DNA-protein interactions. Destabilizing activity of sheep pancreatic RNAase]

Evidence is presented that ovine pancreatic ribonuclease, a protein strictly homologous to bovine RNAase A but with one positive charge less, has a definite 'destabilizing' activity (quite similar to that of the bovine enzyme) on double-stranded DNA. This action of sheep pancreas RNAase has been measured by differential spectrophotometry and determining the thermal-transition profiles of the protein-DNA complexes.     

Purification and characterization of a ribonuclease from human liver

The major ribonuclease of human liver has been isolated in a four-step procedure. The protein appears homogeneous by several criteria. The amino acid composition and the amino-terminal sequence of the enzyme indicate that the protein is related to human pancreatic ribonuclease and to angiogenin, and that it may be identical with an eosinophil-derived neurotoxin and to a ribonuclease that has been isolated from urine. The catalytic activity of the liver ribonuclease and its sensitivity to iodoacetic acid inactivation also relate the enzyme to the pancreatic RNases, but the liver protein is clearly differentiated by immunological measurements. Antibodies to the liver ribonuclease inhibit its activity, but not that of the human pancreatic enzyme; cross-reactivity in a radioimmunological assay is small but measurable. Immunochemical measurements have been used to examine the distribution of the liver-type protein in other tissues. Inhibition of enzyme activity by anti-liver ribonuclease shows that a cross-reactive enzyme is predominant in extracts of spleen and is a significant component in kidney preparations, while the liver-type protein is almost absent in brain or pancreas homogenates. Cross-reactive ribonuclease is present in serum, but levels are not correlated with any of the disease states examined.     

Acetyl-coenzyme A hydrolase,an artifact? The conversion of acetyl-coenzyme A into acetate by the combined action of carnitine acetyltransferase and acetylcarnitine hydrolase

1. The nature of the acetyl-CoA hydrolase (EC 3.1.2.1) reaction in rat and sheep liver homogenates was investigated. 2. The activity determined in an incubated system was 5.10 and 3.28nmol/min per mg of protein for rat and sheep liver homogenate respectively. This activity was not affected by the addition of l-carnitine, but was decreased by the addition of d-carnitine. 3. No acetyl-CoA hydrolase activity could be detected in rat or sheep liver homogenates first treated with Sephadex G-25. This treatment decreased the carnitine concentrations of the homogenates to about one-twentieth. Subsequent addition of l-carnitine, but not d-carnitine, restored the apparent acetyl-CoA hydrolase activity. 4. Sephadex treatment did not affect acetyl-carnitine hydrolase activity of the homogenates, which was 5.8 and 8.1nmol/min per mg of protein respectively for rat and sheep liver. 5. Direct spectrophotometric assay of acetyl-CoA hydrolase, based on the reaction of CoA released with 5,5'-dithiobis-(2-nitrobenzoic acid), clearly demonstrated that after Sephadex treatment no activity could be measured. 6. Carnitine acetyltransferase (EC 2.3.1.7) activity measured in the same assay system in response to added l-carnitine was very low in normal rat liver homogenates, owing to the apparent high acetyl-CoA hydrolase activity, but was increased markedly after Sephadex treatment. The V(max.) for this enzyme in rat liver homogenates was increased from 3.4 to 14.8nmol/min per mg of protein whereas the K(m) for l-carnitine was decreased from 936 to 32mum after Sephadex treatment. 7. Acetyl-CoA hydrolase activity could be demonstrated in disrupted rat liver mitochondria but not in separated outer or inner mitochondrial membrane fractions. Activity could be demonstrated after recombination of outer and inner mitochondrial membrane fractions. The outer mitochondrial membrane fraction showed acetylcarnitine hydrolase activity and the inner mitochondrial membrane fraction showed carnitine acetyltransferase activity. 8. The results presented here demonstrate that acetyl-CoA hydrolase activity in rat and sheep liver is an artifact and the activity is due to the combined activity of carnitine acetyltransferase and acetylcarnitine hydrolase.     

