Biochemical characterization of density-separated human erythrocytes.

A simple, reproducible method for the separation of human erythrocytes, described recently (Murphy, J. R. (1973) J. Lab. Clin. Med. 82, 334-341) has been utilized for the purpose of obtaining a wide range of biochemical data on these cells. Using phthalate ester density centrifugation of the fractions obtained by Murphy's method, we established that the cells were separated exclusively on the basis of their densities. Data on a wide range of biochemical and hematological parameters, when compared with previously reported density separation procedures showed that this simple technique can be used to fractionate the cells according to their densities (age) in their own plasma. Cells of increasing density consistently and reproducibly exhibited an increase in hemoglobin concentration, a moderate elevation in Na+ and a decrease in the following: K+, acetylcholinesterase, sialic acid, membrane protein, 2,3-diphosphoglycerate, ATP, cholesterol, phospholipid, mean corpuscular volume and critical hemolytic volume, However, no change in mean corpuscular hemoglobin was evident. The observed differences were not artifacts of the centrifugation process. This was determined in recentrifuged top fractions from which new top and bottom cells were obtained. The latter cells resembled the top fraction from which they were obtained, rather than the original bottom fraction. Whereas the parameters mentioned above exhibited consistency and reproducibility, such was not the case with the ATPase values. Depending on the cell density group examined and/or buffer as well as other conditions, significant variability in the activity levels of the ouabain sensitive, as well as the Ca2+ -stimulated ATPase, was observed. Use of these enzyme activities as indicators of cell age must be viewed with caution.

Ethanol feeding produces deficiencies in left ventricle total RNA, total DNA and mitochondrial ribosomal RNA.

Chronic alcoholism causes a variety of ultrastructural, biochemical and functional alterations in the myocardium, but the underlying mechanisms are not well understood. Molecular changes that developed in the left ventricles of rats fed for 1 to 24 weeks on liquid diets containing ethanol as 36% of total calories were analyzed. Total tissue RNA and DNA were chemically extracted and measured by spectroscopic methods; mitochondrial DNA and mitochondrially-coded ribosomal RNA were measured at the 12s rRNA region by a quantitative polymerase chain reaction method; mitochondrial protein and enzyme activities were assayed. Ethanol-fed rats had 83.9 +/- 2.9% (mean +/- S.E.M.) as much DNA/g tissue and 74.7 +/- 3.9% as much total left ventricle DNA as pair-fed controls (P < 0.001). The alcoholics had 71.4 +/- 1.7% as much RNA/g tissue and 64.4 +/- 2.7% as much total left ventricle RNA as controls (P < 0.001). Mitochondrially-coded 12s rRNA was a lower proportion of total left ventricle RNA in all of the alcoholics; it was only 59.9 +/- 4.6% of control values (P < 0.001). Total left ventricle 12s rRNA was < 40% of normal. There was little or no change in mitochondrial DNA levels measured at the 12s location. Mitochondrial cytochrome contents were reduced 26-38% in the ethanol-fed rats, but only after 24 weeks. This study shows that experimental alcoholism produces rapid and sustained decreases in left ventricle total RNA and DNA and mitochondrial ribosomal RNA. The observed effects would be expected to have a major impact on left ventricle structural integrity and functional capacity.

Low-dose vincristine-associated bilateral vocal cord paralysis.

BACKGROUND: Vincristine-associated peripheral neuropathy is a well-described entity. We describe a case of vincristine-induced vocal cord paralysis, which is a rare complication of this drug. We report herein the second case of bilateral vocal cord paralysis in a patient receiving conventional doses of vincristine. OBJECTIVE: To present a case report of vincristine-associated vocal cord paralysis and to review the relevant English language literature on this subject. DESIGN: Report and review of the literature. SETTING: Outpatient community cancer center. PATIENT: A 58-year-old female with a diffuse large cell lymphoma stage IV receiving cyclophosphamide, doxorubicin, vincristine, and prednisone. RESULTS: Bilateral vocal cord paralysis occurred in this patient receiving vincristine as part of her chemotherapy regimen. In addition to this case there have been a total of 25 prior reports, which are reviewed in the text. CONCLUSION: The incidence of bilateral vocal cord paralysis in patients receiving vincristine on the usual low-dose schedule is low. Prompt withdrawal of the offending agent results in prompt recovery without untoward long-lasting sequela.

