Subcellular distribution and properties of guanylate cyclase in rat cerebellum.

In rat cerebellum the major portion of guanylate cyclase was found to be particulate-bound. The properties of particulate and supernatant guanylate cyclases from the cerebellum were comparatively examined. Both enzymes required the same optimal concentration of Mn2+ and were stimulated by Ca2+ in the presence of a low concentration of Mn2+. But dispersion of the particulate enzyme with Triton X-100 altered the Mn2+ concentration producing maximum activity and the inhibitory effect of Ca2+. The subcellular distributions of guanylate and adenylate cyclases were also studied in rat cerebellum. The major portions of the two cyclases were found in the mitochondrial fraction. The submitochondrial fractions separated by sucrose gradient showed that the major activities of both cyclases were concentrated in the fraction containing mainly nerve ending particles.     

Enzymatic synthesis of poly-β-hydroxybutyrate inZoogloea ramigera

The enzyme activity synthesizing poly--hydroxybutyrate (PHB) was mainly localized in the PHB-containing particulate fraction ofZoogloea ramigera I-16-M, when it grew flocculatedly in a medium supplemented with glucose. On the other hand, the enzyme activity remained in the soluble fraction, when the bacterium grew dispersedly in a glucose-starved medium.The soluble PHB synthase activity became associated with the particulate fraction as PHB synthesis was initiated on the addition of glucose to the dispersed culture. Conversely, the enzyme activity was released from the PHB-containing granules to the soluble fraction when the flocculated culture was kept incubated without supplementing the medium with glucose.PHB synthase was also incorporated into the newly formed PHB fraction when partially purified soluble PHB synthase was incubated withd(-)--hydroxybutyryl CoA in vitro.Although attempts to solubilize the particulate enzyme were unsuccessful, and the soluble enzyme became extremely unstable in advanced stages of purification, both PHB synthases had the same strict substrate specificity ford(-)--hydroxybutyryl CoA, and showed the same pH optimum at 7.0.Non-Standard Abbreviations PHB poly--hydroxybutyrate     

The size of the corpus luteum during pseudopregnancy and pregnancy in the rat.

The corpus luteum in mature Sprague Dawley rats was weighted at the various stages of pseudopregnancy and pregancy. The average size of these corpora lutea was 1.0 +/- 0.10 mg, 1.61 +/- 0.69 mg, 1.90 +/- 0.25 mg, 3.69 +/- 0.36 mg, and 4.37 +/- 0.50 mg on day 2 of diestrus, on days 10-15 of psuedopregnancy, on days 9-10, 14, and 20 of pregnancy, respectively. The fact that the average size of the corpus luteum on days 10-15 of pseudopregnancy was larger than that on day 2 of diestrus is thought to drive from prolonged exposure of the corpus luteum to prolactin. The average size of the corpus luteum on days 9-10 of pregnancy had a tendency to be larger than that on days 10-15 of pseudopregnancy and this seems to demonstrate that the placenta secreted placental lactogen by this stage of pregnancy. The average size of the corpus luteum on day 14 of pregnancy was larger than that on days 9-10 of pregnancy. This phenomenon might be attributed to the presence of large amounts of placental lactogen secreted from the placenta between days 10 and 14 of pregnancy. Furthermore, it was noted that the size of the corpus luteum on day 20 of pregnancy was larger than that of day 14, which suggests that further secretion of placental lactogen continued after day 14 of pregnancy. As there was a remarkable decrease in the number of fetuses on day 20 of pregnancy when overiectomy was performed on day 14 of pregnancy, the ovary was considered indispensable in maintaining pregnancy in the rat.     

Transplacental transmission of BK virus in human.

The prevalance and distribution of BK virus antibody in women during pregnancy and the occurrence of transplacental transmission of BK virus was determined by measurement of IgM antibody in the serum. Sera were collected from 63 nonpregnant women, 71 women who had experienced spontaneous abortion, 80 in the first trimester of pregnancy and the same 80 at delivery. Umbilical cord blood was also taken at delivery. Hemagglutination inhibition (HI) tests for BK virus used the micromethod of Gardner. Results indicate that a significant level of HI antibody was present in 70% of sera from all 4 experimental groups. This showed that BK virus infection was not limited to cases of spontaneous abortion. Of the 80 pregnant mothers, 6 showed a 4-fold or greater HI antibody seroconversion to BK virus after delivery. Of these 6 seroconversion patients, sensitive antibody was detected in 3 umbilical cords. Umbilical cords of those without seroconversion had no sensitive antibody. As evidenced by 2-NE-sensitive antibody, BK virus infections were also recognized in 6 of 71 women who aborted, 4 of 80 in the first trimester of pregnancy and 2 collected after delivery. The 2-ME-sensitive antibody was not found in any of 63 samples from nonpregnant women. Data indicate that 2-ME-sensitive antibody was present only in sera of women during pregnancy and after abortion. It may be possible that BK virus persists in a latent form in many healthy women and becomes activated during pregnancy.     

Phosphatidohydrolase activity in a solubilized preparation from rat brain particulate fraction.