Phytochrome Control of the Level of Extractable Ribonuclease Activity in Etiolated Hypocotyls

AN increase in RNA degrading activity of hypocotyl homo-genates can be detected 6–10 h after transferring 5 day old, dark-grown seedlings of Lupinus albus L. to continuous white light¹. This increase is concomitant with the onset of inhibition of hypocotyl extension^{1, 2}. The ribonuclease component whose level of activity is affected by light treatment (tentatively identified as a ribonucleate nucleotido-2′-transferase (cyclizing) (EC 2.7.7.17)) is associated with the ribosomes (and polysomes)¹ in hypocotyl homogenates. This is in contrast to the bulk of RNA degrading activity in homogenates which is associated with the soluble fraction¹. Isolation of the ribosomal fraction therefore makes it possible to follow changes in the level of activity of this ribonuclease which would be too small to detect in unfractionated cell homogenates. The results reported here indicate that the photoconversion of the red-absorbing form of phytochrome (P_r) to the physiologically-active far-red-absorbing form (P_fr) leads to a specific increase in ribosome-bound ribonuclease activity.     

Purification and properties of sulfhydryl oxidase from bovine pancreas

Immunofluorescent studies showed that antibodies prepared against bovine milk sulfhydryl oxidase reacted with acinar cells of porcine and bovine pancreas. A close inspection of the specific location within bovine pancreatic cells revealed that the zymogen granules, themselves, bound the fluorescent antibody. Bovine pancreatic tissue was homogenized in 0.3 M sucrose, then separated into the zymogen granule fraction by differential centrifugation. The intact zymogen granules were immunofluorescent positive when incubated with antibodies to bovine milk sulfhydryl oxidase, and glutathione-oxidizing activity was detected under standard assay conditions. Pancreatic sulfhydryl oxidase was purified from the zymogen fraction by precipitation with 50% saturated ammonium sulfate, followed by Sepharose CL-6B column chromatography. Active fractions were pooled and subjected to covalent affinity chromatography on cysteinylsuccinamidopropyl-glass using 2 mM glutathione as eluant at 37 degrees C. The specific activity of bovine pancreatic sulfhydryl oxidase thus isolated was 10-20 units/mg protein using 0.8 mM glutathione as substrate. Ouchterlony double-diffusion studies showed that antibody directed against the purified bovine milk enzyme reacted identically with pancreatic sulfhydryl oxidase. The antibody also immunoprecipitated glutathione-oxidizing activity from crude pancreatic homogenates. Western blotting analysis indicated a 90,000 Mr antigen-reactive band in both bovine milk and pancreatic fractions while sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single silver-staining protein with an apparent Mr 300,000. Thus, we believe that sulfhydryl oxidase may exist in an aggregated molecular form. Bovine pancreatic sulfhydryl oxidase catalyzes the oxidation of low-molecular-weight thiols such as glutathione, N-acetyl-L-cysteine, and glycylglycyl-L-cysteine, as well as that of a high-molecular-weight protein substrate, reductively denatured pancreatic ribonuclease A.     

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.     

Hepatic nucleases. 1. Methods for the specific determination and characterization in rat liver.

With a view to the study of the subcellular localization of nucleases, methods ensuring the homogenates. The ribonuclease activity of rat liver is due to the three enzymes with different pH optimun. For acid ribonuclease (pH optimun 5.3), it is possible to avoid interference from the other ribonucleases by performing the incubation at pH 5. Neutral ribonuclease (pH optimum 7.6) is differentiated by relying on its sensitivity to the natural inhibitor from the supernatant of liver homogenate. Comparison of activities before and after pretreatment at 50 degrees C in acid medium permits the specific measurement of alkaline ribonuclease (pH optimum 8.8). The optimal conditions for the determination in liver homogenates of two deoxyribonucleases and of an enzyme acting on polyriboadenylate are also described. The activity of these various nucleases is compared and some of their properties are investigated.     

Developmental Pattern for Deoxyhypusine Hydroxylase in Rat Brain

Deoxyhypusine hydroxylase catalyzes the formation of hypusine from deoxyhypusine in a precursor form of eukaryotic initiation factor 4D (eIF-4D). The enzymatic activity was examined in mammalian brain homogenates and the results were consistent with the existence of deoxyhypusine hydroxylase levels comparable to those occurring in other mammalian tissues. Interspecies differences in the enzyme distribution were quite limited, with the highest specific activity values observed in cow brain (1.82 units/ mg of protein). In the rat the enzyme was found to be unevenly distributed among various brain regions. The parietal cortex contained the highest specific activity (2.1 units/mg of protein). Rat brain deoxyhypusine hydroxylase was mainly present in the postmicrosomal supernatant (81% of the total activity). The highest specific activity (3 units/mg of protein) was observed in the rat brain during the first few days of life. Thereafter the activity started to decline, and continued to do so for 15 days, remaining throughout the rest of life at levels of less than one-half that of newborn.     

