Rabbit Liver Extracts Research Articles

First pass effect of pinazepam in the rabbit liver.

The liver first pass effect of pinazepam in four anaesthetized rabbits was determined by measuring the concentrations of this compound and those of its metabolite N-desmethyldiazepam in the plasma from the portal and hepatic veins. Anaesthesia was induced with an i.p. injection of urethane (1.5 g/kg). Portal and hepatic veins were exposed by midline incision. Pinazepam (20 mg/kg) was orally administered in a single dose by stomach intubation. Blood was simultaneously drawn from the portal and hepatic veins of each rabbit at different intervals after dosing. Pinazepam plasma levels were significantly higher in portal than hepatic vein. While N-desmethyldiazepam concentrations were significantly higher in the hepatic than portal vein plasma. Therefore, it has been demonstrated that the rabbit liver extracts pinazepam from the blood and converts this compound into N-desmethyldiazepam.

Inactivation of liver aldolase in fasting rabbits: Evidence for the accumulation of two different immunoreactive forms

During prolonged starvation the activity of aldolase in crude rabbit liver extracts decreases to less than one-half the value observed in extracts of livers from fed animals. The specific activity of the enzyme purified by adsorption on phosphocellulose and elution with substrate is also approximately one-half that of the purified native enzyme. Both the level of enzyme activity and the specific activity are restored to normal within 36 h of refeeding. After removal of active aldolase from the liver extracts by adsorption on phosphocellulose an additional immunoreactive protein can be isolated by adsorption on antialdolase-Sepharose and elution with 4 m MgCl 2. This protein is devoid of catalytic activity and in livers of fasted rabbits accounts for nearly 40% of the total immunoreactive material. It has also been detected in extracts prepared from livers of fed rabbits, where it accounts for 10–20% of the total protein adsorbed by antialdolase-Sepharose. The low-activity enzyme isolated from livers of fasted rabbits cannot be reactivated by sulfhydryl compounds; it shows similar sensitivity to heat and denaturing agents as the enzyme isolated from livers of fed rabbits. The activity ratios with fructose 1,6-bisphosphate, fructose 1-phosphate, and triose phosphate are similar to those observed for the native liver enzyme.

Isolation and characterization of L-fucose dehydrogenase from rabbit liver.

L-Fucose dehydrogenase [EC 1.1.1.122] was isolated from a rabbit liver extract and purified about 390-fold with a yield of approximately 13%. The purification procedures included treatment with protamine, ammonium sulfate fractionation, treatment with acid, DE-32 celluose colum chromatography, gel filtration on Sephadex G-100, preparative polyacrylamide gel electrophoresis, and affinity chromatography on 5' AMP-Sepharose 4B. The last procedure, affinity chromatography on 5' AMP-Sephadex 4B, was useful for the removal of other dehydrogenases. The eznyme which was homogeneous, as shown by polyacrylamide gel electrophoresis, had a molecular weight of about 92,000. The optimum pH was at 10.0 and isoelectric point at 5.2. The enzyme accepted both L-fucose and D-arabinose as substrate, but was specific for NAD+ as coenzyme. Km values were 0.15 mM, 1.4 mM, and 0.7 mM for L-fucose, D-arabinose, and NAD+, respectively. A single enzyme catalyzed the oxidation of L-fucose and D-arabinose, which had the same configurations of hydroxyl groups from C-2 to C-4. The reaction products obtained with L-fucose as substrate were L-fucono-lactone and L-fuconic acid. The L-fucono-lactone was an immediate product of oxidation and was hydrolyzed to L-fuconic acid spontaneously. This reaction was irreversible. Therefore, it is likely that L-fucose dehydrogenase is involved in the initial step of the catabolic pathway of L-fucose in rabbit liver.