Phospholipase D activity (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) was demonstrated in vitro in a solubilized preparation from rat brain particulate fraction which also possessed the transphosphatidylation activity. The preparation attacked a phosphatidylcholine microdispersion and cleaved the terminal phosphate diester bond of this phospholipid resulting in the formation of phosphatidic acid. The pH optimum for the phosphatidohydrolase activity was broad with an apparent peak aroung 6.0 whereas the transphosphatidylation showed a sharp pH optimum at 7.2 Ca²⁺ was not essential for the hydrolysis, but merely stimulated slightly with an optimum about 5 mm, however, it could be replaced by Mg²⁺. The hydrolysis of phosphatidylcholine by the enzyme was almost completely inhibited in the presence of either diethyl ether (20% by volume) or p-chloromercuriophenyl sulfonate (6 × 10 ^?5m). The latter inhibition was reduced by the addition of dithiothreitol (6 × 10^?4m). The result suggests an essential role of sulfhydryl group in the formation of the enzyme-substrate complex.The apparent K_m for phsophatidylcholine for the phosphatidohydrolase activity was about 8.3 × 10 ^?4m.     

Circadian rhythms in digestive enzymes in the small intestine of rats. I. Patterns of the rhythms in various regions of the small intestine.

The activities of the digestive enzymes, maltase [EC 3.2.1.20], sucrase [EC 3.2.1.26], trehalase [EC 3.2.1.28], Leucine aminopeptidase [EC 3.4.11.1], and alkaline phosphatase [EC 3.1.3.1] were measured in various regions of the small intestine of rats. The activities of all these enzymes were much higher in the jejunum than in the ileum, and in the distal regions of the ileum no sucrase, trehalase or alkaline phosphatase activity was detected. In the jejunum, the activities of all the enzymes tested exhibited clear circadian variations with the highest activity at 0000-0400 h and the lowest at 1200 h when the rats were fed ad libitum. In the ileum, maltase and sucrase also exhibited circadian variations, but the amplitude of the rhythm was smaller than that in the jejenum. Trehalase and alkaline phosphatase did not show any circadian variation in the ileum. Leucine aminopeptidase showed a circadian variation in the ileum with the same amplitude as in the jejunum. The phase of the circadian variations shifted about half a day when the rats were fed in the daytime, but the amplitude of the rhythm did not change.     

Studies on a phospholipase B from Penicillium notatum. Purification, properties, and mode of action.

1. Phospholipase B which hydrolyzes both the acyl ester bonds of diacylphospholipids (diacyl-hydrolase) and the acyl ester bond of monoacylphospholipids or lysophospholipids, [monoacyl-hydrolase or lysophospholipase, EC 3.1.1.5] was purified from Penicillium notatum about 2000-fold over the crude extract. The final preparation was homogeneous on disc electrophoresis. The apparent molecular weight, determined by gel filtration on Sephadex G-200, was about 116,000. The isoelectric point was pH 4.0. 2. The purified enzyme was a glycoprotein. The carbohydrate content was approximately 30%, consisting of mannose, glucose, and glucosamine. The amino acid composition was also determined. 3. The ratio of monoacyl-hydrolase to diacyl-hydrolase activities was influenced by the physical state of the substrate in the assay system. It was about 1 : 1 or 100 : 1 in the presence of absence of Triton X-100, respectively, and the latter value remained constant throughout the purification procedures. 4. Both enzyme activities had the same pH optimum, 4.0, and were heat-labile. None of the metals tested had any effect on either activity except for Fe2+ and Fe3+. Diisopropyl fluorophosphate at relatively high concentrations completely inhibited both enzyme activities. 5. The Michaelis-Menten constants (Km) of the enzyme for egg lecithin were about 1.5 and 25 mM in the absence and presence of Triton X-100, respectively. The Km value for dicaproyllecithin was 9.8 mM in the absence of Triton X-100. 6. Using a mixture of 1-[14C]stearoyl-lecithin and 2-[14C]oleoyl-lecithin in the presence of Triton X-100 as a substrate, it was found that the P. notatum phospholipase B attacked the acyl ester bonds sequentially, first the 2-acyl and then 1-acyl groups.     

Mutagenicity of dimethylnitrosamine to mammalian cells as determined by the use of mouse liver microsomes.

The mutagenicity of dimethylnitrosamine (DMN) to mammalian cells was investigated using a metabolic activation system. Mutation from 8-azaguanine (8AG) sensitivity to resistance in FM3A cells, a cell line derived from C3H mouse mammary carcinoma, was found only in the presence of dimethylnitrosamine, mouse liver microsomes and cofactors. The different inducibility of the mutation was shown by the use of liver microsomes from different strains of mouse.     

DNA strand scission by neocarzinostatin: molecular recognition process responsible for site-specificity

A series of hexanucleotides possessing A-T, G-C, inosine (I)-C and 2-aminoadenine (ANH2)-T base pairs at 5'-side of the target thymine were prepared and their selectivity for C-5' and C4' oxidation in the NCS-mediated degradation was investigated. Quantitative product analysis indicated that preferential C5' oxidation of deoxyribose moiety of the target T occurs at -5'-AT- and 5'-IT- sites, whereas C5' and C4' oxidation occurs competitively at T of -5'-GT- and -5'-ANH2T- sites. Based on the experimental results, an intercalation model that permits competitive hydrogen abstraction from C5' and C4' of deoxyribose moiety has been proposed.