The protein composition of the fibrinoid material at the human uteroplacental interface.
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Carbohydrates↗
Biomedical subjects
R G Sutcliffe
Publications and source records attributed to R G Sutcliffe.
Studies on the concentration of pregnancy-associated plasma protein-A during normal and complicated pregnancy.
Pregnancy-associated plasma protein-A was assayed in the blood of 347 women during pregnancy, using a new primary standard of PAPP-A as reference. The protein was assayed by antibody-antigen crossed electrophoresis with the lower limit of confident assay being 9.5 micrograms PAPP-A/ml (13 pmol/ml). PAPP-A was first detected at 14 weeks of gestation; by term it had risen to within the range 20 to 320 micrograms/ml. There was an indication that pregnancies involving a male baby had higher PAPP-A levels in blood than did those involving female babies. In 51 blood samples from 30 patients with gestational diabetes (taken between 28 weeks of pregnancy and term) there was no significant alteration in PAPP-A values compared with controls. In 35 blood samples from 15 patients with insulin-dependent diabetes, levels of PAPP-A were significantly lower than in controls or in gestational diabetes. In 43 blood samples from 35 patients with babies affected with intrauterine growth retardation (between 28 weeks and term), there was no significant difference in PAPP-A levels compared with controls. The effect of insulin on the blood levels of PAPP-A suggests that the concentration of PAPP-A is capable of altering significantly in response to certain physiological changes associated with the control of carbohydrate metabolism.
Birth Weight↗
Pregnancy-associated plasma protein A: purification under mild conditions, peptide mapping and tests for possible interactions with trypsin, plasmin and complement.
Pregnancy-associated plasma protein A (PAPP-A) has been obtained in an enriched state under mild conditions of purification involving cibacron blue dye-ligand chromatography, negative antibody-affinity chromatography and gel filtration. The product was a mixture of PAPP-A and alpha-2-macroglobulin (alpha 2M). The ability of these proteins to bind 125I-trypsin and 125I-plasmin was studied. In contrast to previous studies by others, there was no evidence that PAPP-A could bind either protease. It is pointed out that protease-inhibitory activity in samples of PAPP-A can be due to the presence of very small amounts of contaminating alpha 2M in the preparations of PAPP-A. In further experiments PAPP-A did not inhibit the complement-induced lysis of sensitized red cells. Finally, cyanogen bromide peptide mapping experiments failed to yield evidence of structural homologies between PAPP-A and alpha 2M.
Chromatography, Affinity↗
Purification and analysis of the microvillous (M) and A forms of placental alkaline phosphatase.
Microvillous M-PAP and A-PAP were purified to homogeneity from butanol extracts of syncytiotrophoblastic microvillous membranes using DEAE Sepharose CL-6B chromatography. The two isoenzymes had indistinguishable pH optima and Km values. Under reducing conditions in SDS-polyacrylamide gels, both forms showed a similar subunit molecular weight of 69 000. In the absence of reduction M-PAP was present in a dimeric form similar to A-PAP. However, the mobility of the dimer of M-PAP corresponded to a molecular weight of 116 000 compared with 130 000 for A-PAP. It was concluded that this difference is due to the A and M dimers being in different conformations. In SDS-polyacrylamide gels the M form also showed a higher molecular weight species corresponding to a mobility of 180 000, but species corresponding to the native molecular weight of 725 000 wer not found. Under different alkylating conditions the polypeptide subunits of both forms showed similar isoelectric banding patterns. The M-PAP showed some extra bands with a relatively high pI value. The hydrophobicity of the different forms of PAP was investigated by testing their affinities for columns of various hydrocarbon-coated agaroses. Unalkylated agarose did not bind the enzyme; C6 alkyl chains bound A, B and M-PAP. C2 alkyl chains bound M-PAP, but not A or B-PAP. This provided a potentially new and simple method of purifying M-PAP, and supports the view that M-PAP is hydrophobic relative to the A and B forms. The structural implications of this hydrophobicity are discussed.
