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Three distinct forms of D-glucosamine 6-D (Gm 6-P):N-acetyltransferases (EC 2.3.1.4) were partially purified from human placental homogenates by carboxy methyl-Sephadex chromatography. Purification of forms I and II were 13.5-fold, while that of form III was 114-fold. All three forms had a pH optimum value of 9.7 in glycine-NaOH buffer. Enzymes II and III had a K(m) value for Gm 6-P of 3.0 mM, which was less than half of that observed for form I (7.1 mM). The corresponding K(m) values for acetyl CoA were 0.157 (form I), 0.187 (form II) and 0.280 mM (form III), respectively. Activities of all three forms were inhibited at high concentrations of either substrate. These enzymes were inhibited from 82 to 92% by 2.5 mM p-chloromercuribenzoate. The inhibition was largely reversible by inclusion of 2.5 mM dithiothreitol in the incubation mixtures. There was no requirement for divalent cations, as demonstrated by lack of inhibition of enzyme activity by ethylene diamine tetraacetate. The results are discussed in terms of differences among the enzyme properties of human placental, rodent and porcine liver forms.

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Injections of an aqueous extract of winter cherry fruits (Physalis alkekengi) to adult female cycling rats by an intraperitoneal route resulted in the diminution of the pituitary lysyl-aminopeptidase (Lys-AP) activity by 50% and that of the basomedial hypothalamus (BMH) by 45%. Administration of daily doses of 3.75, 7.5, and 15 micrograms beta-estradiol for a period of 5-8 days to such animals increased pituitary Lys-AP activity from 31% to 61.5% and that of BMH from 20% to 87%, respectively. Administration of the same doses of beta-estradiol along with a given dose of the aqueous extract for 7-8 days diminished Lys-AP inhibitory effect of the extract in both the pituitary and BMH and eventually, at the highest dose of beta-estradiol, increased the pituitary enzyme activity by 9% and that of BMH by 5%. It is concluded that Lys-AP enzymes of both tissues, being estrogen-induced proteins, are inhibited by the estrogen antagonistic principle of the winter cherry aqueous extract. It is further suggested that BMH Lys-AP activity may be used as an enzyme marker for the action of beta-estradiol in hypothalamus.

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Cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices was purified 22-fold by QAE- and SP-Sephadex chromatography. The pH optimum of the enzyme was 8.0 in either Tris-HCI or barbital buffer. The Km values of oxaloacetate and NADH were 0.400 +/- 0.018 and 0.410 +/- 0.038 mM, respectively. The enzyme lost about 90% of its activity when heated for 2 min at 65 degrees C. A 61.4% inhibition of the enzyme was noted at 4 mM concentration of diethyl pyrocarbonate. A 3 mM concentration of fructose 1,6-diphosphate inhibited the enzyme by 76.5%. The inhibition was non-competitive with respect to NADH with a Ki value of 0.85 mM. A 75% inhibition of the enzyme was noted at 1 mM concentration of mebendazole that inhibited the enzyme upon competing with NADH with a Ki value of 0.176 mM. A 2-mM concentration of citrate almost doubled the enzyme activity. The enzyme was inhibited at high concentrations of either substrate. The enzyme was not inhibited by p-hydroxymercuribenzoate or fumarate. The enzyme was absolutely specific for NADH as a cofactor. The properties of this enzyme are compared with those of the enzyme from the host liver, the cyst fluid and some other animal sources. The results are discussed in terms of the differences among the properties of the host liver, the cyst fluid and the protoscolices enzymes. The biochemical basis for the use of mebendazole in the treatment of echinococcosis is also elucidated.

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Intraperitoneal injections of an aqueous extract of winter cherry fruits (Physalis alkekengi) to adult normal cycling female rats produced 100% diestrus and diminished uterine glucose 6-P dehydrogenase activity (an estrogen-induced protein) by 52%. Daily doses of 1.88, 3.75 and 7.5 micrograms 17-beta-estradiol administered intraperitoneally to adult female rats for a period of 6-8 days prolonged proestrus or estrus and increased uterine glucose 6-P dehydrogenase activity by 11.5%, 26.9% and 82.1%, respectively. Combined intraperitoneal injections of a given dose of the aqueous extract together with the above doses of 17-beta-estradiol for 8 consecutive days shortened the time spent in diestrus proportional to the dose employed and proportionately reduced the uterine glucose 6-P dehydrogenase inhibitory power of the aqueous extract (1.88 micrograms estradiol, 33.9% inhibition; 3.75 micrograms estradiol, 27% inhibition; and 7.5 micrograms estradiol, 6.0% activation). The data obtained clearly demonstrate the presence of an estrogen antagonist in the aqueous extract of Physalis alkekengi fruits.

