Extracts Of Human Placenta Research Articles

Purification and identification of multiple forms of dynorphin in human placenta

Immunoreactive dynorphin (ir-Dyn) and opiate-like peptides (OLP) were measured in acid (HCl) extracts of human placenta by the use of an antibody to synthetic Dyn-(1–13) and of the displacement of [ 3H]-naloxone binding to rat brain homogenates, respectively. The placenta contained 57.6 pmoles per g of ir-Dyn and 134.4 pmoles per g of naloxone binding equivalents. After passage of the extract through cartridges of Sep Pak C18, half of the OLP was eluted with ir-Dyn at 35% acetonitrile (ACN), the rest being eluted at 60% ACN. Both fractions obtained from Sep Pak were chromatographed separately on Sephadex G-50, the OLP of the 35% ACN fraction coeluting with the ir-Dyn peak and that of the 60% ACN fraction being eluted at the same volume as synthetic β-endorphin. Conversely, the fraction of OLP coeluting with synthetic leucine-enkephalin (Leu-Enk) in these two chromatographies was minimal. The Dyn-immunoreactive material was further purified by high pressure liquid chromatography on reverse phase μ-Bondapak C18 columns to give three distinct peaks corresponding to synthetic Dyn-(1–11), Dyn-(1–13) and Dyn-(1–12), respectively. Our results indicate that the human placenta contains several forms of ir-Dyn which account for about half of its endogenous OLP.

Corticotropin-releasing factor-like activity in human placental extracts.

Corticotropin-releasing factor (C R F)-like activity, which was first found in the hypothalamus, was detected in the extracts of human placentas obtained at full term from spontaneous deliveries. The molecular size of the placental CRF-like substance is slightly larger than that of adrenocorticotropin. In cultures of rat anterior pituitary cells, the placental CRF-like material shows bioactivity similar to that of the partially purified rat hypothalamic CRF and synthetic ovine C R F on the release of adrenocorticotropin and (β-endorphin. Furthermore, the CRF-like activity from human placenta was attenuated by trypsin digestion. Thus the human placenta may elaborate a substance similar to hypothalamic CRF.

Human term placenta contains transforming growth factors

Growth factors of apparent molecular weights of 6,000, 10,000, 20,000 and one in excess of 30,000 daltons can be isolated from acid-ethanol extracts of human term placentas. Each size class of growth factor resembles transforming growth factor (TGF) in that it stimulates anchorage independent growth of normal rat kidney cells and competes with EGF for binding to EGF membrane receptors. The 6,000, 10,000 and 20,000 molecular weight polypeptides also resemble TGF in their acid and heat stability, and their requirement for intact disulfide bonds for growth promoting activity. By homologous radioimmunoassay, neither the 6,000 nor 10,000 dalton polypeptide is related to human epidermal growth factor (hEGF). The presence of these TGFs in ample concentrations (approximately 100 ng of EGF equivalents per term placenta for the 10,000 dalton polypeptide) indicates the usefulness of this tissue source for study of human TGFs.

A monoclonal antibody to intestinal alkaline phosphatase made against D98/AH-2 (HeLa) cells.

A monoclonal antibody (AAP1) to human intestinal alkaline phosphatase (ALP) was produced by immunizing a mouse with D98/AH-2 (HeLa) cells, which produce the enzyme ectopically. The antibody, which did not inhibit enzyme activity using p-nitrophenyl phosphate as the substrate, was of the IgG2A class and did not show complement-dependent cytotoxicity. In trace binding assays AAP1 bound only to cells that expressed an intestinal-like form of human ALP, including some human intraspecific (D98/AG-2 x human lymphocyte or fibroblast) hybrids. Immunoprecipitation of immune complexes from cell-free extracts of D98/AG-2 cells, using protein A containing S. aureus and AAP1 antibody, resulted in precipitation of all the ALP activity. The precipitated material had a subunit molecular weight of 80,000 daltons, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. In non-denaturing conditions, AAP1 antibody prevented the migration of ALP activity into the gel when cell-free extracts were made from human adult or fetal intestine, or D98/AH-2 cells. Similarly, AAP1 could be used to precipitate ALP activity from these extracts but not from extracts of human liver, kidney or placenta.

