Effect of pressure upon the fluorescence of various flavodoxins.

The effects of hydrostatic pressure in the range of 10(-3) to 11 kbar on the fluorescence of flavodoxins from Peptostreptococcus elsdenii, Desulfovibrio vulgaris, Azotobacter vinelandii, and Clostridium MP were investigated. The first three flavoproteins showed under high pressure enhancements of flavin fluorescence of over 50 times resulting from the release of flavin mononucleotide from the protein complex. The Clostridial flavodoxin showed a very much smaller fluorescence change. At pH 7.5 the high-pressure fluorescence changes of the flavodoxins of D. vulgaris and P. elsdenii were not reversed by decompression, but in A. Vinelandii the pressure changes were over 80% reversible. At pH 5 over 80% reversibility was restored to the flavodoxins of D. vulgaris and P. elsdenii, although the pressure dependence of the fluorescence changes was very similar in the reversible and irreversible cases. The midpoint pressures in the reversible reactions were 4.7 kbar (D. vulgaris), 8.7 kbar (P. elsdenii), and 10.6 kbar (A. vinelandii) indicating specific differences in the flavin binding regions. Apparent volume changes in these reactions were 65-75 mL/mol indicating participation of a large fraction of the protein in the pressure-induced changes. The irreversible changes are not related to protein aggregation and are believed to result from a pressure-dependent covalent modification, not yet characterized, of the flavin binding region of the protein.     

Thyroxine-induced hyperthyroidism upon the reproductive performance of female rats.

L-Thyroxine (L-T4) was injected into mature rats daily at levels of 0, 3, 6, 12, 24, 48 and 96 mug/100 g body weight through estrous cycling, conception, pregnancy, parturition and 20 days of lactation. Mothers which lactated to 20 days were killed and mammary glands measured for DNA and RNA content. Those which were not able to maintain their pups were killed on the day when all pups died. Estrous cycling and pregnancy were not markedly effected by exogenous L-T4 at levels 3-96 times the normal thyroxine secretion rate in rats. After parturition the mothers on L-T4 above 12 mug did not allow the pups to suckle. As a result the pups died within 2-7 days after birth. Levels of L-T4 from 3 to 12 mug allowed lactation to progress, but survival of pups to day 20 of lactation was significantly lower than in the normal control group. The results of this study indicate that high levels of L-T4 inhibit mammary growth and mild secretion. This, in turn, results in the loss of pups, probably through depletion of prolactin as a result of higher metabolic rate due to hyperthyroidism.     

Diazepam effect upon the microscopic structure of the mouse placenta.

CD-1 strain, female mice, aged 5 to 7 months, were mated with males of the same age. Females presenting vaginal plug were separated and randomly distributed in two groups to be treated from the 6th to 17th day of gestation. One group received single daily diazepam doses (2.7 mg/kg i.p.), the other, 0.9% saline in equivalent volumes. Females were killed on 18th day, the placentas removed and fixed in 10% formaldehyde, pH 7.3, dehydrated and embedded in paraplast; 3 microns thick sections were stained with hematoxylin-eosin and Weigert hematoxylin and analyzed under light microscopy. Placentas of the diazepam-treated females presented dilated chorion vessels and intervillous spaces. Trophoblastic cell nuclei presented chromatin in coarse granules, atypically distributed in the karyolymph, which had lesser staining affinity. Giant cells showed vacuolized cytoplasm and coarsely granulated chromatin. Results indicate that diazepam causes structural changes, possibly placental and fetal physiology.     

Influence of prostaglandins upon adenosine release and of adenosine upon prostaglandin release in the isolated rabbit heart.

Prostaglandin (PG) E1 with its high effect of coronary dilation causes a distinct increase in the adenosine release from the isolated rabbit heart (Langendorff technique). Compared to this, an equal dose of PGF2alpha increases the release of adenosine only to a small extent. Conversely, application of adenosine results in a considerable release of PG-like substances from the rabbit heart in vitro. The present investigations support the hypothesis that, apart from adenosine, PG's too, are involved in coronary regulation as modulators.     

Effects of phenobarbital upon triacylglycerol metabolism in the rabbit.

