Characterization of three aminopeptidases purified from maternal serum.

The biochemical characteristics of aminopeptidase A (EC 3.4.11.7), oxytocinase (EC 3.4.11.3) and alanyl aminopeptidase (EC 3.4.11.2) purified from serum of pregnant women were compared. Aminopeptidase A hydrolysed only acidic amino acid derivatives, whereas oxytocinase and alanyl aminopeptidase had partially overlapping broad substrate specificities. Oxytocinase showed the highest Vmax value with LeuNA but the lowest Km value with ArgNA (Km 0.059 +/- 0.08 mmol/l). Alanyl aminopeptidase hydrolysed AlaNA most rapidly, but showed the highest affinity for LysNA (Km 0.054 +/- 0.006 mmol/l). The enzymes were sensitive to EDTA. Co2+, Ni2+ and Zn2+ were able to reactivate all suppressed enzymes, but Mn2+ reactivated only aminopeptidase A after EDTA inhibition. The alkaline earth metals were activators of aminopeptidase A, while Co2+ activated only alanyl aminopeptidase. This enzyme was the most sensitive to L-amino acids. Acidic amino acids inhibited aminopeptidase A but had no effect on the two other enzymes. Oxytocinase was most sensitive to thermal treatment. Amastatin did not inhibit oxytocinase, whereas aminopeptidase A was more resistant than alanyl aminopeptidase to this effector.

Purification and partial characterization of aminopeptidase A from the serum of pregnant and non-pregnant women.

Aminopeptidase A (L-alpha-aspartyl(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7) was purified from human maternal and control sera using CM-cellulose chromatography, DEAE-Sephacel chromatography, Sephacryl S-300 gel filtration and hydroxyapatite chromatography. The purification coefficients were 3069 and 5210 and the yields 6.3 and 6.1% for the maternal and control serum, respectively. The purified enzymes appeared free from other serum aminopeptidases in polyacrylamide gel electrophoresis. The biochemical and physical characteristics of the enzymes from maternal and control sera were similar. A molecular weight of 260 000, an optimum at pH 6.75-7.25 and a fairly good stability of the enzymes at 4 and -18 degrees C were recorded. The alkaline earth metals (Ca2+, Ba2+, Sr2+) were the activators of alpha-L-glutamyl-beta-naphthylamide hydrolysis, while alpha-L-aspartyl-beta-naphthylamide hydrolysis was markedly potentiated with Ca2+ but not with Ba2+ at all. The most rapid hydrolysis was shown with GluNA (Km with Ba2+ 0.156 +/- 0.014 mM and 0.136 +/- 0.009 mM in maternal and control serum, respectively), while only minimal hydrolysis of some neutral and basic amino-acid-beta-naphthylamides were observed. The contribution of the placenta to the elevated aminopeptidase A levels in the pregnancy plasma could not be solved on the basis of the present observations.

Biochemical studies on dipeptidyl peptidases I to IV of the human placenta.

Homogenates of human term placentae were utilized to demonstrate the presence of four dipeptidyl peptidases (DPP I to IV) after sequential fractionations with gel filtration and anion-exchange chromatography. DPP I was assayed with SerTyrNA as substrate and showed the characteristics of a thiol-dependent serine enzyme with optimum at pH 4.5 and a molecular weight of 210,000. DPP II, analysed with LysAlaNA as substrate, had an optimum at pH 5.5 and a molecular weight of 130,000 with no dependence on thiol groups or divalent ions. DPP III was studied with ArgArgNA as substrate. It was optimally hydrolysed at pH 8.8 and had a molecular weight of 84,000. The enzyme was highly suppressed by chelating agents but could be reactivated by Co2+ and some other divalent ions. DPP IV, with GlyProNA as substrate, displayed an optimum at pH 8.2. The fractionations displayed multiple forms of the enzyme, possibly indicating the presence of proto- and polymeric DPP IV activities. The protomer had a molecular weight of 260,000 and showed no dependence on thiol groups but was sensitive to Zn2+.

Characterization of three aminopeptidases purified from human placenta.