Effect of stagnant hypoxia on acid ribonuclease activity in the rat prosencephalon during ontogenesis.

In the rat prosencephalon it proved possible to differentiate lysosomal ribonuclease from alkaline ribonuclease activity, which could be detected only in the presence of p-chlormercuribenzoate. Acid RNase activity related to the amount of protein in the prosencephalon fell during ontogenesis. It was not significantly affected by four hours' stagnant hypoxia induced by ligation of both carotids. Its release from the lysosomes rose, however (when isotonic homogenates were spun at 20,000 g, acid ribonuclease activity in the supernatants was elevated). The absence of correlation between this activation and the degree of maturity of the nervous tissue refutes the hypothesis that regulation of this enzyme is per se responsible for the known changes induced by hypoxia in the RNA content of the prosencephalon of rats of different ages. On the contrary, the results indirectly support studies which demonstrate changes in the extent of RNA synthesis after hypoxia.     

Triacylglycerol biosynthesis in bovine liver and subcutaneous adipose tissue.

1. Adipose tissue from Angus and Brahman steers incubated with [1-14C]palmitate in the absence and presence of glucose exhibited a greater rate of lipid production than liver (P < 0.05). 2. Homogenates of adipose tissue used in the glycerol-3-phosphate acyltransferase assay exhibited a greater glycerolipid specific activity (nmol lipid/mg protein/30 min) when compared to liver (P < 0.05). 3. The inverse was true for liver homogenates when calculated for tissue activity (nmol lipid/g tissue/30 min). 4. Lysophosphatidate was produced in greater (P < 0.05) amounts than all other glycerolipids in the glycerol-3-phosphate acyltransferase assay. 5. The activity of phosphatidate phosphohydrolase in liver homogenates displayed greater rates than their respective adipose tissue homogenates. 6. Diacylglycerol acyltransferase activity was greater in adipose tissue homogenates compared to liver homogenates.     

Enzymic hydrolysis of acetylcarnitine in liver from rats, sheep and cows

1. The enzymic utilization of O-acetyl-l-carnitine other than via carnitine acetyltransferase (EC 2.3.1.7) was investigated in liver homogenates from rats, sheep and dry cows. 2. An enzymic utilization of O-acetyl-l-carnitine via hydrolysis of the ester bond to yield stoicheiometric quantities of acetate and l-carnitine was demonstrated; 0.55, 0.53 and 0.30mumol of acetyl-l-carnitine were utilized/min per g fresh wt. of liver homogenates from rats, sheep and dry cows respectively. 3. The acetylcarnitine hydrolysis activity was not due to a non-specific esterase or non-specific cholinesterase. O-Acetyl-d-carnitine was not utilized. 4. The activity was associated with the enriched outer mitochondrial membrane fraction from rat liver. Isolation of this fraction resulted in an eightfold purification of acetylcarnitine hydrolase activity. 4. The K(m) for this acetylcarnitine utilization was 2mm and 1.5mm for rat and sheep liver homogenates respectively. 6. There was a significant increase in acetylcarnitine hydrolase in rats on starvation and cows on lactation and a significant decrease in sheep that were severely alloxan-diabetic. 7. The physiological role of an acetylcarnitine hydrolase is discussed in relation to coupling with carnitine acetyltransferase for the relief of ;acetyl pressure'.     

大鼠、小鼠胰蛋白酶的分离纯化及动力学性质

采用STI-Sepharose 4B亲和层析的方法,从鼠新鲜胰脏中分离得到纯的胰蛋白酶。大鼠胰蛋白酶的比活为24 615BAEEU/mg蛋白,总活性回收率47％,小鼠胰蛋白酶的比活为32 768BAEEU/mg蛋白,总活性回收率55％。经SDS-聚丙烯酰胺凝胶电泳鉴定,大鼠、小鼠胰蛋白酶均呈现单一蛋白带,两者的分子量都是24kD。用等电聚焦电泳测定,二者的等电点均为p19.5以上。对它们的动力学性质作了研究,大鼠胰蛋白酶的Km值为2.33×10~(-4)mol/L,K,值为0.92×10~(-5)mol/L,小鼠胰蛋白酶的Km值为5.60×10~(-4)mol/L,K:值为1.27×10~(-5)mol/L。     

Tissue distribution of adenosine diphosphatase activity in the rat

The specific activity of adenosine diphosphatase (ADPase) was determined in rat tissue homogenates using a specific radioassay. 14 different tissues were sampled and activity was found in most homogenates examined. The levels ranged from very little activity in the pancreas to high levels in the duodenum and ileum. High levels of ADPase activity found in the gastrointestinal tract could be partially attributed to non-specific alkaline phosphatase. It is concluded that ADPase activity is widely distributed in rat tissues.     