1. The association between hepatic microsomal enzyme induction and triacylglycerol metabolism was examined in fasting male rabbits (2kg body wt.) injected intra-peritoneally with 50 mg of phenobarbital per kg for 10 days. 2. Occurrence of enzyme induction was established by a significant increase in hepatic aminopyrine N-demethylase activity and cytochrome P-450 content, as well as a doubling of microsomal protein per g of liver and a 54% increase in liver weight. Parallel increments in hepatic gamma-glutamyltransferase (EC 2.3.2.2) activity occurred; these were more pronounced in the whole homogenate than in the microsomes, which only accounted for 12.5% of the total enzyme activity in the controls and 17.0% in the animals given phenobarbital. Increased activity of gamma-glutamyltransferase activity was also observed in the blood serum of the test animals. 3. The rabbits given phenobarbital manifested increased hepatic triacylglycerol content and the triacylglycerol concentration of blood serum was also elevated. These changes were accompanied by a significantly enhanced ability of cell-free fractions of liver from the test animals (postmitochondrial supernatant and microsomal fractions) to synthesize glycerolipids in vitro from sn-[14C] glycerol 3-phosphate and fatty acids, when expressed per whole liver. Relative to the protein content of the fraction, glycerolipid synthesis in vitro was significantly decreased in the microsomes, presumably consequent upon the dramatic increase in their total protein content, whereas no change occurred in the postmitochondrial supernatant, possibly due to the protective effect of cytosolic factors present in this fraction and known to enhance glycerolipid synthesis. 4. Microsomal phosphatidate phosphohydrolase accounted for 85% of the total liver activity of this enzyme and its specific activity was 20-fold higher than that of the cytosolic phosphatidate phosphohydrolase (EC 3.1.3.4), when each was measured under optimal conditions. A significant increase in the activity of both enzymes per whole liver occurred in the rabbits given phenobarbital. A closer correlation between hepatic triacylglycerol content and and microsomal phosphatidate phosphohydrolase, as well as the above observation, suggest that this, rather than the cytosolic enzyme, may be rate-limiting for triacylglycerol synthesis in rabbit liver. 5. Significant correlations were observed between the various factors of hepatic microsomal-enzyme induction (aminopyrine N-demethylase and gamma-glutamyltransferase activity as well as cytochrome P-450 content) and hepatic triacylglycerol content, suggesting that that microsomal enzyme induction may promote hepatic triacylglycerol synthesis and consequently hypertriglyceridaemia in the rabbit.     

Effects of cysteine upon tumor cells.

Cysteine had been reported to increase survival time in thymoma-bearing mice and the interpretation suggested was that this was due to inhibition of a collagenase activity associated with some tumor cells by a chelating action of cysteine. In the present work it was shown that cysteine was a particularly potent inhibitor of amino acid transport into S37 ascites tumor cells, raising another possible interpretation of the earlier data. Sarcomas have previously been reported to lack collagenase activity; a survival study using S37 cells was therefore undertaken in an attempt to distinguish between possible interpretations of the earlier data involving thymomas. A null result was obtained with either cysteine or EDTA, reinforcing the earlier interpretation that survival enhancement with thymoma-bearing mice was due to an effect on collagenase. Other sulfhydryl analogs were found to inhibit transport also, and the effect was more pronounced with system L than system A. The reason for cysteine's particularly potent action on amino acid transport may be associated either with chelation of a metal ion involved in transport, or the involvement of the gamma-glutamyl cycle in the support of amino acid transport.     

Solvation effects upon the thermodynamic substrate activity; correlation with the kinetics of enzyme catalyzed reactions. I. Effects of added reagents such as methanol upon alpha-chymotrypsin.

Solvents, detergents, etc., have often been added to the medium to study the kinetics of enzyme action and for binding studies. They have been employed for diverse reasons such as solubilization of substrates or to stabilize an enzyme that was originally membrane bound. Thermodynamic considerations dictate that any added substance, such as methanol, which is present in significant quantity must affect the thermodynamic activities of the enzyme, enzyme-substrate complex, substrate and any other intermediates although cancellation effects may occur in this regard. The influence upon substrate activities is the only one that is easily experimentally accessible. These effects are shown, from the data of Bernard and Laidler, to be large in the case of the alpha-chymotrypsin catalyzed hydrolysis of methylhydrocinnamate. The variation of the Michaelis-Menten constant is quantitatively explainable in terms of the alteration of the thermodynamic activity of the substrate by methanol.     

Influence of the freezing process upon fluoride binding to hemeproteins.

Fluoride association with ferric myoglobins and hemoglobins in aqueous buffers above freezing has been well studied. We chose this reaction to investigate the feasibility of observing titration intermediates and estimating dissociation constants at the freezing temperature by electron paramagnetic resonance spectroscopy at cryogenic temperatures. Dependence of apparent dissociation constant upon protein concentration was observed, a factor of four decrease in protein accompanied by about a fourfold increase in the apparent tightness of binding in the range of protein concentration studied. Binding was also found to depend upon cooling rate and concentration of additives (serum albumin, sucrose, glycerol). These effects appear to be associated with freezing-induced concentration of ligand, a process described in the literature. Bands of high concentration of electrolyte accompany solute rejection during ice growth, sweeping by slowing moving macromolecules. Thus, just before being trapped in the solid, the protein can experience a greater concentration of salt than in the original liquid. A mathematical model of this process, based upon simplifying assumptions about nucleation and ice-crystal growth rates in super-cooled solution, shows how the average concentration of mobile solute species can depend upon the concentration of all species present. Semiquantitative computer simulations of the actual, more complex, freezing are also presented and lead to estimates of ice particle size which are then compared with estimates from the former model.