Intracellular localization of the mRNAs of argininosuccinate synthetase and argininosuccinate lyase around liver mitochondria, visualized by high-resolution in situ reverse transcription-polymerase chain reaction.

Argininosuccinate synthetase and argininosuccinate lyase, two cytoplasmic enzymes of the urea cycle, are released into the soluble phase in the absence of detergent when cells are disrupted. Yet previous biochemical studies, as well as immunocytochemistry at the electron microscope level, have shown that these enzymes are localized around mitochondria in situ. Such intracellular localization of soluble enzymes requires mechanisms to deliver the proteins to the appropriate sites, where they may then be anchored by specific protein-protein interactions. A method was developed to examine the intracellular distribution of the mRNA of argininosuccinate synthetase and argininosuccinate lyase in intact rat liver at the ultrastructural level by in situ reverse transcription and the polymerase chain reaction, using primers targeting regions of the coding sequences of the rat enzymes, digoxigenin-dUTP as the label, and anti-digoxigenin/1 nm [corrected] gold plus silver enhancement as the detection method. The tissue was fixed in 4% paraformaldehyde/0.1% glutaraldehyde and embedded in Lowicryl. Examination of the numbers and the location of the silver grains, coupled with morphometric analysis of the electron micrographs, permitted the calculation of the silver "enrichment ratio" for each type of cell structure. These ratios showed that the mRNAs for argininosuccinate synthetase and argininosuccinate lyase were located next to the cytoplasmic side of the mitochondrial membrane and in the nearby endoplasmic reticulum. Most of the silver grains that were observed in the endoplasmic reticulum were within 200 nm of the mitochondria; it was not possible, however, to determine if those grains were actually associated with the reticular membranes. These studies demonstrate that the mRNAs of these two soluble cytoplasmic proteins are localized to the same limited regions where the proteins are situated. Translation of the proteins, therefore, must occur at these specific sites. The targeting of argininosuccinate synthetase and argininosuccinate lyase mRNAs to the immediate vicinity of the mitochondria may be the first step of the mechanisms by which the spatial organization of these soluble proteins in situ is accomplished. The targeting of mRNAs for soluble cytoplasmic proteins of organized metabolic pathways has not been demonstrated previously. These studies also show that in situ reverse transcription and the polymerase chain reaction at the ultrastructural level, which has not been previously reported, can be used to detect specific mRNAs; it should be extremely valuable for the intracellular detection of low-abundance mRNAs.

N-acetylglutamate-independent activity of carbamyl phosphate synthetase (ammonia): implications for the kinetic assay of acetylglutamate.

In the presence of Mn2+, carbamyl phosphate synthetase (ammonia) catalyzes considerable carbamyl phosphate synthesis in the absence of the allosteric activator, N-acetylglutamate. Under standard conditions, the acetylglutamate-independent activity of a purified carbamyl phosphate synthetase preparation was 8 to 10% of the Vmax observed at saturating (1 mM) acetylglutamate. The product formed in the reaction was identified unequivocally as carbamyl phosphate. Standard conditions included 5 mM ATPMn and 1.5 mM excess Mn2+. The highest rate of acetylglutamate-independent activity was observed at [excess Mn2+] of 1.5 mM; increasing the [ATPMn] from 5 to 20 mM doubled the acetylglutamate-independent activity, to 18% of Vmax. Only 1/20 as much acetylglutamate-independent activity was observed when Mg2+ was substituted for Mn2+. When both Mn2+ and Mg2+ were present, the acetylglutamate-independent activity was less than when Mn2+ alone was present. Measurement of acetylglutamate-dependent activity of carbamyl phosphate synthetase (ammonia) revealed that one-half Vmax with Mn2+ was achieved at 17 microM acetylglutamate (about one-fifth of the value reported with Mg2+), and the Vmax with Mn2+ under standard conditions was only 60% of that observed with Mg2+. The high affinity of carbamyl phosphate synthetase for acetylglutamate in the presence of Mn2+ has been used in the development of sensitive, accurate method for the measurement of acetylglutamate in small quantities of mitochondrial extracts. This method is described in detail.