Studies on immunoreactive ACTH from human term placenta. (I) Detection of a high molecular weight-immunoreactive ACTH in term placenta (author's transl)

Extracts of human term placenta were fractionated by Sephadex G-75 gel filtration and assayed for immunoreactive ACTH. Both high and low molecular weight protein fractions were detected to be immunologically reactive toward anti-human ACTH (1--39 alpha) antibody. For the extraction of low molecular weight ACTH from human term placenta (pl. -ACTH), a glacial acetic acid-acetone mixture was employed, while a pH 3.0-HCl solution was used for high molecular weight immunoreactive ACTH. The high molecular weight immunoreactive ACTH fraction (F-I), co-eluted with horse hemoglobin from a Sephadex G-75.column in 0.1M acetic acid, was essentially devoid of low molecular weight materials as revealed by polyacrylamide gel disc electrophoresis at pHs 9.5 and 4.3. Tryptic digestion of F-1 at pH 8.1 and 37 degrees C for 4 hr with E/S of 1/100, followed by fractionation with a Sephadex G-75, resulted in the formation of lower molecular weight fragments. One fragment was eluted at the same position as that of porcine ACTH with a recovery of 86% of immunoreactivity of F-I. Another fragment which was eluted last exhibited positive beta-endorphin receptor binding activity. These results suggest the presence of a common precursor protein to ACTH and beta-endorphin in human term placenta.

High-molecular-weight immunoreactive beta-endorphin in extracts of human placenta is a fragment of immunoglobulin G.

A high-molecular-weight protein with beta-endorphin- and adrenocorticotropin-immunoreactivities was isolated from extracts of human placenta after several purification steps, including immunoadsorption with a well-characterized antiserum raised to beta-endorphin. This protein was identified as the heavy chain of the human immunoglobulin class IgG1. These results have led to the recognition of homologies in the amino acid sequences of these physiologically unrelated molecules. They also suggest caution in accepting immunological competence as the sole criterion of the chemical identity of a ligand.

Thyrotropin-releasing hormone-like bioactivity in placenta: evidence for the existence of substances other than Pyroglu-His-Pro-NH2 (TRH) capable of stimulating pituitary thyrotropin release.

The purpose of this study was to investigate the chemical nature of the TRH-like bioactivity and immunoreactivity in extracts of human placenta. Methanolic extracts of human placenta contained nearly 36 ng TRH-like bioactivity/g dried tissue. The immunoreactivity of this extract was only 3 ng/g dried tissue. Two molar acetic acid extracts of human placenta yielded 9 ng TRH-like bioactivity/g dried tissue. The immunoreactivity of these acetic acid extracts, however, was 5.5 ng/g dried tissue. When these placental extracts were subjected to gel filtration chromatography, the bioactivity was found to reside in two fractions which were distinct from synthetic TRH. Furthermore, the immunoreactivity present in these placental extracts was also chromatographically distinct from that of synthetic TRH. In conclusion, these experiments confirm the presence of substantial quantities of materials in placenta which possess TRH-like immunoreactivity and bioactivity. These results also argue that the immunoreactive fractions are not bioactive and provide firm evidence that neither the TSH-releasing substances nor the TRH-like immunoreactivity found in human placenta are identical to pyroglu-His-Pro-NH2.

Inhibition of platelet aggregation by placental extracts.

Abstract Extracts of human placenta and of human umbilical cord vessels were examined for anti-platelet aggregating activity. Placental extracts progressively inhibited aggregation induced by ADP but had little effect on that produced by adrenaline, collagen or ristocetin. In contrast, umbilical vessel extracts and PGI2 had an almost immediate inhibitory effect on aggregation induced by all four reagents. The effect of placental extracts was shown to be mediated through the degradation of ADP and was mimicked by purified human placental alkaline phosphatase. Since human placental alkaline phosphatase activity is known to be low in pregnancies associated with toxaemia, an alteration in this ADPase activity of the placenta may contribute to the pathogenesis of disseminated intravascular coagulation in eclampsia of pregnancy.

