope proteins and had a buoyant density of 1.2502 g/cm3. The protein profile of the outer membrane determined by SDS-PAGE resolved into 12 distinct protein bands, 3 of which represented major proteins.