Three aminopeptidases purified from the human placenta were characterized and compared with each other. Aminopeptidase II1 preferred L-arginine- and L-lysine-beta-naphthylamides or p-nitroanilides as substrate, with low or negligible hydrolysis of other amino acid derivatives. It was inhibited by L-arginine, L-lysine and L-methionine. This enzyme activity was highly sensitive to heat treatment, N-ethylmaleimide, p-chloromercuribenzoate, puromycin, bestatin, epsilon-amino-n-caproic acid (EACA) and EDTA. After EDTA, this enzyme could be reactivated by Co2+. It is concluded that aminopeptidase II1 is identical with arginine aminopeptidase (EC 3.4.11.6) from other mammalian tissues. Aminopeptidase II2 preferred L-alanine-beta-naphthylamide and p-nitroanilide as substrates. It was also able to hydrolyse L-leucine, L-arginine, L-methionine and L-lysine derivatives but only very weakly L-cystine and Bz-L-cysteine substrates. This enzyme was inhibited by L-arginine, L-alanine, L-lysine and most strongly by L-leucine and L-methionine. It was resistant to bestatin and heat treatment but sensitive to EACA. EDTA caused a marked suppression, which could be prevented by Co2+ and Zn2+. These characteristics are reminiscent of those of alanine aminopeptidase (EC 3.4.11.-) found in other tissues. The third enzyme was the only one clearly particle bound and was therefore called PB-aminopeptidase. It preferred L-leucine derivatives as substrate but also readily hydrolysed other amino acid-beta-naphthylamides and p-nitroanilides including L-cystine and Bz-L-cysteine substrates. Among the amino acids L-cysteine, L-leucine and L-methionine were inhibitory. Bestatin and thiol reagents were without effect and EACA was only moderately inhibitory. EDTA caused a strong suppression, which could be prevented by Co2+ and Zn2+. These properties are equal to those previously described for the placental cystine aminopeptidase (oxytocinase) (EC 3.4.11.3). All three enzymes had an optimum close to neutral pH but apparent differences in their Km and Vmax values with various substrates. These findings suggest that the three purified aminopeptidases are distinct enzymes. Two of these (aminopeptidases II1 and II2) have not previously been isolated and characterized in the human placenta.

Purification and partial characterization of human placental particle-bound aminopeptidase.

Particle-bound aminopeptidase was purified from the human term placenta by separation of the particles from the soluble fraction by centrifugation and solubilization of the particle fraction with 0.5 per cent (v/v) Triton X-100 followed by CM-cellulose chromatography, Sepharose 6B gel filtration, DE-cellulose and Con A-Sepharose 4B affinity chromatography. Polyacrylamide gel electrophoresis indicated enzymatic and protein homogeneity of the purified enzyme. The enzyme seemed to be a glycoprotein with a molecular weight of 320 000. The purified enzyme did not endure freezing but was fairly stable at 4 degrees C. At 60 degrees C more than half and at 65 degrees C all enzyme activity disappeared in 15 min. The pH dependence of the purified enzyme varied between 6.75 and 7.5, and 6.0 and 6.5, depending on the substrate used. The enzyme hydrolysed most rapidly LeuNA (Km 0.095 +/- 0.008 mmol) followed by beta-naphthylamide derivatives of alanine (Km 0.222 +/- 0.02 mmol), arginine (Km 0.041 +/- 0.002 mmol), lysine (Km 0.084 +/- 0.005 mmol), methionine (Km 0.085 +/- 0.004 mmol) and cystine (Km 0.048 +/- 0.004 mmol). Thus LeuNA was the most specific among the substrates for the enzyme. The purification process revealed, however, that CysNA was the most selective substrate for the particulate enzyme, which readily hydrolysed also Leu-p-na (Km 0.865 +/- 0.023 mmol) and Bz-Cys-p-na (Km 0.248 +/- 0.034 mmol).

Effects of human placental S9 and induced rat liver S9 on the mutagenicity of drinking waters processed from humus-rich surface waters.

The mutagenicity of chlorinated drinking waters processed from humus-rich surface waters has been shown to be very high. The effect of placental S9 on the mutagenicity of drinking waters has not been studied previously. The purpose of this study was to compare the effects of human placental and rat liver microsomal fractions on the mutagenicity of drinking waters processed from humus-rich surface waters. The samples of 34 drinking and two raw waters from 26 localities in Finland were tested for mutagenicity in Ames Salmonella typhimurium tester strain TA100 with and without metabolic activations. Between the drinking water samples, clear differences were recorded in the presence of placental and rat liver S9, suggesting different mutagens in the drinking waters. Rat liver S9 decreased the mutagenicities of drinking water concentrates, but placental S9 increased, decreased, or had no effect. It is not known if placental mutagenicity enhancing system might cause any health hazard to a developing fetus.

Binding of the strong bacterial mutagen, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) to bovine serum albumin.

