The regulation of carbamoyl phosphate synthase activity in rat liver mitochondria.

The rate at which isolated rat liver mitochondria synthesized citrulline with NH4C1 as nitrogen source was markedly dependent on the protein content of the diet. 2. Citrulline synthesis was not rate-limited by substrate concentration, substrate transport or ornithine transcarbamoylase activity under the conditions used. 3. The intramitochondrial content of an activator of carbamoyl phosphate synthase, assumed to be N-acetyl-glutamate, varied markedly with dietary protein content. The variation in the concentration of this activator was sufficient to account for the observed variation in the rates of citrulline synthesis if this synthesis were rate-limited by the activity of carbamoyl phosphate synthase. 4. The rates of urea formation from NH4Cl as nitrogen source in isolated liver cells showed variations in response to diet that closely paralleled the variations in the rates of citrulline synthesis observed in isolated mitochondria. 5. These results are consistent with the postulate that when NH4Cl plus ornithine are present in an excess, the rate of urea synthesis is regulated at the level of carbamoyl phosphate synthase activity.     

The molecular weights of two forms of carbamoyl phosphate synthase from rat liver

1. N-Acetylglutamate-dependent carbamoyl phosphate synthase from rat liver was centrifuged in sucrose density gradients. The concentration-dependence of s was consistent with a chemical equilibrium existing between the 11S and 7.5S forms of the enzyme. 2. Under conditions favouring the 11S form, the properties of the enzyme in ultra-short-column equilibrium experiments suggest a molecular weight of 316000+/-42000 for the 11S form. 3. Under conditions favouring the 7.5S form, high-speed equilibrium-sedimentation measurements gave a value of 160000+/-10000 as the molecular weight of the 7.5S form of the enzyme.     

Control of pyrimidine biosynthesis in the perfused liver. Feedback inhibition of glutamine-dependent carbamoyl phosphate synthetase.

The site of feedback inhibition of the biosynthesis of pyrimidine nucleotides de novo was investigated in the isolated perfused rat liver. Hepatic uridine phosphate contents were specifically depleted by use of D-galactosamine. The effective activities of enzymes involved in the synthetic pathway were deduced from the rats of incorporation of labeled precursors into the acid-soluble uracil nucleotide pool and into some intermediates of the pathway. The labeling of hepatic urea was also monitored. When the uridine phosphate contents were less than 20% of controls, the incorporation of [14-C]-bicarbonate was stimulated about 20-fold. Label from [U-14C]oxaloacetate used as permeable precursor of intrace-lular aspartate was introduced into the uridylates to the same extent in normal and UTP-depleted livers. Similar results were obtained with labeled carbamoyl phosphate although the uptake of this compound by the liver was rather low. The lack of labeling of urea from exogenous carbamoyl phosphate does not indicate a free exchange of extra- and intramitochondrial carbamoyl phosphate. [ureido-14C]Ureidosuccinate produced in normal and D-galactosamine-treated livers almost identical labeling patterns of dihydroorotate, orotate and orotidine 5'-phosphate. The steady state concentrations of these intermediates were all below 15 nmol/g liver wet weight.     

Immunoferritin location of carbamoyl phosphate synthetase in rat liver.

Experiments were carried out to locate carbamoyl phosphate synthetase (CPS) in rat liver by direct immunoferritin labeling. By using Epon sections treated with sodium methoxide, homogenates or mitochondrial and mitoplast fractions, carbamoyl phosphate synthetase was found homogeneously distributed in the mitochondrial matrix. Immunoferritin was detected with high resolution which permits the identification of individual molecules. Measurements were made of the number of ferritin particles per square micron of mitochondrial surface, providing a novel and independent assessment of the carbamoyl phosphate synthetase concentration.     

Effects of dimethyl sulfoxide on cultured rat hepatocytes in sandwich configuration.

A recently developed sandwich culture system, in which hepatocytes are sandwiched between two layers of collagen, has been shown to be capable of maintaining long-term expression of hepatocellular function (J. C. Y. Dunn et al., Biotechnol. Prog. 7, 237-245, 1991). The development of an adequate technique for the cryopreservation of hepatocytes in such a stable culture configuration would ensure a ready supply of hepatocytes for use in bioreactors or bioartificial liver support devices. This report describes the effects of exposing hepatocytes in sandwich culture to different concentrations of the cryoprotectant dimethyl sulfoxide (Me2SO) at 22 degrees C on Day 7 of culture. Cell function, morphology, and cytoskeletal organization were followed for 14 days after exposure. Hepatocellular morphology and albumin secretion remained normal when cultures were exposed for up to 120 min to predicted final Me2SO concentrations up to 1.33 M. Exposure for less than 60 min to equilibrium concentrations of up to 3.33 M Me2SO did not adversely affect cell morphology or albumin secretion rate, but at the highest concentration (3.33 M), increase of the exposure time to 60 or 120 min resulted in dramatic, irreversible cell damage and loss of function. Actin filament organization was shown to be undisturbed when the cells were exposed to 1.33 M Me2SO for 60 min, but was irreversibly disrupted by exposure to 3.33 M for 120 min. Based on these results, a simple and safe procedure is suggested for the addition of Me2SO to hepatocytes in a sandwich culture configuration and its subsequent removal, which will be valuable for studies on hepatocyte cryopreservation.     

