Activity In Pregnancy Serum Research Articles

Pregnancy-associated plasma protein-A accounts for the insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) proteolytic activity in human pregnancy serum and enhances the mitogenic activity of IGF by degrading IGFBP-4 in vitro.

Pregnancy-associated plasma protein-A (PAPP-A) has been identified as the insulin-like growth factor (IGF)-dependent IGF-binding protein-4 (IGFBP-4) protease produced by human fibroblasts. Recently, we found that serum proteases induced during human pregnancy cleaved IGFBP-4 in both an IGF-II-dependent and an IGF-II-independent fashion. This study sought to determine whether PAPP-A is the predominant IGFBP-4 protease in human pregnancy serum (PS) and to assess the in vitro role of serum PAPP-A. Immunoprecipitation with PAPP-A antibody effectively depleted PAPP-A from the PS and completely abolished both IGF-II-dependent and IGF-II-independent IGFBP-4 proteolytic activity in PS. Direct addition of PAPP-A antibody to PS completely blocked IGFBP-4 proteolysis and partially blocked IGFBP-5 proteolysis, but had no effect on IGFBP-3 proteolysis. To evaluate the role of serum PAPP-A, we tested whether PAPP-A in PS modulated the inhibitory activity of IGFBP-4 on IGF-II-induced cell proliferation in human osteosarcoma MG63 cells. The wild-type IGFBP-4 (WTBP-4; 200 ng/mL) failed to inhibit proliferation of the cells treated with PS (0.1% or 0.3%) alone or in combination with IGF-II (40 ng/mL), whereas the inhibitory effect of WTBP-4 was observed in the cells treated with nonpregnancy serum alone or in combination with IGF-II (P < 0.05). In contrast to WTBP-4, a protease-resistant IGFBP-4 was able to inhibit proliferation of the cells treated with PS alone or in combination with IGF-II (P < 0.05). In the presence of PAPP-A neutralizing antibody, the inhibitory effect of WTBP-4 on proliferation of the cells treated with IGF-II and PS was restored. In summary, these data demonstrate 1) that PAPP-A represents the predominant IGFBP-4 protease in PS; 2) that PAPP-A may in part contribute to IGFBP-5, but not IGFBP-3, proteolytic activity in PS; and 3) that PAPP-A enhances the bioactivity of IGFs in vitro by degrading IGFBP-4.

Expression of Recombinant Human Pregnancy-associated Plasma Protein-A and Identification of the Proform of Eosinophil Major Basic Protein as Its Physiological Inhibitor

Pregnancy-associated plasma protein-A (PAPP-A), originally known from human pregnancy serum, has recently been demonstrated to be a metzincin superfamily metalloproteinase involved in normal and pathological insulin-like growth factor (IGF) physiology. PAPP-A specifically cleaves IGF-binding protein (IGFBP)-4, one of six antagonists of IGF action, which results in release of IGF bound to IGFBP-4. IGFBP-4 is the only known PAPP-A substrate. Its cleavage by PAPP-A uniquely depends on the presence of IGF. We here report mammalian expression and purification of recombinant 1547-residue PAPP-A (rPAPP-A). The recombinant protein is secreted as a homodimer of about 400 kDa composed of two 200-kDa disulfide-bound subunits. Antigenically and functionally, rPAPP-A behaves like the native protein. In human pregnancy, PAPP-A is known to circulate as a 500-kDa disulfide-bound 2:2 complex with the proform of eosinophil major basic protein (proMBP), PAPP-A/proMBP. A comparison between rPAPP-A and pregnancy serum PAPP-A/proMBP complex surprisingly reveals a difference greater than 100-fold in proteolytic activity, showing that proMBP functions as a proteinase inhibitor in vivo. We find that polyclonal antibodies against PAPP-A abrogate all detectable IGFBP-4 proteolytic activity in pregnancy serum, pointing at PAPP-A as the dominating, if not the only, IGFBP-4 proteinase present in the circulation. We further show that pregnancy serum and plasma contain traces (<1%) of uncomplexed PAPP-A with a much higher specific activity than the PAPP-A/proMBP complex. The measurable activity of the PAPP-A/proMBP complex probably results from the presence of a minor subpopulation of partly inhibited PAPP-A that exists in a 2:1 complex with proMBP. Inhibition of PAPP-A by proMBP represents a novel inhibitory mechanism with the enzyme irreversibly bound to its inhibitor by disulfide bonds.

Serum Insulin-Like Growth Factor-I, IGF Binding Protein-3 and IGFBP-3 Protease Activity after Cranial Irradiation