Specificity of the serine protease inhibitor, phenylmethylsulfonyl fluoride

Clarified cell-free extracts were prepared from rapidly dividing Bacillus subtilis cells and from rabbit liver cells. These extracts were treated with [ 3H]-phenylmethylsulfonyl fluoride (PMSF) and analyzed by electrophoresis in isoelectric focusing polyacrylamide gels or detergent gels. Not less than 14 proteins in the B. subtilis extracts and not less than 15 proteins in rabbit liver extracts reacted covalently with PMSF. These results suggest that PMSF is not as specific for serine proteases as sometimes supposed, and its effects in physiological experiments should be interpreted with caution.

Mechanism of glucose transfer to steroids by rabbit liver microsomes.

The formation of the 3-glucosides of estrone, 17alpha-estradiol, or 17beta-estradiol by washed rabbit liver microsomes in the absence of UDP glucose (UDPG) was increased by the addition of crude lipid extracts of rabbit or pig liver and of dolichol or retinol. The reaction was stimulated by Mn2+, Mg2+, and Ca2+. Direct transfer of glucose to the steroid from added UDPG also occurred by a mechanism which was inhibited by metal ions. The two transferases can be separated from each other into the 105000 g pellet and supernatant respectively by treatment with detergents.

The Presence of Liver Auto-Antibodies Induced by Entamoeba Histolytica in the Sera from Both Naturally Infected Humans and Immunized Rabbits

Auto-antibodies against normal human liver have been detected in the sera of humans with highly positive indirect hemagglutination (IHA) amebiasis titers and with clinically-proven amebic liver abscess. Sera of amebiasis patients and rabbits immunized with killed Entamoeba histolytica were tested for anti-amebic antibodies by the IHA test and for auto-antibodies by the complement fixation test, using the antigens prepared from extracts of human liver and rabbit liver. A direct correlation was found to exist between high anti-Entamoeba antibody titers and the presence of anti-liver antibody in the serum. It is proposed that, in addition to direct parasite damage to host tissue, immunological damage could result from the attachment of circulating antigen to the cell surfaces of host tissues such as the liver.

Purification and properties of a heat-stable protein inhibitor of phosphoprotein phosphatase from rabbit liver.

A heat-stable protein inhibitor of phosphoprotein phosphatase has been purified to homogeneity from rabbit liver extract by heating to 95 degrees followed by ion exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The purified inhibitor showed a single band when examined by gel electrophoresis S20, w and Stokes radius values were 1.45 and 25.5, respectively. Using these two values, the molecular weight and frictional ratio was calculated to be 15,500 and 3.40, respectively. The molecular weight determined by sodium dodecyl sulfate-gel electrophoresis was found to be 14,200. The inhibition of phosphoprotein phosphatase was linear up to 40% inhibition with respect to inhibitor was constant with time of incubation for at least 30 min. The optimum pH for the inhibition was between 6.8 and 7.6. A kinetic analysis of the effect of the inhibitor on the dephosphorylation of [32P]phosphorylase a by rabbit liver phosphoprotein phosphatase indicated a noncompetitive inhibition with respect to phosphorylase a. Purified liver inhibitor inhibited the phosphoprotein phosphatase activity in all rat tissues examined. Utilizing purified rabbit liver phosphoprotein phosphatase, the presence of inhibitor activity was also demonstrated in all rat tissues tested.

Studies on the role of adenosine 3‘:5‘-monophosphate in the activation of liver phosphorylase.

Crude extracts of rabbit liver, preincubated to promote the dephosphorylation of enzymes or other regulatory proteins, were used to study the role of cyclic AMP in the activation of glycogen phosphorylase. Inasmuch as endogenous liver phosphorylase was irreversibly altered by the preincubation procedure, crystalline skeletal muscle phosphorylase was used as the substrate in these studies. In the presence of magnesium ions and ATP, phosphorylase b was converted to phosphorylase a, and in an apparent biphasic process the phosphorylase a formed was subsequently converted to phosphorylase b. In the presence of adenosine 3':5'-monophosphate the rate of phosphorylase a formation and the maximal amount of phosphorylase a formed were increased. The cyclic AMP effect was enhanced by glucose-6-P and required the presence of glycogen. The catalytic subunit of cyclic AMP-dependent protein kinase could replace cyclic AMP in the stimulation of phosphorylase a formation. The effects of cyclic AMP or the catalytic subunit were shown to be due to stimulation of phosphorylase kinase rather than to inhibition of phosphorylase phosphatase. Preliminary fractionation experiments showed that it is possible to separate phosphorylase kinase catalytic activity from a factor or factors required for stimulation of its activation by the catalytic subunit.