Argininosuccinate synthetase and argininosuccinate lyase are localized around mitochondria: an immunocytochemical study.

Argininosuccinate synthetase and argininosuccinate lyase are soluble cytoplasmic enzymes of the urea cycle. Previous biochemical studies using permeabilized hepatocytes showed that these enzymes are organized in situ, and function as if they are located next to the outer membrane of mitochondria. We have now confirmed and extended those observations in intact liver by means of immunocytochemistry at the electron microscope level. Morphometric analysis of the electron micrographs shows that argininosuccinate synthetase and argininosuccinate lyase are located in the immediate vicinity of the mitochondria, predominantly next to the cytoplasmic surface of the outer membrane. Some immuno-specific protein is also observed in the endoplasmic reticulum in the immediate vicinity of the mitochondria. These results support our previous biochemical findings, and additionally suggest that virtually all of the argininosuccinate synthetase and argininosuccinate lyase of the liver parenchymal cell are located just outside the mitochondria.

Stimulation of erythropoiesis in mice by adenosine 3',5'-monophosphate and prostaglandin E1.

Adenosine 3',5'-monophosphate, 2 to 16 mg, and prostaglandin E1, 0.05 to 0.10 mg, stimulate erythropoiesis in ex-hypoxic, polycythemic mice. The stimulation can be prevented by the simultaneous administration of an antiserum to erythropoietin. India ink penetration shows that at these doses blood circulation is blocked in the kidney. Tissue hypoxia, especially in the kidney, causes production of erythropietin. The strong effect on the kidney circulation suggests that the major part of the stimulation of erythropoiesis by these agents is through their effect on renal circulation.

Multiple forms of cytochrome b in Mycobacterium phlei: kinetics of reduction.

The kinetics of reduction of the b-type cytochromes in the electron transport particles (ETP) from Mycobacterium phlei were studied with nicotinamide adenine dinucleotide, reduced form (NADH) or succinate as electron donors. There appeared to be three active cytochromes b in the ETP,bS563 and bS559, which were reducible by either substrate, and bN563, which was reducible by NADH but not by succinate. In the presence of adenosine 5'-triphosphate, a substantial increase in b563 reduction was observed with succinate at anaerobiosis. This was followed by a decrease in absorption. Adenosine 5'-triphosphate did not effect an increase in cytochrome b563 reduction at transition with NADH, but the occurrence of a secondary decrease in absorption was reflected in a decrease in total enzymatic reduction. The adenosine 5'-triphosphate effect was altered in trypsin-treated ETP, and abolished by uncoupling agents or by removal of the coupling factor-latent adenosine triphosphatase. In the presence of a supernatant fraction obtained during the preparation of the ETP, b563 reduction with succinate was greatly increased. A smaller increase was observed with NADH. Cytochrome b reduction was also studied in ETP inhibited by 2-n-nonylhydroxyquinoline-N-oxide, which appears to inhibit at bS563. On the basis of these data the interrelationships among the b-type cytochromes can be described in relation to the M. phlei electron transport chain.

Differential effects of N-acetylglutamate on citrulline synthesis by coupled and uncoupled mitochondria.

