Exploring Redox Modulation of Plant UDP-Glucose Pyrophosphorylase.

UDP-glucose (UDPG) pyrophosphorylase (UGPase) catalyzes a reversible reaction, producing UDPG, which serves as an essential precursor for hundreds of glycosyltransferases in all organisms. In this study, activities of purified UGPases from sugarcane and barley were found to be reversibly redox modulated in vitro through oxidation by hydrogen peroxide or oxidized glutathione (GSSG) and through reduction by dithiothreitol or glutathione. Generally, while oxidative treatment decreased UGPase activity, a subsequent reduction restored the activity. The oxidized enzyme had increased Km values with substrates, especially pyrophosphate. The increased Km values were also observed, regardless of redox status, for UGPase cysteine mutants (Cys102Ser and Cys99Ser for sugarcane and barley UGPases, respectively). However, activities and substrate affinities (Kms) of sugarcane Cys102Ser mutant, but not barley Cys99Ser, were still prone to redox modulation. The data suggest that plant UGPase is subject to redox control primarily via changes in the redox status of a single cysteine. Other cysteines may also, to some extent, contribute to UGPase redox status, as seen for sugarcane enzymes. The results are discussed with respect to earlier reported details of redox modulation of eukaryotic UGPases and regarding the structure/function properties of these proteins.

Plasminogen Receptors and Fibrinolysis.

The ability of cells to promote plasminogen activation on their surfaces is now well recognized, and several distinct cell surface proteins have been demonstrated to function as plasminogen receptors. Here, we review studies demonstrating that plasminogen bound to cells, in addition to plasminogen directly bound to fibrin, plays a major role in regulating fibrin surveillance. We focus on the ability of specific plasminogen receptors on eukaryotic cells to promote fibrinolysis in the in vivo setting by reviewing data obtained predominantly in murine models. Roles for distinct plasminogen receptors in fibrin surveillance in intravascular fibrinolysis, immune cell recruitment in the inflammatory response, wound healing, and lactational development are discussed.

The plasminogen receptor, Plg-RKT, plays a role in inflammation and fibrinolysis during cutaneous wound healing in mice.

Wound healing is a complex physiologic process that proceeds in overlapping, sequential steps. Plasminogen promotes fibrinolysis and potentiates the inflammatory response during wound healing. We have tested the hypothesis that the novel plasminogen receptor, Plg-RKT, regulates key steps in wound healing. Standardized burn wounds were induced in mice and time dependence of wound closure was quantified. Healing in Plg-RKT-/- mice was significantly delayed during the proliferation phase. Expression of inflammatory cytokines was dysregulated in Plg-RKT-/- wound tissue. Consistent with dysregulated cytokine expression, a significant delay in wound healing during the proliferation phase was observed in mice in which Plg-RKT was specifically deleted in myeloid cells. Following wound closure, the epidermal thickness was less in Plg-RKT-/- wound tissue. Paradoxically, deletion of Plg-RKT, specifically in keratinocytes, significantly accelerated the rate of healing during the proliferation phase. Mechanistically, only two genes were upregulated in Plg-RKT-/- compared with Plg-RKT+/+ wound tissue, filaggrin, and caspase 14. Both filaggrin and caspase 14 promote epidermal differentiation and decrease proliferation, consistent with more rapid wound closure and decreased epidermal thickness during the remodeling phase. Fibrin clearance was significantly impaired in Plg-RKT-/- wound tissue. Genetic reduction of fibrinogen levels to 50% completely abrogated the effect of Plg-RKT deletion on the healing of burn wounds. Remarkably, the effects of Plg-RKT deletion on cytokine expression were modulated by reducing fibrinogen levels. In summary, Plg-RKT is a new regulator participating in different phases of cutaneous burn wound healing, which coordinately plays a role in the interrelated responses of inflammation, keratinocyte migration, and fibrinolysis.

Plasminogen is a master regulator and a potential drug candidate for the healing of radiation wounds.

Around 95% of cancer patients undergoing radiotherapy experience cutaneous side effects, and some develop radiation wounds or fibrosis. Currently, there is no effective treatment for these indications. We show here that plasminogen administration enhanced the healing of radiation wounds via pleiotropic effects on gene expression. Using RNA sequencing, we found that plasminogen downregulated the expression of genes in the TLR, TNF, WNT, MAPK, and TGF-ß signaling pathways, and enhanced the anti-inflammatory effect of arachidonic acid, leading to significantly decreased inflammation and improved remodeling of granulation tissue compared with placebo treatment. In addition, plasminogen induced metabolic changes, including decreased glycolysis. Importantly, many of the factors downregulated by plasminogen are pro-fibrotic. Therefore, in radiation wounds with excessive inflammation, plasminogen is able to enhance and redirect the healing process, such that it more closely resembles physiological healing with significantly reduced risk for developing fibrosis. This makes plasminogen an attractive drug candidate for the treatment of radiation wounds in cancer patients.

Plasminogen activation is required for the development of radiation-induced dermatitis.

