J.C. Robinson

Enzymes of normal and malignant trophoblast: Phosphoglucose isomerase, phosphoglucomutase, hexokinase, lactate dehydrogenase, and alkaline phosphatase

In this study we compare the specific activities and isoenzyme patterns of five enzymes--phosphoglucose isomerase, phosphoglucomutase, hexokinase, lactate dehydrogenase, and alkaline phosphatase--in term placenta with the analogous enzymes in a clone of choriocarcinoma cells grown in culture. Phosphoglucose isomerase, phosphoglucomutase, and lactate dehydrogenase specific activities of the choriocarcinoma did not differ by more than two or three times from the mean activities of the comparable enzymes in placenta; the specific activity of hexokinase in the choriocarcinoma amounted to 14 per cent of the mean value for placenta. In contrast, the mean specific activity of heat-stable alkaline phosphatase in the choriocarcinoma amounted to only 1 per cent of the mean value for placenta. By growing the cells in 5-bromodeoxyuridine, 20 mug per milliliter, we were able to increase alkaline phosphatase activity to 68 per cent of the mean value for placenta. For both extracts, phosphoglucose isomerase zymograms were similar and phosphoglucomutase zymograms were similar. The hexokinase zymogram of term placenta showed two isoenzymes which stained more intensely with 0.5 mM. glucose than with 0.1M glucose. A hexokinase isoenzyme was observed in zymograms of both extracts which stained more intensely with 0.1M glucose than with 0.5 mM glucose. Lactate dehydrogenase exhibited an extra isoenzyme in the choriocarcinoma extract. When the cells were cultivated in medium containing 5 mug per milliliter of 5-bromodeoxyuridine, the induced phosphatase in the cell line was electrophoretically similar to placental phosphatase. At higher concentrations of 5-bromodeoxyuridine, the most anodal isoenzyme was 0.5 cm. slower in mobility than the comparable placental isoenzyme.

Electrophoretic characterization of glucose dehydrogenase, β-glucuronidase, and N-acetyl-β-glucosaminidase from placenta and gestational serum

Abstract Methods were devised for demonstrating isoenzyme patterns of 3 enzymes from human placenta: glucose dehydrogenase, β-glucuronidase, and N-acetyl-β-glucosaminidase. Starch gel electrophoresis of extracts from 12 placentas revealed electrophoretic variants of all 3 enzymes. A single isoenzyme of β-glucuronidase and one of N-acetyl-β-glucosaminidase were found to be increased in gestational serum. The placenta did not seem to be the source of either elevated serum level. In contrast to increases seen in both isoenzymes during pregnancy, an increase in only the serum β-glucuronidase isoenzyme was noted in women taking an oral contraceptive preparation.

Preparation of starch gel zymograms: Peroxide-producing enzymes and ceruloplasmin

Abstract Glucose oxidase, galactose oxidase, xanthine oxidase, l -amino acid oxidase, d -amino acid oxidase, and diamine oxidase form hydrogen peroxide as reaction product. Electrophoretic components of these enzymes can be demonstrated following incubation of the gels in a mixture containing substrate, peroxidase, and a chromogen, 3-amino-9-ethylcarbazole. The method is not sufficiently sensitive for the routine detection of the pregnancy-associated plasma diamine oxidase. The chromogen is a substrate for ceruloplasmin; hence this plasma protein can be detected directly without the use of the coupled peroxidase system.

Placental enzymes: Specific activities and isoenzyme patterns during early and late gestation

Abstract The specific activities and the isoenzyme patterns of 6 enzymes—lactate dehydrogenase, glucose dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, β-glucuronidase, and N-acetyl-β-glucosaminidase—were studied in extracts from first-trimester, second-trimester, and term placentas. All 6 enzymes were present in all specimens. Significant decreases were noted in the specific activity of glucose-6-phosphate dehydrogenase in the second trimester relative to the first, of 6-phosphogluconate dehydrogenase in the second trimester relative to both the first trimester and term, and of N-acetyl-β-glucosaminidase at term relative to both the first and second trimesters. Isoenzyme patterns of 5 of the enzymes were basically unchanged from the first trimester to term. Lactate dehydrogenase showed an additional isoenzyme in first-trimester zymograms; it also exhibited marked shifts in relative isoenzyme intensities between the first trimester and term.