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Intraperitoneal injections of an aqueous extract of winter cherry fruits (Physalis alkekengi) to new-born, weanling and adult female rats and to weanling and adult male rats had no effect on body weight, liver weight and liver cytosol protein content. The specific activities of hepatic glucose 6-P dehydrogenase (an estrogen induced protein) in rats of different age and sex groups in terms of mU/mg protein were: treated new-born females, 15.9 +/- 0.5; control, 29.1 +/- 0.6; treated weanling females, 14.9 +/- 0.3; control, 24.8 +/- 0.7; treated adult females, 25.7 +/- 0.5; control, 26.1 +/- 0.5; treated weanling males, 7.9 +/- 0.2; control, 7.9 +/- 0.1; treated adult males, 9.6 +/- 0.4; and control, 9.7 +/- 0.3. Treatment of new-born and weanling female rats with the extract resulted in 40-45% reduction in hepatic G6PD activity. However, treatment of adult females, and weanling and adult males produced no significant change in the activity of this enzyme. The data are discussed both in terms of the increase in the capacity of rodent liver to metabolize steroidal compounds with age and the presence of low levels of circulating estradiol necessary for enzyme induction in male rats.

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Human placental cytoplasmic aspartate transaminase was purified 404-fold by heat treatment, ammonium sulfate fractionation, dialysis and DEAE-Sephadex chromatography. The pH optimum of the enzyme was 6.8 in either phosphate or cacodylate buffer. The Km values of alpha-ketoglutarate and L-aspartate were 2.06 and 22.5 mM, respectively. A 78% inhibition of the enzyme was noted at 4 mM concentration of maleate which inhibited the enzyme upon competing with alpha-ketoglutarate with a Ki value of 1.72 mM. The kinetic properties of this enzyme are compared with those of the enzyme from various mammalian and other sources. The data are discussed in terms of the probable effectiveness of this enzyme in catabolizing L-aspartate in placenta especially after the consumption of a high protein diet by the pregnant mother.

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Ovine liver Echinococcus granulosus hydatid cyst fluid and cytoplasmic healthy ovine liver malate dehydrogenases were purified 24- and 30-fold by Sephadex G-200 and DEAE-Sephadex chromatography. Both enzymes were eluted with the same elution volume and the same salt concentration from the respective columns. The pH optimum of the enzymes from both sources was 8.4 in either Tris-HCl or barbital buffer. The Km values for oxaloacetate were 0.211 and 0.200 mM for hydatid cyst fluid and healthy ovine liver enzymes, respectively. The Km values for NADH were 0.220 and 0.213 mM for hydatid cyst fluid and healthy ovine liver enzymes, respectively. Enzyme from both sources demonstrated similar heat denaturation patterns. Both enzyme preparations were inhibited at high concentrations of either substrate. Neither enzyme was inhibited by para-hydroxymercuribenzoate or fumarate, and both enzyme preparations were specific for NADH as a cofactor. The results are discussed in terms of the possible infiltration of the host enzyme into the cyst fluid.

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Intraperitoneal injections of the aqueous extract of winter cherry fruits (Physalis alkekengi) to female rats produced 100% diestrus. The rats resumed their normal estrus cycle upon withdrawal of this extract. Although there was no significant decrease in the number of implantation sites, the number of pups born to rats decreased by 96% with extract administration. Treatment with this extract had no effect on body weight, uterus weight, plasma protein level or plasma total creatine kinase activity. However, the level of plasma progesterone was diminished by 44%. In addition, uterine creatine kinase BB-isozyme (an estrogen-induced protein) showed a time-dependent inhibition of activity from 55% to 82%.

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A deficiency of vitamin B6 has been reported to enhance oestrogen responsiveness of the uterus in rats whereas zinc deficiency provokes a syndrome suggestive of a diminution in oestrogen sensitivity. In this study [3H]oestrogen uptake by the uterus was increased in rats deficient in either nutrient and the differences were additive in the dually deficient animals. The total number of oestrogen receptors per g tissue was unaffected by either nutrient but the proportion of the receptors recovered from the nuclear fraction increased from about 6 to 74% when both nutrients were withheld. The results are consistent with the hypothesis that both zinc and pyridoxal phosphate play important metabolic roles in end-organ responsiveness to oestrogen.

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Cultured adult rat hepatocytes incubated in media containing fructose exhibit increased levels of cytochrome P-450, relative to cells incubated with equimolar glucose, and the effect of fructose is proportional to its concentration between 2 and 10 mM. For investigating the mechanism of the effect of fructose on cytochrome P-450 in cultured cells, [U-14C]fructose or [U-14C]glucose were added to the incubation medium, and their uptake and utilization were compared. While the uptake kinetics of the two hexoses were similar, the rate of phosphorylation of fructose was more than 10-fold that of glucose. Similarly, the appearance of fructose carbon in metabolic pools, as well as its conversion to CO2 and cellular glycerolipid, was increased. The latter finding suggested that fructose might alter cytochrome P-450 by stimulating glycerolipid synthesis, since the stability of the cytochrome is lipid-dependent. However, the changes in glycerolipid formation failed to parallel changes in the level of cytochrome P-450 in fructose-treated cells. Moreover, the relative distribution of 14C into specific lipids was similar for both hexoses, suggesting that an increased carbon flux in cells incubated with fructose did not directly impose a qualitative change in cellular lipid synthesis. We conclude that the fructose-mediated alteration of cytochrome P-450 in cultured rat hepatocytes reflects a process other than increased incorporation of fructose carbon into metabolic pools.