Binding of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) to bovine serum albumin (BSA) was studied. MX bound mainly reversibly to BSA but, for a minor part, also irreversibly. It was possible to extract the main part of the reversibly bound MX with ethyl acetate and the extractable compound was chromatographically identical to MX. The affinity-binding characteristics of the interaction with albumin were K = 1.6 x 10(7) M-1, n = 3.4. Furthermore, mutagenicity studies indicated that reversibly bound MX remained mutagenic but that irreversibly bound MX was no longer mutagenic in the Ames test. These results suggest that the binding of MX to albumin is an important factor for both the toxicological effects and the toxicokinetics of MX.

Mutagenic compounds in wood-chip drying fumes.

The mutagenicity of fumes from the heating of freshly cut spruce and birch chips was measured with Salmonella typhimurium strains TA98, TA100 and TA102. The bacteria were exposed directly and indirectly to the fumes. Wood chips were also extracted with solvents. No mutagenicity was found in wood extracts or the fume samples measured indirectly. The results from the direct exposure experiments indicate, however, that drying spruce and birch at 170 degrees C emits mutagenic compounds, which are short-lived and/or volatile. One of the mutagenic compounds of the fumes is probably 3-carene. These results are consistent with previous epidemiological findings, which suggest that these fumes are carcinogenic.

Partial purification and characterization of four aminopeptidases from the soluble fraction of human placenta.

Four aminopeptidases hydrolysing amino acid-beta-naphthylamides were partially purified from human placental soluble fraction. Separation of the activities, Con-pools I, II1, II2 and III, have not been previously reported. They had apparent molecular weights of 80,000, 100,000, 210,000, and 120,000, respectively. Con-pool II2 seemed to be a glycoprotein. Con-pools I and III were activated by sulphydryl modifiers and inhibited by p-chloromercuribenzoate (p-CMB), properties which indicate their dependence on thiol-groups. Con-pool II1 hydrolysed only arginine- and lysine-beta-naphthylamides. It was inhibited by bestatin and p-CMB. Its special characteristic was activation by monovalent anions, notably chloride ions. These results suggest that Con-pool II1 is aminopeptidase B.

Fractionation and characterization of cystine aminopeptidase (oxytocinase) and arylamidase of the human placenta.

Three activity peaks hydrolysing L-cystine-di-beta-naphthylamide (CysNA) and two activities hydrolysing L-leucine-beta-naphthylamide (LeuNA) were separated by gel filtration on Sepharose 6B from human placental tissue. The enzyme activities in the void volume and the solubilized enzyme activities with both substrates apparently are bound and free forms of the same enzymes (I) since detergent treatment caused a total disappearance of the activities in the void volume. The second distinct enzyme (II) was highly soluble and detected only with CysNA. The particle-bound enzyme(s) had a pH optimum at 6.5 with CysNA and at about 7.5 with LeuNA. They were highly sensitive to EDTA, could be reactivated by Co2+ and Zn2+ and were more sensitive to Ni2+ and L-methionine than the soluble enzyme II. The former enzyme(s) tolerated thermal treatment better than the soluble enzyme II. The solubilized free enzyme(s) I had a molecular weight of about 309,000. The soluble enzyme II was resistant to EDTA. Its optimum was at pH 6.0 and an estimate of 76,000 for the molecular weight was obtained.

Fractionation and characterization of cystine aminopeptidase (oxytocinase) and arylamidase of human serum during pregnancy.

Cystine aminopeptidase and arylamidase activities in human serum were determined by enzymic hydrolysis of L-cystine-di-beta-naphthylamide (CysNA) and L-leucine-beta-naphthylamide (LeuNA), respectively. The activities of both enzymes increased during pregnancy, cystine aminopeptidase 12.5-fold and arylamidase 8.3-fold. Serum CysNA and LeuNA hydrolysing aminopeptidases were separated by gel filtration on Sepharose 6B. Serum from non-pregnant women (control) contained arylamidase (Ic), which hydrolysed LeuNA and (weakly) CysNA, and cystine aminopeptidase II, hydrolysing only CysNA. During pregnancy a new enzyme appeared in maternal serum and showed cystine aminopeptidase and arylamidase activity (Im). Maternal serum Enzyme(s) I had higher pH optima (6.5 with CysNA; 7.5 with LeuNA) and higher molecular weights (309,000) than arylamidase Ic (pH optima at 5.52-5.5 with CysNA and 7.0 with LeuN; mol.wt approximately 130,000). Arylamidase Ic was more sensitive to L-methionine, but more resistant to heat than maternal serum Enzyme(s) I. Both control and maternal serum Enzyme(s) I were inhibited by EDTA, but were re-activated by Zn2+ and Co2+ with LeuNA and CysNA as substrates and by Ni2+ with CysNA. Cystine aminopeptidase Im and arylamidase Im may be a single enzyme although differences were obtained in pH optima and reactivation by Ni2+ after EDTA treatment. Since maternal serum Enzyme(s) I had biochemical characteristics similar to those of placental aminopeptidase(s) I, it is suggested that the activities are of placental origin. Cystine aminopeptidase II appeared in all sera. It differed clearly from both maternal and control serum enzyme(s) I: it had the lowest molecular weight (76,000), a different pH optimum (6.0) and was resistant to EDTA and L-methionine. It was not as effectively inhibited by Ni2+ as was Enzyme(s) I.