The effects of dimethyl sulfoxide on the osmotic properties of a rat megakaryocytopoietic cell line

Dimethyl sulfoxide (DMSO) at 0.6 M alters the osmotic state of intracellular water in a line of rat megakaryocytopoietic cells. The response is a function of temperature. Maximal structuring of water occurs at 24.7 degrees C, while disorganization occurs at 30 and 37 degrees C. Little effect occurs at 3 and at 18 degrees C. Parallel measurements of membrane permeability to water indicate that DMSO inhibits the osmotic exit of water at all temperatures. Maximal inhibition occurs at 24.7 degrees C, the same temperature at which maximal organization of water occurs. These findings emphasize the possible role which water may play in explaining the diverse functions of DMSO as cryogenic substance, promoter of cell fusion, and stimulator of cell differentiation.     

Citrulline synthesis in rat tissues and liver content of carbamoyl phosphate and ornithine

Rat liver ornithine carbamoyltransferase appears to be located exclusively in the mitochondria; the activity that is found in the soluble fraction is indistinguishable from mitochondrial ornithine carbamoyltransferase by simple kinetic criteria, and seems to result from breakage of mitochondria during homogenization. Of several rat tissues studied, only the liver and the mucosa of small intestine contain significant amounts of ornithine carbamoyltransferase; the activity in intestinal mucosa is less than one thousandth of that in liver. Qualitatively, this distribution coincides with that of carbamoyl phosphate synthetase I and its cofactor, acetylglutamate. The rat liver contents of carbamoyl phosphate and ornithine were 0.1 and 0.15mumol/g wet wt. of tissue respectively. On the basis of these values, it is proposed that in vivo the ornithine carbamoyltransferase activity of liver may be much lower than its maximal activity in vitro might suggest.     

Assay and properties of digitonin-activated bilirubin uridine diphosphate glucuronyltransferase from rat liver

1. The bilirubin UDP-glucuronyltransferase assay described by Van Roy & Heirwegh (1968) has been improved. 2. Extraction of final azo-derivatives is rendered more simple and efficient by thorough emulsification and by cooling. 3. Pretreatment of homogenates and cell fractions with digitonin increases the sensitivity of the assays and gives less variable results than those with untreated preparations. The activation procedure is flexible. 4. Blank values (obtained from incubation mixtures from which activating bivalent metal ion and UDP-glucuronic acid were omitted) are low. No endogenous conjugate formation could be detected except with untreated, fresh liver homogenates. Control incubation mixtures containing the latter preparations are preferably kept at 0 degrees C. 5. With activated microsomal preparations, rates of breakdown of UDP-glucuronic acid (as monitored by release of P(i)) were low. Little if any increase in enzyme activity was found when UDP-N-acetylglucosamine was included in the incubation mixtures. 6. Slight deviation from Michaelis-Menten kinetics with respect to bilirubin observed at low substrate concentrations is probably related to the use of binding protein in the assay mixtures. Michaelis-Menten kinetics were followed with respect to UDP-glucuronic acid. Part of the enzyme in microsomal preparations from rat liver functioned independently of added bivalent metal ions. Mn(2+) was slightly more, and Ca(2+) somewhat less, stimulatory than Mg(2+). The Mg(2+)-dependent fraction showed Michaelis-Menten kinetics with respect to the added Mg(2+). 7. The enzyme activities found were higher than values reported in the literature for untreated or purified preparations from rat liver. They were above reported values of the maximal biliary excretion rate of bilirubin.     

Acetohydroxyacid synthase isozyme I from Escherichia coli has unique catalytic and regulatory properties

AHAS I is an isozyme of acetohydroxyacid synthase which is apparently unique to enterobacteria. It has been known for over 20 years that it has many properties which are quite different from those of the other two enterobacterial AHASs isozymes, as well as from those of "typical" AHASs which are single enzymes in a given organism. These include a unique mechanism for regulation of expression and the absence of a preference for forming acetohydroxybutyrate. We have cloned the two subunits, ilvB and ilvN, of this Escherichia coli isoenzyme and examined the enzymatic properties of the purified holoenzyme and the enzyme reconstituted from purified subunits. Unlike other AHASs, AHAS I demonstrates cooperative feedback inhibition by valine, and the kinetics fit closely to an exclusive binding model. The formation of acetolactate by AHAS I is readily reversible and acetolactate can act as substrate for alternative AHAS I-catalyzed reactions.     