Alkaline Phosphatase↗
A high molecular weight species of placental alkaline phosphatase in human syncytiotrophoblast microvilli.
A high molecular weight form of human placental alkaline phosphatase has been detected in extracts of placenta at term by electrophoresis in starch gels containing 0.5 per cent Triton X-100. The enzyme has a mobility intermediate between the previously described A and B forms of the enzyme and has been called the 'M' form of placental alkaline phosphatase. The M form is the major form of the enzyme found in microvilli extracted from syncytiotrophoblast, though trace amounts of membrane-associated M form can be found in extracts of placentae which had previously been experimentally depleted of microvilli. The M form is present in both of the two recently described subfractions of placental microvilli (see Davies, Parry and Sutcliffe, 1981; Truman, Wakefield and Ford, 1981). A variety of experiments show that the M form is not an artefact of extraction. The characteristic mobility of the M form in starch/Triton gels is the same, whether the microvilli are extracted in butanol, chloroform/methanol, Nonidet P40, Triton X-100 or Na deoxycholate. Serological, heat-stability and genetic studies showed that the A and M forms contain the same enzymatic polypeptide. Gel filtration of butanol/H2O and butanol/saline extracts of microvilli provided an estimated molecular weight of the A form of 127000 and of the M form of 725000; these values were unaffected by the presence of Triton in the medium.
Alkaline Phosphatase↗
Placental alkaline phosphatase integrates via its carboxy-terminus into the microvillous membrane: its allotypes differ in conformation.
Human placental microvillous alkaline phosphatase (M-PLAP) was extracted from microvilli either by butanol extraction or subtilisin proteolysis. The data indicate that subtilisin cleavage of PLAP removes a membrane-binding domain of approximately 2000 molecular weight, leaving the catalytic site intact and the protein in solution. Sequencing studies on the N-terminal 13 amino acids of both the subtilisin-cleaved and uncleaved forms of M-PLAP indicate that the enzyme is anchored to the plasma membranes by its carboxy-terminus. The N-terminal 13 amino acids of A-PLAP were the same as those of M-PLAP. Trypsin solubilization failed to release M-PLAP from these membranes and it appears to cleave a portion of molecular weight of about 9K from the amino terminus, leaving an enzymatically active portion of PLAP associated with the membrane. On SDS gels, subtilisin-cleaved M-PLAP showed an apparent dimeric molecular size larger than that of the original uncleaved enzyme, presumably due to the generation of a less compact conformational state. On starch gels, cleaved M-PLAP showed a single zone of enzyme activity with a mobility sightly greater than that of A-PLAP, which did not require the presence of Triton X-100 to enter the gel. Variations in the apparent molecular sizes of the different allelic forms of PLAP were also observed.
Alkaline Phosphatase↗
Variation in the expression of human 3 beta-hydroxysteroid dehydrogenase.
Polymorphic genetic variation shows that the genes for human 3 beta-hydroxysteroid dehydrogenases (3 beta-HSD) types I and II are closely linked. The type II mutations A82T, S100N and L173R are associated with male pseudohermaphroditism and A82T is associated, with variable penetrance, with female premature puberty. When expressed in vitro A82T showed less than 5% of normal activity and L173R showed a 30-50% reduction in activity. PCR experiments and direct genomic cloning show that there is a larger family of 3 beta-HSD sequences which require to be tested for expression. The phenomenon of epitopic heterogeneity of 3 beta-HSD is discussed and is now shown to apply to testicular Leydig cells as well as extrauterine trophoblast. RT-PCR analyses indicate that the phenomenon is most likely to be due to post-translational modification affecting the carboxytermini 3 beta-HSD types I and II. This phenomenon may reflect a further level at which enzyme activity is regulated.
3-Hydroxysteroid Dehydrogenases↗