Regional distribution of isozymes of D-amino-acid oxidase and acetylesterase in developing primate brain

Abstract Isozymes of d -amino-acid oxidase and acetylesterase in different regions of the brain of the developing rhesus monkey were separated by starch gel electrophoresis. Activity of d -amino-acid oxidase was identified by the use of d -phenylalanine as substrate in a coupled peroxidase system, and acetylesterase activity was identified by using alpha-naphthyl acetate as substrate in the presence of Diazo Blue. d -amino-acid oxidase isozymes in the brain of the juvenile (2- to 3-year-old) monkey were localized primarily in brain stem, cord, and midline and lateral cerebellum but were also demonstrable in zymograms of subcortical white matter, corpus callosum, thalamus, hypothalamus, pituitary gland, and globus pallidus. Although isozymes of acetylesterase were more generally distributed throughout all brain areas, one band (band 1) was much more prominent in specimens from caudate nucleus and putamen than in specimens from any other area of brain. Isozymes of d -amino-acid oxidase were not generally detected until after birth, but then all major bands appeared fairly rapidly. By 20 weeks of postnatal age patterns of zymograms were similar to those of the adult brain. Although zymograms of the isozymes of acetylesterase in the brain of the first trimester fetus were very similar to those in regions of brain of the 2- to 3-year-old animal, one band (band 6) was much more pronounced in the zymograms of fetal brain than in those from brains at any later age. The principal contribution of these studies is the demonstration of ( a ) an enormously higher concentration, relative to other brain regions, of one of the acetylesterase isozymes (band 1) in extracts of caudate nucleus and putamen, ( b ) the progressive decline of one of the acetylesterase isozymes (band 6) during fetal development, and ( c ) the occurrence of two different patterns of distribution and development of isozymes in the primate central nervous system.

An investigation of the gestational increase in serum hexosaminidase B

Abstract Serum hexosaminidase B, which is known to be increased during pregnancy, was found not to be increased during the administration of oral contraceptives or of estrogens for replacement therapy. Sera from patients with augmented gonadotropins, whether due to exogenous administration or to choriocarcinoma, likewise showed no increase in hexosaminidase B. This serum isoenzyme was, however, increased in 4 patients with hepatitis; this suggests that the gestational increase may be of hepatic origin. No detectable differences in the level of hexosaminidase A were found in any of the sera examined.

Cystine aminopeptidase and leucine aminopeptidase of choriocarcinoma cells grown in culture

Cystine animopeptidase (CAP) and leucine aminopeptidase (LAP) in choriocarcinoma cells grown in culture were assayed and characterized electrophoretically. The specific activity of CAP in choriocarcinoma was about 1.5-fold greater than the specific activity of term placenta. The extract of choriocarcinoma contained an enzyme which is similar to one of the pregnancy-specific serum aminopeptidases in electrophoretic mobility and in resistance to inhibition by 0.1M methionine. The CAP activity was not increased in either the cells or the medium by growing the cells in the presence of oxytocin, estrogen, progesterone, or prostaglandin F-2alpha. It was tentatively concluded that choriocarcinoma cells produce CAP and that it is not regulated by any of these hormones.

Multiple enzyme deficiencies in placental tissue from two blighted-ovum abortions

Abstract Specific activities and isoenzyme patterns of 6 enzymes in placental homogenates from 2 blighted-ovum pregnancies (Specimens A and B) and from 4 normal firsttrimester pregnancies were compared. All placentas were obtained by abdominal hysterectomy from clinically normal pregnancies. In Specimen A, lactate dehydrogenase, 6-phosphogluconate dehydrogenase, and N-acetyl-β-glucosaminidase specific activities were outside the normal ranges; in Specimen B, 6-phosphogluconate dehydrogenase and β-glucuronidase specific activities were outside the normal ranges. Only the reductions of lactate dehydrogenase in Specimen A and 6-phosphogluconate dehydrogenase in Specimen B were statistically significant ( p