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Purification of a neutral proteinase, associated with the actomyosin complex, from uterine myometrium.

ABSTRACT: We have purified and characterized a neutral proteinase activity from pig uterine myometrium. The proteinase co-purified with the actomyosin complex and could only be separated from it by a high concentration of a chaotropic ion, 3M-NaBr. The proteinase was further purified by gel filtration and affinity chromatography. The purified protein showed a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis corresponding to an Mr of 28 000. Gel filtration on Sephadex G-100 in a buffer containing 3M-NaBr gave an Mr of 27 500. Without the addition of the chaotropic Br- ion, the proteinase aggregates to high-Mr forms of more than 10(6)Da. The proteinase has optimum hydrolytic activity with casein as substrate at pH 7.5-8.0. The thiol-group-blocking reagents p-chloromercuribenzoate, p-chloromercuribenzenesulphonate and Hg2+, as well as soya-bean trypsin inhibitor and 4-aminobenzamidine, inhibited the proteinase. Other bivalent cations, chelating agents and the serine-specific reagents 7-amino-1-chloro-3-tosylamido-L-heptan-2-one and phenylmethanesulphonyl fluoride were without any effect on proteinase activity. The proteinase degraded myosin very rapidly at a molar ratio of proteinase to myosin of 1:50, concomitant with the rate of loss of the ATPase activity. Compared with myosin, actin was only a poor substrate and was degraded at a much lower rate, even at a high molar ratio of the proteinase to actin.

SUBMITTER: Barth R

PROVIDER: S-EPMC1144221 | biostudies-other | 1984 Sep

REPOSITORIES: biostudies-other

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Proteinase and proteinase-inhibitor activities of rat uterine myometrium during pregnancy and involution.

Project description:A supernatant fraction was prepared from rat uterine myometrium by homogenization, sonication and centrifugation. In this supernatant the protein concentration and the activities of an acid proteinase, an acid phosphatase and a proteinase inhibitor were measured. From the fibrous sediment, after washing with 0.5% Triton X-100 and with water, an actomyosin-containing solution was obtained by extraction with 0.6M-NaCl, and in this extract the protein concentration and a neutral proteinase activity were measured. The myometrial wet weight and the activities of the acid proteinase, acid phosphatase and proteinase inhibitor increased by factors of 3-15 during pregnancy and decreased to the same or a greater extent during involution. The amount of protein extracted with 0.6M-NaCl increased by a factor of only 2.3 and the neutral proteinase activity remained essentially constant during pregnancy and involution. The pH optimum of the neutral proteinase, and its pattern of activity compared with those of the lysosomal enzymes, show that the neutral proteinase is not of lysosomal origin. Actomyosin is degraded by the neutral proteinase activity in vitro. Since actomyosin is rapidly broken down only after parturition, the action of the neutral proteinase activity on actomyosin, if this occurs in vivo, must be regulated in some way. The proteinase-inhibitor activity measured in the first supernatant varied in a manner which suggested that it could be involved in this control.

| S-EPMC1186343 | biostudies-other

Partial purification and characterization of a novel neutral proteinase from human uterine cervix.

Project description:1. Human uterine cervical stroma was found to contain a Ca(2+)-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein-Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4-8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4x10(5) by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10mum) or leupeptin (10mum) was also found to be inhibitory, but chymostatin (40mug/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein, Bz-dl-Arg-Nan and heat-denatured collagen, but was inactive towards native collagen and several synthetic substrates, such as 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, 3-carboxypropionyl-Ala-Ala-Ala p-nitroanilide and 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, and also proteoglycan. The enzyme did not act as a plasminogen activator. 6. These properties suggested that a neutral proteinase in the human uterine cervix was different from enzymes previously reported.

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RAC1 is involved in uterine myometrium contraction in the inflammation-associated preterm birth.

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| S-EPMC9513643 | biostudies-literature

Actomyosin Complex.