Multiple forms of 2-deoxy-d-glucoside-2-sulphamate sulphohydrolase from human placenta

2-Deoxyglucoside-2-sulphamate sulphohydrolase was purified about 10 000-fold from the soluble extract of human placenta by using as substrate [N-sulpho-35S]heparin. Differently charged enzyme forms were observed on chromatography on DEAE-cellulose, all of which had an apparent mol.wt. of 110 000 as determined by gel filtration. By using immobilized heparan sulphate as affinity matrix the sulphamate sulphohydrolase could be separated into two forms, a minor one with low and a major one with high affinity for the adsorbent. When tested with [N-sulpho-35S]heparan sulphate the low-affinity form had a Km of 0.2 mM, and the high-affinity form a Km of 0.03 mM. Both forms exhibited the same Km of 10 microM towards [N-sulpho-35S]heparin and were equally well adsorbed to immobilized heparin. The two forms could be distinguished by their pH-optima and by the influence of KCl on heparan sulphate sulphohydrolase activity.

Purification and properties of N-acetylgalactosamine 6-sulphate sulphatase from human placenta.

1. N-Acetylgalactosamine 6-sulphate sulphatase was purified about 20000-fold from the soluble extract of human placenta with N-acetylgalactosamine 6-sulphate-glucuronic acid-N-acetyl[1-(3)H]galactosaminitol 6-sulphate as substrate in the activity assay. The enzyme appears to be a glycoprotein with a mol.wt. of about 100000 as determined by gel filtration. On gel electrophoresis in the presence of sodium dodecyl sulphate the major protein band had a mol.wt. of 78000. Variable charge heterogeneity was observed in several enzyme preparations. 2. The purified enzyme released up to one sulphate molecule from the disulphated trisaccharide. It was active towards N-acetylgalactosamine 6-sulphate and exhibited no measurable N-acetylglucosamine 6-sulphate sulphatase or any other known lysosomal sulphatase activity. Hydrolysis of [1-(3)H]galactitol 6-sulphate was achieved by incubation neither with a crude nor with a purified enzyme preparation. Chondroitin 6-sulphate and keratan sulphate, as well as heparin and heparan sulphate, served as competitive inhibitors of the enzyme. 3. Purified N-acetylgalactosamine 6-sulphate sulphatase activity was optimal at pH4.9 and 4.4 when assayed in 0.02m-sodium acetate buffer and at pH4.2 and 5.2 in 0.1m-sodium acetate buffer. A single pH-optimum at pH4.8 was observed for the crude enzyme and for the purified enzyme after mild periodate treatment. The sulphatase activity was inhibited by a variety of anions and cations and activated by thiol-specific and thiol reagents.

Thyrotropin-releasing hormone activity in the human placenta.

TRH immunological and biological activities were found in extracts of human placentas. Eight term placentas delivered by cesarean section were extracted with 2 N acetic acid, followed by glacial acetic acid. Lyophilized samples were resuspended in phosphate-buffered saline. TRH activity, identified in each placental extract by specific RIA, averaged 19.8 +/- 3.3 pg/mg protein. TRH in the placental extracts was similar to synthetic TRH by four criteria. 1) Serial dilutions of placental extracts defined an immunoassay inhibition curve with a slope (-1.93) identical to synthetic TRH (-1.88). 2) When placental extract or synthetic TRH was chromatographed on Sephadex G-10, TRH immunoreactivity was found in similar fractions of the eluate. 3) Both placental extract and synthetic TRH stimulated TSH release from rat pituitaries in vitro. 4) Human serum degraded placental extract and synthetic TRH in a qualitatively similar manner. Placental minces did not accumulate exogenous [3H]TRH, suggesting that the placental TRH may originate endogenously.

S-adenosylhomocysteine hydrolase is an adenosine-binding protein: a target for adenosine toxicity.

When adenosine deaminase activity is inhibited, low concentrations of adenosine are toxic to human lymphoblast mutants that are unable to convert adenosine to intracellular nucleotides. In order to identify the mediator of this cytotoxicity, we searched for a cytoplasmic protein capable of binding adenosine with high affinity. Such a protein was identified in extracts of human lymphoblasts and placenta as the enzyme S-adenosylhomocysteine hydrolase.