When rats were placed on a low-protein (5%) diet for 24 h or less, liver mitochondrial acetylglutamate decreased rapidly, carbamyl phosphate synthetase (ammonia) and ornithine transcarbamylase decreased little, and carbamyl phosphate synthesis (measured as citrulline) by isolated mitochondria occurred at very low rates. The matrix acetylglutamate content of these mitochondria, whether coupled or uncoupled, was increased similarly by preincubating them with added acetylglutamate, but citrulline synthesis increased from less than 1 to 2.3 nmol min-1 mg-1 in the coupled state, and from less than 1 to 35 nmol min-1 mg-1 in the uncoupled state. However, when coupled mitochondria were incubated with the substrates required for the synthesis of acetylglutamate in the matrix, citrulline synthesis increased to 48 nmol min-1 mg-1; this rate was similar to that of mitochondria from control rats (fed a normal diet). When mitochondria from controls were incubated with up to 5mM acetylglutamate, citrulline synthesis by coupled mitochondria was increased by 10 to 40%, while synthesis by uncoupled mitochondria was 1.5 to 4 times higher than that observed with the coupled mitochondria; matrix acetylglutamate in both conditions rose to levels similar to those in the medium. The reason for the different behavior of carbamyl phosphate synthetase (ammonia) in coupled and uncoupled mitochondria was not apparent; neither oxidative phosphorylation nor ornithine transport were limiting in the coupled system. These observations are an example of the restrictions imposed upon enzymatic systems by the conditions existing in the mitochondrial matrix, and of the different behavior of carbamyl phosphate synthetase in situ and in solution. In addition, they show that conclusions about the characteristics of the enzyme in coupled mitochondria based on observations made in uncoupled mitochondria are not necessarily justified.

Measurements of mitochondrial volumes are affected by the amount of mitochondria used in the determinations.

Mitochondrial water spaces were determined by centrifugal filtration, by using 3H2O and [14C]-sucrose, -mannitol, -inulin and -dextran. The volume (in microliter/mg of mitochondrial protein) of each of the spaces was inversely proportional to the amount of mitochondria (mg of protein) centrifuged. The dextran space (representing extramitochondrial water carried down with the mitochondria) decreased the most, and accounted for most of the changes observed in the other spaces. However, the calculated matrix and intermembrane spaces also decreased when increasing amounts of mitochondria were centrifuged. For each space, the same value was obtained when centrifugal filtration was done at 8000 and at 15,600 g, and when the mitochondria were incubated with the markers for 15 s to 5 min, indicating that sucrose, mannitol and inulin do not penetrate the matrix, nor does dextran penetrate the intermembrane space, under the incubation and centrifugation conditions generally used to measure mitochondrial spaces.

Channeling of extramitochondrial ornithine to matrix ornithine transcarbamylase.

In the presence of citrulline synthesis, we made the following observations. External ornithine is channeled between its transporter and ornithine transcarbamylase; mitochondria preloaded with cold ornithine, then incubated with [3H]ornithine, produced citrulline of the same specific radioactivity as that of external ornithine, while matrix ornithine remained essentially unlabeled. The channeling of ornithine suggests that some soluble enzymes are organized within the mitochondrial matrix. The rate of ornithine transport can be greater than 80 nmol/min/mg. At rates of carbamyl phosphate synthesis of 10-50 nmol/min/mg, the rate of citrulline synthesis is controlled by external ornithine in the range 0.03-0.2 mM; at greater than or equal to 0.2 mM ornithine, transport is not limiting for citrulline synthesis. At external ornithine concentrations less than or equal to 1 mM, i.e. within the physiological range, this amino acid is undetectable in the matrix. Given the rates of citrulline and urea synthesis which occur in vivo and the concentrations of ornithine present in the liver, our findings indicate that ornithine may contribute to the physiological regulation of urea synthesis. Preliminary reports of parts of this work have been published (Raijman, L., Cheung, C-W., and Cohen, N. S. (1984) Fed. Proc. 43, 1831; Cohen, N. S., Cheung, C-W., and Raijman, L. (1986) Fed. Proc. 45, 2677).