Project description:Formation of cross-bridges between actin and myosin occurs ubiquitously in eukaryotic cells and mediates muscle contraction, intracellular cargo transport, and cytoskeletal remodeling. Myosin motors repeatedly bind to and dissociate from actin filaments in a cycle that transduces the chemical energy from ATP hydrolysis into mechanical force generation. While the general layout of surface elements within the actin-binding interface is conserved among myosin classes, sequence divergence within these motifs alters the specific contacts involved in the actomyosin interaction as well as the kinetics of mechanochemical cycle phases. Additionally, diverse lever arm structures influence the motility and force production of myosin molecules during their actin interactions. The structural differences generated by myosin's molecular evolution have fine-tuned the kinetics of its isoforms and adapted them for their individual cellular roles. In this chapter, we will characterize the structural and biochemical basis of the actin-myosin interaction and explain its relationship with myosin's cellular roles, with emphasis on the structural variation among myosin isoforms that enables their functional specialization. We will also discuss the impact of accessory proteins, such as the troponin-tropomyosin complex and myosin-binding protein C, on the formation and regulation of actomyosin cross-bridges.

| S-EPMC9710302 | biostudies-literature

Matrix metalloproteinases 2 and 9 are elevated in human preterm laboring uterine myometrium and exacerbate uterine contractility†.

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| S-EPMC6561860 | biostudies-literature

Estrogen receptor alpha isoform ERdelta7 in myometrium modulates uterine quiescence during pregnancy.

Project description:BackgroundCirculating estrogen (E2) levels are high throughout pregnancy and increase towards term, however its local tissue specific actions vary across gestation. For example, myometrial E2 regulated uterotonic action is disabled until term, whereas it's proliferative function is maintained in the breast. We have identified gestationally regulated splicing events, mediated by hnRNPG and modulated by E2 that generate alternatively spliced estrogen receptor alpha (ER?) variants (ER?7 and ER?46) in the myometrium. These variants allow for differential, gestationally regulated, modulation of the uterotonic action of E2.MethodsHuman myometrium isolated from preterm and term non-laboring and laboring pregnant women were analyzed for ER? isoforms and splice factor levels. Lentiviral mediated shRNA knockdown of hnRNPG and overexpression of ER?7 were performed in human myometrial (hTERT-HM) cells. Functional 3D collagen contraction assays were executed.FindingsER?7 acts as a dominant negative repressor of the uterotonic action of ER?66 and ER?46 isoforms through the regulation of the myometrial gap junction protein GJA1. Elimination of hnRNPG inhibits the generation of ER?7 while overexpression of ER?7 inhibited GJA1 expression. Moreover in vivo human myometrial hnRNPG levels decline at term in an E2 dependent manner resulting in a withdrawal of ER?7 levels and its tocolytic action at term.InterpretationOur findings implicate the unique role of ER?7 as a modulator of myometrial quiescence and define the mechanism of ER?7 generation, through hormonally regulated splicing events. FUND: This study was supported by NIH OPRU U01 supplement (HD047905), University of Pittsburgh and Wayne State University Perinatal Research Initiative (USA).

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Cathepsin D from pig myometrium. Characterization of the proteinase.

Project description:The purification of cathepsin D from pig uterus by two-step affinity chromatography on concanavalin A- and pepstatin-Sepharose was described previously [Afting & Becker (1981) Biochem. J. 197, 519-522]. In this paper, chemical and physical properties of the proteinase are presented. The purified enzyme showed three bands on SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis, one main band corresponding to an Mr of 31 000 and two minor bands with Mr values of 43 000 and 15 000 respectively. Gel filtration on Bio-gel P-150 and sedimentation-diffusion equilibrium studies give an Mr for the main band of about 35 000. The pI of the enzyme was determined to be 7.2. Haemoglobin was the best substrate, with a Km value of 6.4 X 10(-6)M. It was hydrolysed with a pH optimum between 3.0 and 3.3 for a substrate concentration of 100 microM. The proteinase was stable over the pH range of 3.5-6.5. At pH 6 the enzyme showed stability up to a temperature of 50 degrees C; at pH 3 the activity was already decreased below 40 degrees C. Carbohydrate studies resulted in the staining of all three bands on an SDS/polyacrylamide gel by thymol/H2SO4. After treatment with endo-beta-N-acetylglucosaminidase H, all three bands were shifted to a region of lower Mr. Of various inhibitors tested, only pepstatin was strongly inhibiting, with a Ki of 2.1 X 10(-9)M.