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The effects of intravenous administration of the saline extract of the ovine hydatid cyst scoleces on the cardiovascular and respiratory systems were studied in sodium pentobarbital-anesthesized sheep. Scoleces were obtained from the lung hydatid cysts of freshly slaughtered sheep, sonicated in physiologic saline and centrifuged to recover the extract in the supernatant fluid. Administration of 1-4 ml of the saline extract resulted in hypotension, shallow rapid respiration, slight elevation of the central venous pressure and transient electrocardiographic changes. Pretreatment of the animals with atropine, 1 mg/kg S.C., or with the antihistamine antazoline, 5 mg/kg I.V., did not block the responses to the saline extract of hydatid cyst scoleces. Pretreatment with compound 48/80, a histamine releaser, abolished the reactions to the administration of the scoleces extract. It is concluded that ovine hydatid scoleces extract has profound cardiovascular and respiratory effects, and that histamine release seems to be involved in the induction of responses to the extract.

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Echinococcus granulosus protoscolex is the actual larval stage of the cestode causing echinococcosis both in man and animals. In the present report, certain properties of phosphoglucose isomerase from the ovine liver E. granulosus protoscolices have been studied and compared with those of the hydatid cyst fluid and the healthy ovine liver enzymes. The protoscolices enzyme prepared in a manner similar to the hydatid cyst fluid and the ovine liver enzymes exhibited the following properties: (1) pH optimum of 8.2 (2) KM value of 0.23 mM, (3) the enzyme was inhibited in the presence of high concentrations of alpha-D-glucose 6-phosphate, (4) no detectable inhibition of the enzyme was observed in the presence of phosphate ion up to 4.1 mM, (5) the protoscolices enzyme was less thermostable as compared to the hydatid cyst fluid and the ovine liver enzymes, (6) the protoscolices enzyme had a lower Ki value (0.7 mM) as compared to either the hydatid cyst fluid (1.1 mM) or the ovine liver enzymes (4.6 mM) when 6-phosphogluconic acid was used as a competitive inhibitor.

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d-Glucosamine-6-P N-acetyltransferase (EC 2.3.1.4) from mung bean seeds (Phaseolus aureus) was purified 313-fold by protamine sulfate and isoelectric precipitation, ammonium sulfate and acetone fractionation, and CM Sephadex column chromatography. The partially purified enzyme was highly specific for d-glucosamine-6-P. Neither d-glucosamine nor d-galactosamine could replace this substrate. The partially purified enzyme preparation was inhibited up to 50% by 2 x 10(-2)m EDTA, indicating the requirement of a divalent cation. Among divalent metal ions tested, Mg(2+) was required for maximum activity of the enzyme. Mn(2+) and Zn(2+) were inhibitory, while Co(2+) had no effect on the enzyme activity. The pH optimum of the enzyme in sodium acetate and sodium citrate buffers was found to be 5.2. The effect of Mg(2+) on the enzyme in sodium acetate and sodium citrate buffers was particularly noticeable in the range of optimum pH. Km values of 15.1 x 10(-4)m and 7.1 x 10(-4)m were obtained for d-glucosamine-6-P and acetyl CoA, respectively. The enzyme was completely inhibited by 1 x 10(-4)mp-hydroxymercuribenzoate, and this inhibition was partially reversed by l-cysteine; indicating the presence of sulfhydryl groups at or near the active site of the enzyme.

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l-Glutamine d-fructose 6-phosphate amidotransferase (EC 2.6.1.16) was extracted and purified 600-fold by acetone fractionation and diethylaminoethyl cellulose column chromatography from mung bean seeds (Phaseolus aureus). The partially purified enzyme was highly specific for l-glutamine as an amide nitrogen donor, and l-asparagine could not replace it. The enzyme showed a pH optimum in the range of 6.2 to 6.7 in phosphate buffer. Km values of 3.8 mm and 0.5 mm were obtained for d-fructose 6-phosphate and l-glutamine, respectively. The enzyme was competitively inhibited with respect to d-fructose 6-phosphate by uridine diphosphate-N-acetyl-d-glucosamine which had a Ki value of 13 mum. Upon removal of l-glutamine and its replacement by d-fructose 6-phosphate and storage over liquid nitrogen, the enzyme was completely desensitized to inhibition by uridine diphosphate-N-acetyl-d-glucosamine. This indicates that the inhibitor site is distinct from the catalytic site and that uridine diphosphate-N-acetyl-d-glucosamine acts as a feedback inhibitor of the enzyme.

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