Natalie S. Cohen

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.

[22] Properties of energy-transducing systems in different types of membrane preparations from Mycobacterium phlei-preparation, resolution, and reconstitution

Publisher Summary Many bacterial systems have been used to study membrane-related phenomena, such as oxidative phosphorylation, electron transport, active transport of solutes, and membrane topology. However, with most systems, little is known about the nature and composition of membrane components or the vectorial orientation of the membrane structure. In contrast, the membrane structures obtained from Mycobacterium phlei have been characterized with regard to the nature of the respiratory chains, the sequence of electron-transport carriers, sites of phosphorylation, membrane orientation, and active transport of metabolites. The chapter reviews number of types of membrane structures and membrane-associated components that have been resolved from whole cells of M . phlei . The different membrane structures appear to contain similar respiratory chains but differ in size and orientation of the membranes. The different types of membrane structures differ in their ability to carry out oxidative phosphorylation or active transport of amino acids. Thus, a meaningful comparison of these membrane-associated processes with the different types of membrane structures is useful in providing some insight concerning the mechanisms of energy transduction underlying these two processes. Protoplast ghosts obtained from M . phlei are about the same size as whole cells, are largely intact, and represent a membrane population that is oriented right-side-out. Sonication of the ghosts or whole cells results in the formation of membrane vesicles, which are referred to as “electron-transport particles” (ETP). The ETP are chiefly oriented inside-out is not a homogeneous population of vesicles because they are contaminated with a small population of membrane vesicles that are oriented right-side-out.

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/10 nm 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.

Multiple cytochromes b in Mycobacterium phlei

Abstract Electron transport particles from M. phlei contain at least 3 different active forms of cytochrome b, one reduced by NADH, with a λmax at 563 nm (bN563), and the other two reduced by either succinate or NADH, with λmax at 559 and 563 nm (bS559) and (bS563). Low temperature λmax for cytochrome b reduction with NADH or succinate are described. During steady state only bS563 was observed with succinate. In the presence of ATP, succinate reduced an increased amount of a b563. A branching of the NAD+-linked pathway and a convergence at the level of cytochrome c is suggested, with only one branch accessible to succinate.

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.

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.

Metabolite Channelling and Protein—Protein Interactions in the Urea Synthesis Pathway

The pathway of urea synthesis of mammalian liver has proved to be an excellent model for the investigation of the intracellular organization of soluble enzymes. Studies of the behaviour and regulation of the enzymes of this pathway in situ (reviewed by Cohen et al., 1997) have conclusively shown that the latter operates as a tightly organized system, referred to by Srere (1987), as a metabolon. Intermediates are retained within the pathway and are channelled between sequential enzymes at every step, even across the mitochondrial membranes. By channelled we mean that the intermediates do not mix freely throughout the bulk aqueous phase of the cell.