Presence of immunoreactive β-lipotropin and β-endorphin in human placenta

Utilizing a sensitive radioimmunoassay capable of detecting both β-lipotropin (β-LPH) and β-endorphin (β-EP), we demonstrated the existence of immunoreactive β-LPH and β-EP in extracts of human placentas. Gel chromatographic studies revealed that total β-EP immunoreactivity consists of two fractions with elution positions compatible with β-LPH and β-EP, respectively, and a fraction of larger molecular weight. All three fractions showed parallel curves with the standard curve of β-EP in radioimmunoassay. Our observations suggests that β-EP, β-LPH and possibly their precursor exist in human placenta.

Nonspecific Crossreacting Antigen (NCA) in Human Mammary Carcinoma Extracts

Human mammary carcinoma (HMC) homogenates from 28 patients and mammary hyperplasia (MH) homogenates from 20 patients undergoing reduction mammoplasty were used as antigens in preparing antibodies in rabbits. Crossed immunoelectrophoresis of HMC extracts against the corresponding antibodies revealed one major precipitate not present in MH. This antigen was present in separately prepared extracts of breast tissue from 3 out of 10 patients with HMC. Antibody titre was unchanged following absorption with MH, normal human liver, kidney, skin and serum. Immunochemical crossreaction with pregnancy zone proteins, alpha‐1‐fetoprotein, ferritin, lactoferrin, plasma, extracts of human placenta and fetal and adult liver did not occur. The antigen could not be demonstrated by rocket immunoelectrophoresis of sera from patients with HMC. Small amounts of the antigen were found in extracts from human spleen and renal carcinoma and larger amounts in extracts from human peripheral granulocytes, but not in preparations of mononuclear leucocytes (98% pure). Tandem crossed immunoelectrophoresis of extracts from HMC and carcino embryonic antigen against antibodies prepared against HMC revealed a reaction of partial identity between the two antigens, and antisera prepared against carcino embryonic antigen contained precipitating antibodies against the HMC antigen. The HMC antigen was eventually identified as nonspecific crossreacting antigen (NCA) described by other authors.

Detection and partial purification of rapidly sedimenting forms of aminoacyl-transfer ribonucleic acid synthetases from human placenta

The pattern of aggregation of aminoacyl-tRNA synthetases in extracts of human placenta is described. The sedimentation behavior of aminoacyl-tRNA synthetases in crude extracts of human placenta was analyzed by velocity gradient centrifugation. Varying proportions of seven aminoacyl-tRNA synthetase activities (arginyl-, glutaminyl-, glutamyl-, isoleucyl-, leucyl-, lysyl-, and methionyl-tRNA synthetases) sedimented as broad peaks at 17 to 21 S. Twelve other aminoacyl-tRNA synthetases sedimented at 5 to 9 S, a range more typical of individual aminoacyl-tRNA synthetases. The seven aminoacyl-tRNA synthetases which are aggregated in the placenta are the same enzymes which are most often reported to be aggregated in diverse mammalian species and tissues. All seven of the aggregated activities copurified as 16.2 to 18.8 S forms by ammonium sulfate precipitation, fractionation on alumina C γ and calcium phosphate gels, and chromatography on diethylaminoethyl (DEAE) cellulose. Before their gradient elution from DEAE-cellulose, the aggregated aminoacyl-tRNA synthetases could be stored with little loss of activity or change in sedimentation profile. Gradient elution of the aggregated activities from DEAE-cellulose columns resulted in accelerated loss of the activities during storage and in reduced sedimentation rates. Further reductions in sedimentation coefficients of aminoacyl-tRNA synthetases were observed after storage of fractions recovered from DEAE-cellulose columns.

Creatine kinase isoenzyme activity in human placenta and in serum of women in labor.

Creatine kinase (EC 2.7.3.2) isoenzymes in extracts of human placenta and in serum from nonpregnant women and women in labor were separated on columns containing diethylaminoethyl-cellulose and assayed. The distribution of the isoenzymes in placenta (n = 10) was 80% BB (200 +/- 66 U/g (wet weight), 19% MM (49 +/- 30 U/g), and 1% MB (2.6 +/- 1.7 U/g); The geometric mean for the serum BB activity of the nonpregnant women (n = 50) was 0.6 +/- 1.5 U/liter, as compared to 3.0 +/- 1.4 U/liter for patients in labor who had normal deliveries (n = 92). The arithmetic mean for serum BB activity of labor patients with induced labor (n = 20), premature labor (n = 7), cesarian section (n = 6), or hypertension and pre-eclampsia (n = 6) did not differ significantly from the arithmetic mean BB activity for serum of labor patients with normal deliveries. However, the arithmetic mean serum BB activity of patients with stillbirths (n = 7) was significantly smaller than the arithmetic mean for normal labor patients.