The relationship between peak growth hormone (GH), insulin-like growth factor I (IGF-I), IGF-I binding protein 3 (IGFBP-3) and IGFBP-3 protease activity was studied in 28 children and adolescents undergoing investigation of pituitary function 0.4–14.2 years after cranial or craniospinal irradiation for the treatment of CNS tumours distant from the hypothalamic-pituitary axis (n = 16) or prophylaxis against CNS leukaemia (n = 12). Seven out of 15 patients with GH deficiency (GHD) (defined as a peak GH concentration <7.5 ng/ml in a stimulation test) had IGF-I <–2 standard deviation score (SDS). None of the 28 patients had serum IGFBP-3 concentrations measured by radioimmunoassay (RIA) <–1.5 SDS with no difference between those with and without GHD. IGFBP-3 concentrations measured by RIA were strongly correlated to IGFBP-3 band density on Western ligand blot (WLB) (r = 0.71; p < 0.0001). IGFBP-3 protease activity was negatively correlated to IGFBP-3 by RIA (r = –0.55; p < 0.01) and to IGFBP-3 by WLB (r = –0.51; p < 0.01). Twenty-two patients had normal IGFBP-3 protease activity (<30% of the activity in pregnancy serum) indicating that serum IGFBP-3 protease activity does not account for the normal levels of IGFBP-3 in RIA.Low serum IGF-I but normal IGFBP-3 concentrations and in the majority normal IGFBP-3 protease activity was found in patients in the years after CNS irradiation. Neither serum IGF-I nor IGFBP-3 can be used as a reliable index of the development of radiation-induced GHD.

Failure to verify high activities of polyamine oxidase in pregnancy serum

The activity of polyamine oxidase in human pregnancy serum was measured by our new specific method with radioisotopic N1-acetylspermine as substrate. The activity in pregnancy serum was very low and did not increase during gestation; it was not higher than that in nonpregnancy serum. The high polyamine oxidase activities previously reported by others appear to result from contamination with diamine oxidase, the level of which is high in pregnancy serum.

Activity of serum cytidine deaminase during pregnancy

A simple and reliable method for the estimation of cytidine deaminase (cytidine aminohydrolase; EC 3.5.4.5) activity in pregnancy serum is described. This enzyme does not require magnesium for activation and is also more stable than deoxycytidylate deaminase (dCMP deaminase, dCMP aminohydrolase; EC 3.5.4.12). There was excellent correlation between the two enzymes (r = 0.92). Both enzymes showed increased activity in abnormal pregnancy. Both enzymes activities were found to be similar in 1305 maternal serum samples and therefore due to the simplicity of cytidine deaminase estimation it is recommended for the screening of large numbers of antenatal sera for abnormal pregnancies.

Spermidine oxidase in human pregnancy serum. Probable identity with diamine oxidase.

Diamine oxidase was previously measured in human pregnancy serum with putrescine or histamine as substrate. We have now documented the presence of spermidine oxidase activity in pregnancy serum by means of a specific radioactive assay with [14C]spermidine as substrate and Dowex 50 cation-exchange chromatography to separate products from substrate. The apparent Km of a partially purified preparation of this enzyme for spermidine was 10.9 microM and the Ki for aminoguanidine was 0.8 microM. The pH optimum (pH 9.0) and temperature optimum (55 degrees C) were identical with those for diamine oxidase. Spermidine oxidase activity and diamine oxidase activity eluted in a concerted fashion when pregnancy serum was subjected to cadaverine-Sepharose chromatography, gel filtration and ion-exchange chromatography. Spermidine oxidase became detectable in serum during pregnancy in the human approx. 8 weeks after the last menstrual period and increased with gestational age in concert with the increase in diamine oxidase activity, reaching a plateau at 20 weeks of gestation. Foetal-cord serum displayed virtually no activity of either enzyme. A 400-fold-purified preparation of diamine oxidase retained the same diamine oxidase/spermidine oxidase ratio as exhibited by crude pregnancy serum. These data suggest that in pregnancy serum, unlike foetal bovine serum, spermidine oxidase and diamine oxidase activity may be a single enzyme protein.

A possible explanation for the occurrence of increased β-hexosaminidase activity in pregnancy serum

The activities of the lysosomal enzymes β-hexosaminidase, β-glucuronidase, and β-galactosidase were confirmed to be elevated in serum from pregnant women. The increased activity of β-hexosaminidase and β-glucuronidase showed the same changes in the isoenzyme pattern as found in serum from patients with some liver affections. In the light of recent findings, the increased activity of β-hexosaminidase (and some other lysosomal enzymes) in pregnancy and liver disease might depend upon the non-parenchymal liver cells not being able properly to clear these enzymes from the blood.

A rapid method for the determination of deoxycytidylate deaminase activity in pregnancy serum

A method is described for determining the activity of deoxycytidylate deaminase in serum. The ammonia liberated from deoxycytidine monophosphate has been specifically determined by enzymatic amination of α-ketoglutarate using glutamate dehydrogenase. The concurrent oxidation of NADH 2 at 340 nm was proportional to the ammonia liberated from deoxycytidine monophosphate. Using the technique described, a result would be available to the clinician in under 4.5 h. The “normal activity” for deoxycytidine deaminase in normal male, female and pregnancy sera has been determined.

An automated method for the measurement of serum aminopeptidase activity with particular reference to cystine aminopeptidase (oxytocinase)

An automated method of determining the cystine aminopeptidase (oxytocinase) activity of pregnancy serum is described and has been developed so that 40 serum samples can be assayed in one hour. l-Cystine-bis- p-nitroanilide is used as a substrate and the problems of its insolubility at optimum pH were overcome by addition of the detergent, triton X-100. Excellent agreement was achieved between this method and that of the manual method using the same substrate. The usefulness of the automated enzyme determination was established by following the serum oxytocinase levels in a small number of women in the third trimester of pregnancy.