Nicotinamide nucleotide analogues of nicotine and cotinine—enzymic studies

Analogues of NAD and NADP were prepared from cotinine, the major metabolite of nicotine, through the NADase-catalyzed exchange reaction These cotinine analogues and the corresponding nicotine analogues were hydrolyzed by snake venom nucleotide pyrophosphatase to produce the mononucleotides The ribosides of nicotine and cotinine were prepared from the mononucleotides by hydrolysis with snake venom 5'-nucleotidase The mononucleotide was the major product formed when (nicotine)AD or (cotinine)AD was incubated with a rabbit liver microsomal fraction. However, the ribosides were not detected when the mononueleotides were incubated with rabbit liver extract. If the nucleotide analogues are formed in vivo, the possibility exists that nicotine and cotinine mononucleotides could accumulate inside the cell.

Double-ternary complex affinity chromatography: preparation of alcohol dehydrogenases

A general affinity chromatographic method for alcohol dehydrogenase purification has been developed by employing immobilized 4-substituted pyrazole derivatives that isolate the enzyme through formation of a specific ternary complex. Sepharose 4B is activated with 300 mg of cyanogen bromide/ml of packed gel and coupled to 4-[3-(N-6-aminocaproyl)aminopropyl]pyrazole. From crude liver extracts in 50 mM phosphate-0.37 mM nicotinamide adenine dinucleotide, pH 7.5, alcohol dehydrogenase is optimally bound at a capacity of 4-5 mg of enzyme/ml of gel. Addition of ethanol, propanol, or butanol, 500 mM, results in the formation of a second ternary complex, which allows the elution of bound enzyme in high yield and purity. This double-ternary complex affinity chromatography has been applied successfully to human, horse, rat, and rabbit liver extracts to isolate the respective homogeneous alcohol dehydrogenases.

Suppression of the antistreptolysin O response by cholesterol and by lipid extracts of rabbit skin.

Lipids extracted from rabbit skin block the hemolytic capacity of SO and also suppress the neutralizing antibody response to this streptococcal extracellular antigen in rabbith immunized intravenosly. The modification in antibody response is specific for SO; the antibody responses to streptococcal DNase B and to streptococcal NADase are not affected. Cholesterol, a lipid present in abundance in skin, has a similar specific effect on the antigenicity of SO and may be the component responsible for the demonstrated effects of these lipid extracts of skin. In vitro experiments indicate that lipid extracts of rabbit skin have a greater capacity to block the hemolytic capacity of SO than do lipid extracts of rabbit heart, kidney, lung, liver, or spleen. These data support the view that the feeble ASO response observed in patients with streptococcal pyoderma is a result of the abundance of a local lipid inhibitor, such as cholesterol, in the skin. They may also bear on the pathogenesis of rheumatic fever, a complication which apparently does not occur following group A streptococcal pyoderma. Two possible explanations for this remarkable epidemiologic observation, both related to the presence of a local inhibitor, are considered: (a) suppression of the ASO response, the magnitude of which has been correlated with the risk of developing rheumatic fever after streptococcal infection of the throat, and (b) inhibition of the toxicity of SO, which has been shown to have a direct toxic effect on the mammalian heart and on isolated beating myocytes.

Naturally occurring immunosuppressive agents. II. The ability of cell-free liver extracts to modify the rejection of allogeneic skin grafts in the rabbit.