Expression of intermediate filaments in human tongue mucosa.

The specific inflammatory lesions of the human tongue, namely fissured tongue and geographic tongue, have been found to differ clinically and histologically from each other and from healthy appearing tongue (control). In this study we describe expression of cytokeratin, vimentin and desmin intermediate filaments in these tongue forms. The most important findings were seen in fissured tongue; strong positive staining of cytokeratin proteins indicate the incomplete keratinization of the epithelium, vimentin staining was irregular indicating subepithelial edema and desmin expression showed the destruction of the uppermost muscle cells. The corresponding changes of geographic tongue were similar but slight when compared with those of fissured tongue. Different immunohistochemical methods can supplement the information obtained from tongue biopsies by conventional methods and lead to a better understanding of the morphology of the tongue mucosa.

Lectin binding of subunits of placental and serum oxytocinase after electrophoresis and transblotting to nitrocellulose.

1. Oxytocinase enzymes were purified from maternal serum and human placenta, run by SDS-PAGE and transferred onto nitrocellulose. Both enzymes were homogeneous in protein staining with Mr of 145,000. 2. Both serum and placental oxytocinases bound concanavalin A (Con A), limax flavus agglutinin (LFA) and wheat germ agglutinin (WGA). The WGA-binding of the placental enzyme was more strongly inhibited by 0.2 M N-acetylglucosamine than that of the serum enzyme which may indicate a higher sialic acid content in the serum enzyme. 3. Neuraminidase treatment did not affect the binding of Con A but decreased the binding of WGA to serum and placental enzymes. Serum enzyme showed a pl 4.7 on isoelectric focusing.

Monoclonal antibodies recognize a cell surface marker of epithelial differentiation in the rabbit reproductive tract.

Monoclonal antibodies against the cell surface were produced by immunizing mice with endometrial scrapings prepared from 6-day pregnant rabbits. Spleen cells from an immune mouse were fused with myeloma cells and cultured by standard hybridoma technology methods. Hybridoma supernatants were screened for reaction with the apical epithelial surface by immunohistochemistry on frozen sections of uterus from 6-day pregnant rabbits, and positive colonies were cloned by limiting dilution. Ascites fluid was produced in mice from hybridoma clones that gave a consistent pattern of apical epithelial surface staining through 6 sub-clonings. Antibodies in the ascites fluid were tested by immunohistochemistry on frozen sections of uterus, oviduct, lung, liver and kidney from nonpregnant or 6-day pregnant rabbits. At a dilution of 1:5000, the antibodies recognized an antigen that was specific to the apical surface of luminal but not glandular epithelium of the 6-day pregnant uterus and could not be detected in the nonpregnant uterine epithelium. At higher concentrations of antibody (1:100 to 1:1000), crossreaction was seen with antigens in stromal and myometrial cells of pregnant and nonpregnant uterus. At a dilution of 1:5000, the antibody also crossreacted with some components of lung, liver and kidney but without discriminating between the two reproductive states. In the oviduct, staining of the surface epithelium was specific to the pregnant state. We conclude that this monoclonal antibody has a high affinity for a luminal epithelial cell surface antigen in the reproductive tract of the pregnant rabbit and shows multiple organ reactivity with other tissues that is not affected by pregnancy. This antigen will provide a useful cell surface marker of epithelial differentiation in the progestational reproductive tract.

Characterization of two thiol-dependent aminopeptidases partially purified from human placenta.

1. Two thiol-dependent aminopeptidases (I and III) were partially purified from the soluble fraction of human placenta. 2. Aminopeptidase I preferred L-alanine-beta-naphthylamide (AlaNA) as substrate at pH 7.0-7.5. It was sensitive to heat and some divalent metal ions (Co2+, Ni2+, Zn2+) but resistant to EDTA, and some amino acids. 3. Aminopeptidase III hydrolysed equally well AlaNA and ArgNA at pH 6.5-7.0. It was markedly suppressed by EDTA and reactivated by Co2+. It was inhibited by heat, some amino acids, benzamadine and puromycin.

Purification of rabbit endometrial plasma membranes from receptive and non-receptive uteri.

