Ornithine cycle in Nostoc PCC 73102. Occurrence and localization of ornithine carbamoyl transferase, and the effects of external carbon and ornithine on nitrogenase activity and citrulline synthesis

Cells of free-living nitrogen-fixing Nostoc PCC 73102, a filamentous heterocystous cyanobacterium originally isolated from coralloid roots of the cycad Macrozamia. were examined for the presence of ornithine carbamoyl transferase (OCT) by native-PAGE/in situ activity stain, and SDS-PAGE/Western immunoblots. Transmission electron microscopy and immunocytological labeling were used to study the cellular and subcellular distribution of OCT in the Nostoc cells. Moreover, the effects of photoautotrophic and dark heterotrophic growth metabolism on growth, nitrogenase activity and in vivo citrulline synthesis were investigated. PAGE in combination with in situ activity staining demonstrated an in vitro active OCT with a molecular weight of approximately 80 kDa. SDS-PAGE/Western immunoblots revealed that a polypeptide with a molecular weight of approximately 38 kDa was immunologically related to OCT purified from pea (Pisum sativum L. cv. Alaska). Immunolocalization demonstrated that the OCT protein was located both in vegetative cells and heterocysts. Using the particle analysis of an image processor, the labeling associated with the photosynthetic vegetative cells was calculated to be 75.6 (± 5.5) gold particles μm^?2 compared with 62.0 (± 7.5) in the nitrogen-fixing heterocysts. Glucose and fructose stimulated both cyanobacterial growth and nitrogenase activity in light and darkness. Addition of exogenous ornithine decreased nitrogenase activity. In light grown cells, additions of glucose and fructose in combination with ornithine not only stimulated growth and nitrogenase activity but also in vivo citrulline synthesis, measured as ¹⁴CO₂-fixation into [¹⁴C]-citrulline. In darkness no stimulation was observed on in vivo citrulline synthesis. The substantial stimulation of nitrogenase activity by additions of external glucose and fructose, both in the light and in darkness, was not followed by a simultaneous stimulation of in vivo citrulline synthesis.     

Ornithine cycle in Nostoc PCC 73102. Arginase,OCT and arginine deiminase,and the effects of addition of external arginine,ornithine, or citrulline

Arginase, ornithine carbamoyl transferase (OCT) and arginine deiminase activities were found in cell-free extracts of Nostoc PCC 73102, a free-living cyanobacterium originally isolated from the cycad Macrozamia. Addition of either arginine, ornithine or citrulline to the growth medium induced significant changes in their in vitro activities. Moreover, growth in darkness, compared to in light, induced higher in vitro activities. The in vitro activities of arginase and arginine deiminase, two catabolic enzymes primarily involved in the breakdown of arginine, increased substantially by a combination of growth in darkness and addition of either arginine, or ornithine, to the growth medium. The most significant effects on the in vitro OCT activities where observed in cells grown with the addition of ornithine. Cells grown in darkness exhibited about 6% of the in vivo nitrogenase activity observed in cells grown in light. However, addition of external carbon (glucose and fructose) to cells grown in darkness resulted in in vivo nitrogenase activity levels similar to, or even higher than, cells grown in light. Growth with high in vivo nitrogenase activity or in darkness with the addition of external carbon, resulted in repressed levels of in vitro arginase and arginine deiminase activities. It is suggested that nitrogen starvation induces a mobilization of the stored nitrogen, internal release of the amino compound arginine, and an induction of two catabolic enzymes arginase and arginine deiminase. A similar and even more pronunced induction can be observed by addition of external arginine to the growth medium.     

Stimulation of testicular ornithine decarboxylase activity by arginine vasopressin

Intratesticular injection with arginine vasopressin caused stimulation of ornithine decarboxylase activity in the testes of immature rats. The increase in ornithine decarboxylase activity in response to arginine vasopressin was dose and time dependent. Maximal stimulation of ornithine decarboxylase activity occurred at 2 h after injection with 0.1 micrograms of arginine vasopressin. It was observed that stimulation of ornithine decarboxylase activity occurred in seminiferous tubules and in Leydig cells of the testis in response to arginine vasopressin.     

Spontaneous and 5-azacytidine-induced reexpression of ornithine carbamoyl transferase in hepatoma cells.

Rat hepatoma cells that do not synthesize the hepatic enzyme ornithine carbamoyl transferase spontaneously give rise to producing cells at a low frequency. Reexpression of this differentiation trait is strongly increased by 5-azacytidine treatment, suggesting that hypermethylation plays a critical role in the impaired expression of the ornithine carbamoyl transferase gene in hepatoma cells.     

Expression of nuclear genes encoding the urea cycle enzymes, carbamoyl-phosphate synthetase I and ornithine carbamoyl transferase, in rat liver and intestinal mucosa

RNA dot-blot, quantitative electron microscope immunocytochemistry, and electrophoretic immunoblotting techniques were employed to investigate the expression of carbamoyl-phosphate synthetase I (CPS) and ornithine carbamoyl transferase (OCT) genes in rat liver and intestinal mucosa. Comparing only those cell types in the two tissues which express these enzymes, we show that the concentration of CPS and OCT in hepatocyte mitochondria is 2.3-times and 1.2-times greater, respectively, than in intestinal epithelial cell mitochondria. As a percentage of total tissue protein, however, liver homogenates contain 10-20 times more CPS and 5-10 times more OCT than is found in intestinal mucosa. These relatively large differences in enzyme protein levels between the two tissues are not reflected by differences in their mRNA levels. As a percentage of total translational activity in vitro (based on incorporation of [35S]methionine), total liver mRNA directed synthesis of about twice as much precursor CPS (pCPS) and precursor OCT (pOCT) than did equivalent amounts of mRNA from intestinal mucosa. The ratio of pCPS and pOCT mRNA levels between the two tissues (2:1, liver:intestinal mucosa) was confirmed by dot-blot and Northern hybridizations employing specific cDNA probes. The sizes of the respective mRNAs were the same for the two tissues: about 6000 residues for pCPS mRNA and about 1700 residues for pOCT mRNA.     