It has been demonstrated that cell-free extracts of pig, rabbit and human liver, but not of other parenchymal organs, contain a factor or factors capable of suppressing the proliferative responses of allogeneic and xenogeneic lymphoid cells stimulated with PHA, PWM, Con-A and allogeneic lymphocytes. Intravenous injection of the liver extract is capable of modifying significantly the skin allograft rejection reaction in normal adult outbred rabbits. Work is now in progress to isolate this factor and to determine its composition.

An isoelectrofocusing study of rabbit pyruvate kinases

1. 1. Extracts of rabbit thigh muscle, liver, kidney cortex and spleen were subjected to isoelectrofocusing. In some cases isolated activity peaks were refocused. 2. 2. Evidence for six variant forms of the K-isozyme were obtained. They had pI values of ~5.4, ~ 5.9, 7.0, 7.4, 8.3 and 8.8. Evidence was also obtained for the existence of two L-isozyme variants with pI values of 4.7 and 5.0. The M-isozyme had a pI value of 8.9. 3. 3. These data were compared to isoelectrofocusing data obtained for other vertebrate species. The pleomorphism of the K and L-isozymes appears to be the rule.

A protein inhibitor of rabbit liver phosphorylase phosphatase

Extracts of rabbit liver contain a heat-stable, non-dialysable inhibitor of phosphorylase phosphatase. The inhibitory activity is destroyed by trypsin treatment or by ethanol precipitation. The kinetics of the inhibition are non-competitive with respect to phosphorylase a. The inhibitory activity is polydisperse on gel permeation chromatography. The mechanism of the inhibition is due to a direct interaction of the protein inhibitor with the enzyme.

Glycosylation of ribonuclease A catalysed by rabbit liver extracts.

Crude extracts of rabbit liver catalyse in vitro the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to bovine pancreatic ribonuclease A. The enzymic activity is contained in rough endoplasmic reticulum. It has an absolute requirement for a bivalent metal ion: Co-2+ greater than Mn-2+ greater than Ni-2+. Mg-2+ is ineffective. There is enzymic activity in the absence of detergent, but increased activity is observed in the presence of Triton X-100. The site of glycosylation of ribonuclease A is asparagine-34, and glycosylation occurs only at this point. These findings agree with the hypothesis that the sequence Asn-X-Thr(Ser) (where X may be one of a number of types of amino acid) is a necessary, but not sufficient, condition for N-acetylglucosaminylation of a protein-bound asparagine residue.

Nicotine and its metabolites: Radioimmunoassay for γ-(3-Pyridyl)-γ-oxo- N-methylbutyramide

A radioimmunoassay has been developed for the nicotine metabolite γ-(3-pyridyl)-γ-oxo- N-methylbutyramide. The sensitivity and specificity of the assay allow the determination of this compound at the picomole level in the presence of several structurally related molecules including nicotine and seven other nicotine metabolites. The assay has been used to characterize the enzyme system present in rabbit liver extract responsible for the conversion of cotinine to the oxoamide, and to measure oxoamide levels in urine, sera, and amniotic fluid of smokers. High-pressure liquid chromatography was used as an independent method to follow the enzymatic oxidation and in conjunction with the radioimmunoassay to analyze the urine samples.

Rhabditis maupasi: Occurrence in food snails and cultivation

Rhabditis maupasi, a nematode, inhabits the mantle cavity of terrestrial snails, Helix aspersa, that are imported from Morocco and sold in the food markets of New York City. Most of the nematodes found in living snails are stage-3 larvae which mature and reproduce after the snail has died and decayed. The nematodes were isolated from snails and cultivated with a microflora and then in species isolation, i.e., axenically. Sterile slices of rabbit kidney supported by agar slants served as the initial culture system. Subsequently, a liquid medium was used that contained raw rabbit liver extract. When, instead, raw snail extract was substituted in the liquid medium, nematode larvae did not mature unless the snail extract was digested secondarily or inactivated by heating. Nematodes grow abundantly in dead snails and in treated snail extract but not in living snails or in raw snail extract. The reason is probably not the nematodes' lack of suitable digestive enzymes for snail tissue nor the absence of adequate nutrients in living, microbially poor snails, but rather the presence of an inhibitor in snails.