We have developed a method for isolation of plasma membranes from rabbit endometrium, with high yield and purification. Endometrial homogenates are precipitated with calcium chloride and the resulting supernatant is fractionated by centrifugation in a self-forming gradient of 20% Percoll. Before fractionation, the intact luminal epithelial surface was labelled with 125I-labelled soyabean agglutinin. Between buoyant densities of 1.015 and 1.017 g/ml, a discrete peak of surface label was obtained, which coincided with activities for 5'-nucleotidase and alkaline phosphatase, enzyme markers for the plasma membrane. This peak was well separated from the majority of cellular protein, and from marker enzyme activities for mitochondria and microsomes (NADH cytochrome C reductase) and lysosomes (acid phosphatase). Electron microscopy of the purified membranes showed membrane sheets and vesicles free from other cellular organelles. Analysis of detergent-soluble membrane proteins, fractionated by concanavalin A-affinity chromatography, revealed differences in the protein pattern of membranes from uteri of rabbits receptive (Day 6 of pregnancy) and non-receptive (Day 3) for implantation. The method will be useful for generation of immunological and affinity probes for surface antigens involved in ovoimplantation.

Purification of three aminopeptidases from human maternal serum.

Three aminopeptidases (I--III) were purified from maternal serum using sequential chromatographic fractionations. Aminopeptidase I was specific for N-terminal alpha-L-dicarboxylic acid residues and activated by alkaline earth metals (Ba2+, Ca2+, Sr2+). It is concluded that aminopeptidase I is aminopeptidase A (L-alpha-aspartyl-(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7). Aminopeptidase II hydrolysed all tested substrates including L-cystine and Bz-L-cysteine derivatives but preferred L-leucine derivatives. The properties of aminopeptidase II are equal to those described for the cystine aminopeptidase (oxytocinase) (EC 3.4.11.3.). Aminopeptidase III preferred L-alanine derivatives as substrates. It was activated by Co2+, but strongly inhibited by amastatin, puromycin and L-methionine. The characteristics are reminiscent of those of alanine aminopeptidase (EC 3.4.11.-).

Initiation and maintenance of in vitro decidualization are independent of hormonal sensitization in vivo.

The effects of in vivo hormonal sensitization on the competence of uterine stromal (US) cells to decidualize in vitro were assessed. In vitro differentiation of uterine stroma isolated from Day 4 pregnant rats, sensitized to respond to a decidual stimulus, was compared to that in nonsensitized immature, castrated or cycling rats. The initiation of in vitro decidualization--as monitored by the expression of the decidual markers desmin and laminin in rat US cells--was independent of the hormonal status of the animal from which the cells were isolated and occurred in the absence of serum in the medium. Differentiation was accelerated in high-density cultures where contact inhibition suppressed proliferation and decreased the extent of cell growth. The extent to which in vitro decidualization imitates in vivo stromal cell differentiation was assessed by comparing decidualization in the rabbit, a species with only a limited decidual cell response, and in the rat. US cells isolated from nonpregnant rabbits differentiated in vitro by expressing laminin, but not desmin. Indirect immunofluorescence of frozen uterine sections from pregnant and nonpregnant rabbits validated in vitro differentiation as a faithful reflection of the in vivo program of decidualization. Although the program of US cell differentiation may vary between the species, initiation of differentiation in vitro appeared to be independent of hormonal preparation in vivo for both the species examined.

Expression of desmin, laminin and fibronectin during in situ differentiation (decidualization) of rat uterine stromal cells.

Immunocytochemical analysis of frozen rat uterine sections containing decidual tissue, formed in response to normal or artificial stimulation of uteri sensitized by endogenous or exogenous hormonal regimens, demonstrated an elevated expression of the intermediate filament protein desmin in decidual cells. Changes in the expression of extracellular matrix (ECM) components were coordinated with the elevated expression of desmin as stromal cells underwent decidualization. In parallel with the pattern of regional decidualization, as determined by elevated desmin expression, laminin accumulated in ECM of decidual cells while an apparent decrease in fibronectin was associated with altered organization at the decidual cell surface. The in situ observations confirm previous results, which indicated that the expression of desmin in decidual cells formed in vivo or in vitro is a valid marker of their differentiation, and resolve questions unanswered in the previous study: (a) desmin (and laminin) appear to be constitutively expressed in non-decidualized stroma at barely detectable levels, (b) desmin is a valid marker of stromal cell differentiation because it is expressed similarly in decidual cells, irrespective of varying experimental protocols for uterine sensitization and stimulation, and (c) desmin expression follows the same regional progression described for the process of decidualization in morphological and histochemical studies.