Expression and purification of imidazole glycerol phosphate synthase from Saccharomyces cerevisiae

Imidazole glycerol phosphate (IGP) synthase is a glutamine amidotransferase that catalyzes the formation of IGP and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) from N(1)-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-car boxamide ribonucleotide (PRFAR). This enzyme represents a junction between histidine biosynthesis and de novo purine biosynthesis. The recent characterization of the HIS7 gene in the yeast Saccharomyces cerevisiae IGP synthase established that this protein is bifunctional, representing a fusion between the N-terminal HisH domain and a C-terminal HisF domain. Catalytically active yeast HIS7 was expressed in a bacterial system under the control of T7 polymerase promoter. The recombinant enzyme was purified to homogeneity and the native molecular weight and steady-state kinetic constants were determined. The yeast enzyme is distinguished from the Escherichia coli IGP synthase in its utilization of ammonia as a substrate. HIS7 displays a higher K(m) for glutamine and a lower turnover in the ammonia-dependent IGP synthase activity. As observed with the E. coli IGP synthase, HIS7 shows a low basal level glutaminase activity that can be enhanced 1000-fold in the presence of a nucleotide substrate or analog. The purification and characterization of the S. cerevisiae enzyme will enable a more detailed investigation of the biochemical mechanisms that mediate the ammonia-transfer process. The fused structural feature of the HIS7 protein and the development of a high-level production system for the active enzyme elevate the potential for determination of its three-dimensional structure through X-ray crystallography.     

Genetic damage is not produced by normal cryopreservation procedures involving either glycerol or dimethyl sulfoxide: a cautionary note, however, on possible effects of dimethyl sulfoxide

Cryopreservation of chinese hamster ovary cells in tissue culture with either glycerol or dimethyl sulfoxide did not result in chromosome damage as measured by the sister chromatid exchange technique. These results are consistent with earlier negative reports in which the freezing and thawing of mammalian cells did not increase the frequency of micronuclei. No increases in the spontaneous mutation rates of several bacterial strains at different genetic loci were observed during the course of a number of years of storage at -196 degrees C. It is concluded that standard cryopreservation procedures are without genetic hazards. However, the well-documented effects of dimethyl sulfoxide on cell fusion and gene differentiation suggest caution in its use as a cryopreservative for animal and human embryos.     

Tritium isotope effects in the measurement of the glycerol phosphate and dihydroxyacetone phosphate pathways of glycerolipid biosynthesis in rat liver

1. Rat liver slices were employed to study the relative rates of incorporation of a mixture of [2-(3)H]- or [1,3-(3)H]-glycerol and [1-(14)C]glycerol into lipids. 2. With 0.1mm-glycerol approx. 82% of the newly synthesized lipid, calculated from (14)C incorporation, was present as neutral lipid, 13% as phosphatidylcholine and 5% as phosphatidylethanolamine. Increasing the glycerol concentration to 40mm caused a decrease in the percentage of neutral lipid to 59% and a corresponding increase in the percentage of phosphatidylcholine to 36% of the newly synthesized lipid. 3. The (d.p.m. of 2-(3)H)/(d.p.m. of 1-(14)C) ratio in glycerolipid was considerably higher than that in precursor glycerol throughout the range of experimental conditions. In contrast the incorporation of a mixture of [1,3-(3)H]glycerol and [1-(14)C]glycerol into lipid occurred with little or no change in the (3)H/(14)C ratio. 4. Respiring rat liver mitochondria were found to oxidize a mixture of sn-[2-(3)H]- and sn-[1-(14)C]-glycerol 3-phosphate with a resultant increase in the (3)H/(14)C ratio of the remaining sn-glycerol 3-phosphate. This increase is due to a (3)H isotope effect of the mitochondrial sn-glycerol 3-phosphate dehydrogenase (EC 1.1.99.5), which discriminates against sn-[2-(3)H]glycerol 3-phosphate during oxidation. 5. A method is described for the simultaneous determination of the relative contributions of the glycerol phosphate and dihydroxyacetone phosphate pathways of glycerolipid biosynthesis in rat liver slices. The method involves measurement of the (d.p.m. of 2-(3)H)/(d.p.m. of 1-(14)C) ratio in both sn-glycerol 3-phosphate and glycerolipid after incubation of rat liver slices with a mixture of [2-(3)H]glycerol and [1-(14)C]glycerol for various times. 6. By using this method it was shown that 40-50% of the glycerol incorporated into lipid by rat liver slices proceeded via the sn-glycerol 3-phosphate pathway and 50-60% was incorporated via dihydroxyacetone phosphate.