The isoelectric focusing pattern of LDH isozymes

The rabbit liver, heart, and muscle extracts showed isoelectric focusing peak patterns of LDH (Ampholine pH 3.5-10), which considerably differed from the LDH zymograms characteristic to those tissues. Ampholine did not give any significant influence on enzymic activity and electrophoretic mobility (agar gel plate) of the five fractions of purified LDH isozymes. Isoelectric focusing of LDH band 1 (H4) produced an acidic peak but with a very poor recovery of activity (1-2%), while that of LDH band 5 (M4) gave an alkaline peak with a good recovery of activity (88%). LDH bands 2 (H3M), 3 (H2M2), and 4 (HM3) exhibited multiple isoelectric focusing peaks, of which an alkaline peak was dominant. Isoelectric focusing of the DANSylated LDH isozyme, band 3, revealed 3 to 4 fluorescent protein peaks not corresponding to the activity peaks. Dissociaton and partial reactivation of LDH (tetramer), accompanying aggregation-denaturation (inactivation), especially in acidic pH, are responsible for producing the modified activity peak pattern of LDH isozymes in the isoelectric focusing experiments.

Modification of the catalytic properties of rabbit liver fructose diphosphatase by a particulate fraction from liver

Fructose diphsphatase (EC 3.1.3.11) 1 assayed in crude extracts of rabbit liver shows maximum activity in the neutral pH range, in contrast to purified enzyme preparations which show an alkaline pH optimum under the same assay conditions. Incubation of the crude enzyme preparations with a particulate fraction from rabbit liver, or with a soluble fraction derived from the particles, causes a decrease in neutral pH activity, and a concomitant increase in activity at alkaline pH; the result is a pH activity curve which resembles that of the purified enzyme. The properties of fructose diphosphatase in crude extracts are compared with those of the purified enzyme activated by formation of mixed disulfides with CoA, acylcarrier protein, or homocystine, all of which show similar pH optima in the neutral pH range, and similar sensitivity to inhibition by AMP. Crude fructose diphosphatase, which shows the neutral pH optimum, can be further activated by homocystine; the neutral activity in crude extracts cannot be attributed to the presence of the disulfide structure.

Rabbit Liver Phosphofructokinase: COMPARISON OF SOME PROPERTIES WITH THOSE OF MUSCLE PHOSPHOFRUCTIOKINASE

A relatively simple procedure was devised for the purification of phosphofructokinase from rabbit liver extracts. The enzyme was purified more than 2600-fold with a yield of close to 50%. Liver phosphofructokinase migrates faster on zone electrophoresis than rabbit skeletal muscle phosphofructokinase. It also differs from muscle enzyme in stability, molecular weight, and kinetic properties. The liver enzyme behaves on Sepharose chromatography as a polymer of various states of aggregation with the largest aggregate having a particle weight of many millions. A comparison of the kinetic properties of liver and muscle phosphofructokinase revealed similarities in pH optimum, cation activation, and substrate affinity at pH 8.2. On the other hand, striking differences in kinetic control properties were observed at pH 7.0. The liver enzyme was more inhibited by ATP, less sensitive to the deinhibiting action of AMP, ADP, and cyclic 3',5'-AMP, less inhibited by citrate, phosphoenolpyruvate, phosphocreatine, 2-phosphoglycerate, and 3-phosphoglycerate, and more inhibited by 2,3-diphosphoglycerate than muscle phosphofructokinase. The results suggest that liver phosphofructokinase is less suited for anaerobic energy production than the skeletal muscle enzyme and may not be subject to the same control mechanism in vivo.