Arginase from human full-term placenta

Arginase was purified about 1800-fold from extracts of human full-term placenta; the enzyme appeared to be homogenous by disc electrophoresis and molecular-sieve chromatography. The mol. wt. determination by gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis yielded a value of 70000 for the most pure and the partially purified enzyme. The human placenta arginase is a metalloenzyme with an optimum pH of 9.1. The Km for L-arginine is 27 mM. L-Ornithine and L-lysine show competitive inhibition with Ki values of 6.3 and 14 mM respectively.

The effect of active immunity against placental proteins on pregnancy in monkeys

Squirrel monkeys were actively immunized against human placental lactogen (HPL) and/or an extract of human placental tissue, or squirrel monkey placenta. Only half of the monkeys immunized with HPL developed a detectable antibody titer to HPL prior to the subsequent mating season. During the subsequent breeding season, the pregnancy rate was cut in half. The titer developed against HPL did not seem to be related to their ability to become pregnant. When the group of animals which had previously been immunized with HPL were challenged with extracts of human placenta, all animals developed detectable circulating antibody. There was no increase in the effectiveness of this immunization as compared to HPL in pregnancy prevention, however. A third group of monkeys was immunized with extracts from their own placental extracts, and all developed some degree of antibody. The titer obtained did not seem to be related to whether or not they became pregnant. In this group, however, the pregnancy rate was decreased to one fourth of what it had been before immunization.

Purification and properties of the glucose-6-phosphate-dependent form of human placental glycogen synthase

Glycogen synthase in the glucose-6-phosphate (glucose-6-P)-dependent form was purified over 10,000-fold from an extract of term human placenta. The purified enzyme shows a single protein band on polyacry1amide-gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme activity in the presence of glucose-6-P is increased by the single addition of Mg 2+, Ca 2+, or Mn 2+ and is reduced by the addition of either sulfate or phosphate. Addition of either Mg 2+, Ca 2+, or Mn 2+ relieves the inhibition by sulfate or phosphate. The enzyme activity in the absence of glucose 6-P is greatly increased by the addition of MnSO 4, CoSO 4, and NiSO 4 and is increased to a lesser extent by MgSO 4, CaSO 4, and FeSO 4. The activation of the glucose-6-P-dependent form of the enzyme by these metal sulfates in the absence of glucose-6-P has never been reported. MnSO 4, which shows homotropic cooperativity, is the best activator among the various metal sulfates tested. The human placental glucose-6-P-dependent form of glycogen synthase (D form) can be converted to the glucose-6-P-independent form (I form) of the enzyme by incubating the partially purified glycogen synthase, which is copurified with synthase phosphatase, with Mn 2+. This conversion can be reversed by the addition of cyclic AMP-dependent protein kinase. The synthase D to synthase I converting system from human placenta is unique in its stringent requirement for Mn 2+.

Changing patterns of placental hexokinase isozymes during the course of gestation

Hexokinase, an enzyme that is capable of regulating the entry of glucose into metabolic sequences, is known to exist in four isozyme forms in a variety of mammalian tissues. Each of these isozymes possesses different kinetic properties, suggesting that they may serve in different regulatory capacities. In addition, the proportions of the four isozymes are characteristically different for various differentiated tissues, presumably reflecting different metabolic capabilities of the tissues. Extracts of rabbit and human placentas from early gestational age and term pregnancies were chromatographed and assayed for hexokinase activity. Four peaks of activity were observed. Elution positions of the four placental hexokinase isozymes were comparable to those from liver; however, the relative proportions were considerably different. In both rabbit and human placentas, the proportion of Type I hexokinase increased during gestation and Type III decreased, while Types II and IV remained essentially unchanged. Implications of the gestational changes for placental carbohydrate metabolism are discussed.