The effects of ornithine on mitochondrial carbamyl phosphate synthesis.

When carbamyl phosphate is synthesized by isolated liver mitochondria in the absence of ornithine, the following is observed. 1. Carbamyl phosphate synthesis is progressively inhibited during the first 2 min of incubation, after which it reaches a steady rate which is 8% of that observed with ornithine. 2. Within 2 min, carbamyl phosphate accumulates in the matrix to very high levels (16 nmol/microliter) which are inhibitory to carbamyl phosphate synthetase; afterwards the levels decline, and by 15 min they are essentially the same as those found in the presence of ornithine (3 to 4 nmol/microliter). 3. The decrease in matrix carbamyl phosphate is the result of decreased synthesis and of leakage from mitochondria; 90% of the total carbamyl phosphate is in the medium after 10 min. 4. Addition of ornithine after 5 to 15 min results in rates of carbamyl phosphate synthesis essentially identical with those found when ornithine is added at the start of the incubation. 5. ATP synthesis appears to be somewhat inhibited. 6. When the accumulation of carbamyl phosphate in the medium is prevented by converting that compound to carbamyl aspartate, the total synthesis of carbamyl phosphate is not increased. Three factors appears to be involved in the low capacity of mitochondria to synthesize carbamyl phosphate in the absence of ornithine: a. inhibition of carbamyl phosphate synthetase by matrix carbamyl phosphate early in incubations; b. slight inhibition of ATP synthesis throughout the incubations; c. quantitatively most important, a very low activity of carbamyl phosphate synthetase even when matrix carbamyl phosphate is low and ATP is not limiting.

Purification and characteristics of hydrophobic membrane protein(s) required for DCCD sensitivity of ATPase in Mycobacterium phlei.

The energy-transducing N,N'-dicyclohexylcarbodiimide-sensitive (DCCD-sensitive) ATPase complex consists of two parts, a soluble catalytic protein (F1), and an intrinsic membrane protein (F0). The bacterial coupling factor complex, BCF0-BCF1, has recently been purified from Mycobacterium phlei, and used to reconstitute oxidative phosphorylation in detergent-extracted membranes. The BCF0 moiety has been purified by being recovered from the purified BCF0-BCF1 complex by affinity chromatography. BCF0 is a lipoprotein or lipoprotein complex with an approximate molecular weight of 60,000. The preparation contained 0.15 mg of phospholipid per milligram protein. There appear to be three polypeptides, with approximate molecular weights of 24,000, 18,000, and 8,000 as determined by sodium dodecylsulfate acrylamide gel electrophoresis. Purified BCF0 conferred DCCD sensitivity on a purified BCF1 preparation. Reconstitution of oxidative phosphorylation was achieved after incubation of detergent-extracted membranes with purified BCF0 and purified BCF1.

Restoration of oxidative phosphorylation by purified N,N'-dicyclohexylcarbodiimide-sensitive latent adenosinetriphosphatase from Mycobacterium phlei.

The N,N'-dicyclohexylcarbodiimide (DCCD)-sensitive latent adenosinetriphosphatase (ATPase) (EC 3.6.1.3; ATP phosphohydrolase) from Mycobacterium phlei has been purified to homogeneity and used to resotre oxidative phosphorylation to detergent-extracted membranes. The phosphorylation was inhibited by DCCD any by tetraphenylboron and valinomycin. The enzyme was solubilized from the membrane vesicles by treatment with cholate followed by extraction with Triton X-100. After partial purification on a sucrose gradient, the enzyme was purified to homogeneity by affinity chromatography on Sepharose coupled to ADP. The DCCD-sensitive latent ATPase of coupling factor from M. phlei consists of two components, the latent ATPase (Bcf4), which is insensitive to DCCD, and an intrinsic membrane component, BCF0. This hydrophobic portion of the DCCD-sensitive ATPase was partially purified on a sucrose gradient after solubilization with detergents from membrane vesicles that had been first depleted of the BCF4 by washing with 0.25 M sucrose. When BCF0 was combined with purified BCF4, the latent ATPase of the resulting complex was sensitive to DCCD. Moreover, like the purified DCCD-sensitive latent ATPase, the combined BCF4 and BCF0 restored coupled phosphorylation to detergent-extracted membranes.