Glucokinase in chicken (Gallus gallus). Partial cDNA cloning, immunodetection and activity determination

Chickens are more hyperglycaemic and insulin-resistant than mammals, and in efforts to understand their glucose metabolism we investigated whether glucokinase (GK) is present in chicken liver or pancreas. This enzyme plays a major role in glucose-sensing in mammals and we have examined whether it also contributes to glucose homeostasis in chickens. Using RT-PCR, we cloned and sequenced a partial cDNA fragment (750 bp) from liver and pancreas that showed a high degree of identity with mammalian GK. Using antibodies directed towards human GK, we immunodetected a 50 kDa band in chicken liver and pancreas. The molecular mass of the band and its specific interaction with the antibody suggest that this protein corresponds to a chicken homologue of human GK. We also determined by spectrophotometry a glucokinase-like activity in crude liver homogenates with an apparent half saturating concentration for glucose of 8.6 mM. GK gene and protein expression did not differ between fed and 24 h fasted states but GK-like activity was significantly increased in fed chickens. In conclusion, our study provides evidence for the presence of GK gene and protein in chicken liver and pancreas and shows that the liver enzyme is active.     

Purification of a ribonuclease from potato tubers and its use as an antigen in the immunochemical assay of this protein following tuber damage

Summary A method for the purification of a ribonuclease from potato tubers is described. The preparation was free from deoxyribonuclease and phosphodiesterase activities and possessed only slight phosphomonoesterase activity. Specific antibodies against the ribonuclease preparation were raised in rabbits. Two precipitin arcs were observed on Ouchterlony plates and three by the use of immunoelectrophoresis suggesting that the preparation contained three antigens. Development of one of the arcs on the diffusion plates could be prevented by prior absorption of the RNase preparation with an antiserum specific for phosphomonoesterase from potato tubers. Two of the arcs developing upon immunoelectrophoresis, one of which had low electrophoretic mobility and the other which migrated to the anode, corresponded in position to that of ribonuclease fractionated by agar gel electrophoresis. The remaining arc corresponded to the position of that arising when the RNase antigen was cross-reacted with specific antibodies against phosphomonoesterase from potato tubers. It was concluded that the anti-acid RNase antiserum may be useful for the immunochemical assay of RNase protein when used in conjunction with an anti-phosphomonoesterase antiserum and it was used for this purpose with homogenates derived from damaged and undamaged tuber tissue cv. Majestic. The observations suggested that RNase protein did not parallel the increase in ribonuclease activity following tissue damage and it was concluded that the enhanced RNase activity following mechanical damage may be due to activation of the pre-formed enzyme.     

Plant nucleases. IV. Genetic control of ribonuclease activity in corn endosperm

Ribonuclease activity in the endosperms of 14 corn (Zea mays L.) inbreds ranged from 285 to 1305 units/g fresh weight 50 days after pollination. Activity is low in the inbred M14 and high in the inbred WF9. Hybrid endosperms contain intermediate levels of ribonuclease, and segregation occurs in the F₂ generation. The ribonuclease contents of the opaque-2 versions of nine inbred lines ranged from 1288 to 5110 units/g. The floury-2 mutation apparently does not affect ribonuclease content. Two or more genes may be involved in the control of ribonuclease content of developing endosperms.Cooperative investigations of the Plant Science Research Division, Agricultural Research Service, U.S. Department of Agriculture, and the Illinois Agricultural Experiment Station, Urbana, Illinois.     