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Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus.

Project description:Streptomyces strain K1-02, which was identified as a strain of Streptomyces albidoflavus, secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70 degrees C. Its sensitivity to protease inhibitors, its specificity on synthetic substrates, and its remarkably high level of NH2-terminal sequence homology with Streptomyces griseus protease B (SGPB) showed that the new enzyme, designated SAKase, was homologous to SGPB. We tested the activity of SAKase with soluble and fibrous substrates (elastin, keratin, and type I collagen) and found that it was very specific for keratinous substrates compared to SGPB and proteinase K.

| S-EPMC91380 | biostudies-literature

The purification and characterization of a glomerular-basement-membrane-degrading neutral proteinase from rat mesangial cells.

Project description:A neutral proteinase, capable of degrading gelatin, has been found in both an active and a latent form in the medium from the culture of rat mesangial cells. The latent form had an Mr of 80,000-100,000 and could be activated with either 4-aminophenylmercuric acetate or prolonged incubation at neutral pH. The active form of the enzyme was extensively purified. The estimated Mr of the purified enzyme on gel filtration was approximately 200,000, indicating that the active enzyme formed aggregates. However, analysis by SDS/polyacrylamide-gel electrophoresis under reducing conditions showed two protein bands, with Mr 68,000 and 66,000. Both proteins were found to contain proteolytic activity when run on SDS/substrate gels. The enzyme was inhibited by EDTA and 1,10-phenanthroline, but not by inhibitors for cysteine, serine or aspartic proteinases. The enzyme did not digest fibronectin, bovine serum albumin, proteoglycan or interstitial collagen. The enzyme degraded pepsin-solubilized placental type V collagen at 31 degrees C, whereas similarly solubilized type IV collagen was only degraded at higher temperatures. In addition, the neutral proteinase degraded native soluble type IV collagen. It also had activity on insoluble type IV collagen of glomerular basement membrane. The above properties suggest that the mesangial neutral proteinase belongs to the gelatinase group of metalloproteinases and that it may play a role in the normal turnover of extracellular glomerular matrix.

| S-EPMC1149019 | biostudies-other

Altered expression of histone deacetylases, inflammatory cytokines and contractile-associated factors in uterine myometrium of Long Evans rats gestationally exposed to benzo[a]pyrene.

Project description:Etiology of preterm birth (PTB) is multifactorial; therefore, decreasing the incidence of PTB is a major challenge in the field of obstetrics. Epidemiological studies have reported an association between toxicants and PTB. However, there are no studies on the role of benzo[a]pyrene (BaP), an environmental toxicant, in the incidence of PTB. We first assessed the effects of BaP (150 and 300 µg kg(-1) body weight) dosed via gavage from day 14 to 17 of pregnancy on gestation length in Long Evans rats. We further assessed the histopathology of the uterus, expression of inflammatory cytokines, contractile-associated factors, histone deacetylases (HDACs) and NFқB-p65 in myometrium collected on day 22 postpartum versus vehicle-treated controls. In our study, rats exposed to BaP delivered prematurely (P < 0.05) compared to control. Hematoxylin and eosin staining of uterus showed squamous metaplasia, glandular and stromal hyperplasia in BaP-exposed rats versus control. The concentrations of BaP metabolites measured by high-pressure liquid chromatography were higher in uterine myometrium of BaP-exposed rats while they were undetectable in controls. Quantitative real-time polymerase chain reaction showed significant increases in mRNA expression of interleukin-1β and -8, tumor necrosis factor-α, connexin 43, cyclo-oxygenase-2 and prostaglandin F2α receptor as compared to controls (P < 0.05). Western blot analysis revealed that BaP exposure caused decreases in class I HDACs 1 and 3 and increases in class II HDAC 5, cyclo-oxygenase-2 and nuclear translocation of NFκB-p65 relative to controls. Our results suggest that gestational exposure to BaP increases incidence of PTB through epigenetic changes that causes increases in the expression of contractile-associated factors through the NFκB pathway. Copyright © 2015 John Wiley & Sons, Ltd.

| S-EPMC5404737 | biostudies-literature

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