Channeling of urea cycle intermediates in situ in permeabilized hepatocytes.

Preferential use of endogenously generated intermediates by the enzymes of the urea cycle was observed using isolated rat hepatocytes made permeable to low molecular weight compounds with alpha-toxin. The permeabilized cells synthesized [14C]urea from added NH4Cl, [14C]HCO3-, ornithine, and aspartate, using succinate as a respiratory substrate; with all substrates saturating, about 4 nmol of urea were formed per min/mg dry weight of cells. Urea usually accounted for about 40-50% of the total (NH3 + ornithine)-dependent counts, arginine for less than 10%, and citrulline for about 30%. Very tight channeling of arginine between argininosuccinate lyase and arginase was shown by the fact that the addition of a 200-fold excess of unlabeled arginine to the incubations did not decrease the percentage of counts found in urea or increase that found in arginine, even though a substantial amount of the added arginine was hydrolyzed inside the cells. The channeling of argininosuccinate between its synthetase and lyase was demonstrated by similar observations; unlabeled argininosuccinate added in 200-fold excess decreased the percentage of counts in urea by only 25%. Channeling of citrulline from its site of synthesis by ornithine transcarbamylase in the mitochondrial matrix to argininosuccinate synthetase in the cytoplasmic space was also shown. These results strongly suggest that the three "soluble" cytoplasmic enzymes of the urea cycle are grouped around the mitochondria and are spatially organized within the cell in such a way that intermediates can be efficiently transferred between them.

Altered enzyme activities and citrulline synthesis in liver mitochondria from ornithine carbamoyltransferase-deficient sparse-furash mice.

Male mice carrying the spfash mutation have 5-10% of the normal activity of ornithine carbamoyltransferase, yet are only slightly hyperammonaemic and develop quite well. A study of liver mitochondria from normal and spfash males showed that they differ in important ways. (1) The spfash liver contains about 33% more mitochondrial protein per g than does normal liver. (2) The specific activities of carbamoyl-phosphate synthetase (ammonia) and glutamate dehydrogenase are about 15% lower than normal in mitochondria from spfash mice, whereas those of beta-hydroxybutyrate dehydrogenase and cytochrome oxidase are 22% higher and 30% lower respectively. (3) In the presence of 10 mM-ornithine and the substrates for carbamoyl phosphate synthesis, coupled and uncoupled mitochondria from spfash mice synthesize citrulline at unexpectedly high rates, about 25 and 44 nmol/min per mg respectively. Though these are somewhat lower than the corresponding rates obtained with normal mitochondria, the difference does not arise from the deficiency in ornithine carbamoyltransferase, but from the lower carbamoyl-phosphate synthetase activity of the mutant mitochondria. (4) At lower external [ornithine] (less than 2 mM), a smaller fraction of the carbamoyl phosphate synthesized is converted into citrulline in spfash than in normal mitochondria. These studies show that what appears to be a single mutation brings about major adaptations in the mitochondrial component of liver. In addition, they clarify the role of ornithine transport and of protein-protein interactions in citrulline synthesis in normal mitochondria.

Microclimate monitoring of indoor environments using piezoelectric quartz crystal humidity sensors.