Differential methylation of the ornithine carbamoyl transferase gene on active and inactive mouse X chromosomes.

Ornithine carbamoyl transferase (Oct) is an X-linked gene which exhibits tissue-specific expression. To determine whether methylation of specific CpG sequences plays a role in dosage compensation or tissue-specific expression of the gene, 13 potentially methylatable sites were identified over a 30-kilobase (kb) region spanning from approximately 15 kb upstream to beyond exon II. Fragments of the Mus hortulanus Oct gene were used as probes to establish the degree of methylation at each site. By considering the methylation status in liver (expressing tissue) versus kidney (nonexpressing tissue) from male and female mice, the active and inactive genes could be investigated on active and inactive X-chromosome backgrounds. One MspI site, 12 kb 5' of the Oct-coding region, was cleaved by HpaII in liver DNA from males but not in kidney DNA from males and thus exhibited complete correlation with tissue-specific expression of the gene. Six other sites showed partial methylation, reflecting incomplete correlation with tissue-specific expression.     

Stimulation of ornithine decarboxylase by relaxin

Relaxin, a protein hormone of pregnancy, stimulated ornithine decarboxylase activity (EC 4.1.1.17) in two of its target tissues. Both the mouse public symphysis and uterus respond to a single injection of relaxin; within 2–4 hours after hormonal treatment of the mice, ornithine decarboxylase activity was observed to increase 2–8 fold over control levels. This increase in enzymatic activity may represent one step in the mechanism by which relaxin exerts its effects.     

Mouse ornithine decarboxylase-like gene encodes an antizyme inhibitor devoid of ornithine and arginine decarboxylating activity

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is a labile protein that is regulated by interacting with antizymes (AZs), a family of polyamine-induced proteins. Recently, a novel human gene highly homologous to ODC, termed ODC-like or ODC-paralogue (ODCp), was cloned, but the studies aimed to determine its function rendered contradictory results. We have cloned the mouse orthologue of human ODCp and studied its expression and possible function. mRNA of mouse Odcp was found in the brain and testes, showing a conserved expression pattern with regard to the human gene. Transfection of mouse Odcp in HEK 293T cells elicited an increase in ODC activity, but no signs of arginine decarboxylase activity were evident. On the other hand, whereas the ODCp protein was mainly localized in the mitochondrial/membrane fraction, ODC activity was found in the cytosolic fraction and was markedly decreased by small interfering RNA against human ODC. Co-transfection experiments with combinations of Odc, Az1, Az2, Az3, antizyme inhibitor (Azi), and Odcp genes showed that ODCp mimics the action of AZI, rescuing ODC from the effects of AZs and prevented ODC degradation by the proteasome. A direct interaction between ODCp and AZs was detected by immunoprecipitation experiments. We conclude that mouse ODCp has no intrinsic decarboxylase activity, but it acts as a novel antizyme inhibitory protein (AZI2).     

Plant-type and bacterial-type ferredoxins in a nitrogen-fixing cyanobacterium: Nostoc sp. strain PCC 73102

Abstract The cyanobacterium Nostoc sp. strain PCC 73102, cultured under nitrogen-fixing conditions, was investigated for the occurrence of ferrodoxins by SDS-PAGE/Western immunoblots using antisera directed against both a major plant-type and a bacterial-type ferredoxin purified from Anabaena variabilis . Immunocytological labelling and transmission electron microscopy were used to study the distribution of both types of ferredoxins in the Nostoc cells. SDS-PAGE/Western immunoblots revealed two proteins/polypeptides in the Nostoc strain, immunologically related to two soluble ferredoxins purified from Anabaena variabilis : the major plant-type ferredoxin (Fd I) and a bacterial-type ferredoxin (Fd III). Immunolocalization showed a uniform distribution of the plant-type and the bacterial-type ferredoxin in both the photosynthetic vegetative cells and in the nitrogen-fixing heterocysts, with no specific association with any subcellular inclusions. Using the particle analysis of an image processor, the labelling associated with the vegetative cells, expressed as number of gold particles per cell area, was found to be only slightly higher (1.2x) or almost twice as high (1.9x) compared to the heterocysts for the major plant-type and the bacterial-type ferredoxin, respectively.     

Control of a futile urea cycle by arginine feedback inhibition of ornithine carbamoyltransferase in Agrobacterium tumefaciens and Rhizobia

In Agrobacterium tumefaciens and Rhizobia arginine can be used as the sole nitrogenous nutrient via degradation by an inducible arginase. These microorganisms were found to exhibit arginine inhibition of ornithine carbamoyltransferase activity. This inhibition is competitive with respect to ornithine (Km for ornithine = 0.8 mM; Ki for arginine = 0.05 mM). This type of urea cycle regulation has not been observed among other microorganisms which degrade arginine via an arginase. The competitive pattern of this inhibition leads to its being inoperative in ornithine-grown cells, where the intracellular concentration of ornithine is high. In arginine-grown cells, however, the intracellular arginine and ornithine concentrations are compatible with inhibition and ornithine recycling appears to be effectively blocked in vivo.