Soluble and particle-bound trehalase in extracts from larvae, pupae, and adults of Musca domestica

Trehalase activity was measured in tissue homogenates and extracts from the larval, pupal, and adult stages of Musca domestica, the common housefly. The tissue homogenates were separated into soluble and particlebound fractions by differential centrifugation, and the trehalase activities of the fractions were measured. The trehalase specific activity (units of enzyme/mg protein) in homogenates from adult insects was nearly twenty times greater than activity in homogenates of larvae. Homogenates of pupae showed intermediate values. In both the adults and larvae the enzyme activity was approximately evenly distributed between soluble and particle-bound forms, whereas 95 per cent of the trehalase activity in the extract of pupae was in the soluble fraction. The results show that the form and amount of trehalase present during housefly development is adjusted to accommodate the enzyme's physiological rôle of splitting trehalose to glucose for the insect's use as an energy source.     

Purification of rat liver particulate neutral ribonuclease and comparison of properties with pancreas and serum ribonucleases.

Rat liver particulate neutral ribonuclease (EC 3.1.4.22) was extensively purified (up to 40000-fold). It is shown to be an endonuclease, specific for pyrimidine bases, hydrolysing 5'-phosphate ester bonds. The enzyme specificity, Km, pH optimum, stability in acid medium and thermal stability at high temperature are the same as those of rat pancreatic and serum ribonucleases. Like pancreatic and serum neutral ribonucleases, the hepatic enzyme is sensitive to the liver natural inhibitor. This inhibitor was purified 8000-fold; its association with ribonuclease follows zero-order kinetics. These identical properties for ribonuclease of rat liver, pancreas and serum support the hypothesis [Bartholeyns, Peeters-Joris & Baudhuin (1975) Eur. J. Biochem. 60, 385-393] of an extrahepatic origin for the liver enzyme, the plasma ribonuclease of pancreatic origin being taken up by endocytosis in the liver. Neutral ribonuclease activity was detected in all rat organs investigated; its distribution among tissues is different from the distribution of the natural ribonuclear inhibitor.     

Purification and characterization of a high-Mr carbonic anhydrase from sheep parotid gland.

Approximately half the carbonic anhydrase activity of sheep parotid-gland homogenate is derived from a high-Mr protein [Fernley, Wright & Coghlan (1979) FEBS Lett. 105, 299-302]. This enzyme has now been purified to homogeneity, and its properties were compared with those of the well-characterized sheep carbonic anhydrase II. The protein has an apparent Mr of 540,000 as measured by gel filtration under non-denaturing conditions and an apparent subunit Mr of 45,000 as measured by SDS/polyacrylamide-gel electrophoresis. After deglycosylation with the enzyme N-glycanase the protein migrates with an apparent Mr of 36,000 on SDS/polyacrylamide-gel electrophoresis. The CO2-hydrating activity was 340 units/mg compared with 488 units/mg for sheep carbonic anhydrase II measured under identical conditions. This enzyme does not, however, hydrolyse p-nitrophenyl acetate. The enzyme contains 0.8 g-atom of zinc/mol of protein subunit. The peptide maps of the two carbonic anhydrases differ significantly from one another, indicating they are not related closely structurally. Unlike the carbonic anhydrase II isoenzyme, which has a blocked N-terminus, the high-Mr enzyme has a free glycine residue at its N-terminus.     

Mitochondrial development in liver of foetal and newborn rats

THE DEVELOPMENT OF THE INNER MITOCHONDRIAL MEMBRANE IN FOETAL AND NEONATAL RAT LIVER WAS STUDIED BY FOLLOWING THREE PARAMETERS: (1) the activity of several respiratory enzymes in homogenates and purified mitochondria, (2) the spectrophotometric determination of cytochrome content in the mitochondria and (3) the cardiolipin content in both homogenates and purified mitochondria. Respiratory-enzyme activities of homogenates of foetal liver were one-quarter to one-twentieth of those of homogenates of adult liver, and the enzyme specific activities in purified mitochondria from foetal liver were one-half to one-eighth of those in mitochondria from adult liver. The cardiolipin content of liver homogenates increased approximately twofold during the development period, but there was no significant change in the cardiolipin content of purified mitochondria. It is concluded that cell mitochondrial content approximately doubles in the immediate postnatal period. There was no evidence for an increase in the relative amount of cristae protein in mitochondria during this period to account for increases in mitochondrial enzyme specific activity, since cardiolipin and cytochrome concentrations remained unchanged and electron micrographs revealed no differences. The cause of the lower respiratory-enzyme specific activity in foetal liver mitochondria is unclear. Qualitative differences in respiratory units in foetal and mature animals are suggested.