The aim of this paper is to describe indoor microclimate monitoring at two different locations, Sandham Memorial Chapel, in Hampshire, England, and the castle El Alcázar, in Segovia, Spain. Piezoelectric quartz crystal sensors with novel humidity sensitive poly(ethyleneimine) coatings and Pt resistance thermometers were used to measure the relative humidity (RH) and temperature gradients across one of the paintings of the British artist, Stanley Spencer, housed in Sandham Memorial Chapel. The measurement period extended from December 1997 to September 1998. Each coated crystal was set in its own housing from which temperature and RH measuring circuits were connected via a cable to the computer. The 9 month monitoring period incorporated the range in seasons from winter through to autumn. Between December and February the RH at the back of the painting was found to be lower than that at the front. In March and April the reverse was true. Additionally, there were large spikes in the data in some of the months for both RH and temperature, probably caused by direct sunlight falling on the sensors. At the second site monitoring was performed for a shorter period, from December 1997 to early January 1998. It served, however, to show that abrupt changes can occur in the microclimate surrounding the painting. These fluctuations can with time lead to alterations to the paint surface and result eventually in cracking and damage to the art work.

Regulation of urea and citrulline synthesis under physiological conditions.

Information on the regulation of urea synthesis in vivo was obtained by examining the relationship between ureagenesis in vivo, citrulline synthesis in vitro, and two factors currently hypothesized to exert short-term regulation of this pathway: the liver mitochondrial content of N-acetylglutamate (NAG) and substrate availability. Rats meal-fed for 4 h every day (4-20 schedule) or for 8 h every other day (8-40 schedule) were used. (1) The citrulline-synthesizing capacity of mitochondria from livers of rats on the 8-40 schedule exceeded the corresponding velocity of urea synthesis in vivo at all time points studied. (2) Mitochondrial NAG in these livers increased from 127 +/- 32 pmol/mg of protein at 0 h to 486 +/- 205 pmol/mg at 3 h after the start of a meal, and decreased thereafter, but the correlation between NAG content and the velocity of citrulline synthesis was not simple, suggesting that NAG is not the only determinant of the state of activation of carbamoyl phosphate synthase I. (3) In rats on the 4-20 schedule killed 1 h after the start of the meal, the liver content of ornithine, citrulline, arginine, glutamate, alanine and urea increased 2.1-12-fold with respect to the values at 0 h; glutamine decreased by 39%. (4) The combined findings indicate that in vivo, moment-to-moment control of the velocity of urea synthesis is exerted by substrate availability. (5) Digestion limits the supply of substrate to the liver, and prevents its ureagenic capacity from being overwhelmed following a protein-containing meal.

Kinetic properties of carbamoyl-phosphate synthase (ammonia) and ornithine carbamoyltransferase in permeabilized mitochondria.

Previous studies using intact rat liver mitochondria have shown that the soluble matrix enzymes carbamoyl-phosphate synthase (ammonia) (CPS) and ornithine carbamoyltransferase (OCT) display some kinetic properties which would not be observed if they were homogeneously distributed in the matrix. In the present work we have extended these studies, using toluene-treated mitochondria which are fully permeable to substrates and inhibitors, yet retain 90% of their soluble enzymes. The results provide evidence of functional organization of CPS and OCT in situ. The major findings are as follows. (1) The apparent Km values of matrix OCT for carbamoyl phosphate and ornithine are respectively 8 and 2 times those measured for the soluble enzyme. delta-N-Phosphonacetyl-L-ornithine inhibits OCT in situ less than in solution, especially when carbamoyl phosphate is synthesized in the mitochondria rather than added to the medium. (2) During citrulline synthesis from endogenously generated carbamoyl phosphate, the concentration of the latter in permeabilized mitochondria is more than 10 times that in the medium, although the mitochondria are freely permeable to added molecules of this size. (3) Endogenously formed carbamoyl phosphate is used preferentially by OCT in situ; addition of a 200-fold excess of unlabelled carbamoyl phosphate has little effect on the conversion of labelled endogenously formed carbamoyl phosphate into citrulline by matrix OCT. (4) The synthesis de novo of carbamoyl phosphate from NH3, HCO3- and ATPMg is the same in the presence and absence of ornithine. (5) Studies with co-immobilized CPS and OCT gave results concordant with some of the above observations and with previous ones with intact mitochondria.