Skin damage caused by radiation therapy (radiodermatitis) is a severe side effect of radiotherapy in cancer patients, and there is currently a lack of effective strategies to prevent or treat such skin damage. In this work, we show with several lines of evidence that plasminogen, a pro-inflammatory factor, is key for the development of radiodermatitis. After skin irradiation in wild-type (plg+/+) mice, the plasminogen level increased in the irradiated area, leading to severe skin damage such as ulcer formation. However, plasminogen-deficient (plg-/-) mice and mice lacking plasminogen activators were mostly resistant to radiodermatitis. Moreover, treatment with a plasminogen inhibitor, tranexamic acid, decreased radiodermatitis in plg+/+ mice and prevented radiodermatitis in plg+/- mice. Together with studies at the molecular level, we report that plasmin is required for the induction of inflammation after irradiation that leads to radiodermatitis, and we propose that inhibition of plasminogen activation can be a novel treatment strategy to reduce and prevent the occurrence of radiodermatitis in patients.

Novel diagnostic ELISA test for discrimination between infections with Yersinia enterocolitica and Yersinia pseudotuberculosis.

Yersiniosis is a foodborne infection caused by Yersinia enterocolitica or Yersinia pseudotuberculosis. Although yersiniosis is most often self-limiting, some patients develop chronic infections, such as reactive arthritis, glomerulonephritis, or myocarditis, which require an antibiotic treatment. Whereas early infections can be diagnosed by direct detection of bacteria, chronic infections can only be identified by serological tests. At this point, a serological method for differentiation between infections with the two Yersinia species is important since antibiotic susceptibility of these bacteria is different. Traditional immunoassays do not distinguish between infections with Y. enterocolitica and Y. pseudotuberculosis. The only test that allows for this differentiation is Mikrogen's strip test where discrimination between the two types of infection is based on two recombinant bacterial proteins, MyfA and PsaA (specific for Y. enterocolitica and Y. pseudotuberculosis, respectively). Here, we show that Y. enterocolitica and Y. pseudotuberculosis, cultured under the conditions that mimic the natural rout of infection, express surface antigens different from MyfA and PsaA that can also be used in a discrimination test. Further, we describe a new ELISA that is based on the whole bacteria and recombinant MyfA and PsaA as antigens, and that allows the differentiation between infections with Y. enterocolitica and Y. pseudotuberculosis and simultaneous detection of yersiniosis.

Plasminogen is a critical regulator of cutaneous wound healing.

Wound healing is a complicated biological process that consist of partially overlapping inflammatory, proliferation and tissue remodelling phases. A successful wound healing depends on a proper activation and subsequent termination of the inflammatory phase. The failure to terminate the inflammation halts the completion of wound healing and is a known reason for formation of chronic wounds. Previous studies have shown that wound closure is delayed in plasminogen-deficient mice, and a role for plasminogen in dissection of extracellular matrix was suggested. However, our finding that plasminogen is transported to the wound by inflammatory cells early during the healing process, where it potentiates inflammation, indicates that plasminogen may also have other roles in the wound healing process. Here we report that plasminogen-deficient mice have extensive fibrin and neutrophil depositions in the wounded area long after re-epithelialisation, indicating inefficient debridement and chronic inflammation. Delayed formation of granulation tissue suggests that fibroblast function is impaired in the absence of plasminogen. Therefore, in addition to its role in the activation of inflammation, plasminogen is also crucial for subsequent steps, including resolution of inflammation and activation of the proliferation phase. Importantly, supplementation of plasminogen-deficient mice with human plasminogen leads to a restored healing process that is comparable to that in wild-type mice. Besides of being an activator of the inflammatory phase during wound healing, plasminogen is also required for the subsequent termination of inflammation. Based on these results, we propose that plasminogen may be an important future therapeutic agent for wound treatment.

[Cranial ultrasound through posterolateral fontanelle in visualization of posterior fossa abnormalities in preterm and term neonates]. / Ultrasonografia glowy przez ciemiaczko tylno- boczne w ocenie nieprawidlowosci tylnego dolu czaszki u wczesniaków i noworodk>w donoszonych.

OBJECTIVES: The aim of the study was to evaluate the usefulness of US in the diagnosis of posterior fossa abnormalities in neonates by posterolateral fontanelle as compared with the anterior fontanelle approach and MRI. MATERIAL AND METHODS: US studies were performed on 1337 neonates, including 512 preterm infants, through the anterior and posterolateral fontanelles. Abnormalities were detected in 134 patients. Among them, abnormalities in posterior fossa were visualized with the posterolateral approach in 14 neonates. MR images were obtained in that subgrqup. RESULTS: The lesions consisted of cerebellar hemorrhage and congenital cerebellar malformations. Foci of hemorrhage were visualized by US in preterm neonates (n = 5), only through the posterolateral approach and on MRI. Dandy-Walker malformations (n = 2) were detected by US with both approaches and confirmed on MRI. In pontocerebellar hypoplasia (n =2), US with both approaches, showed hypoplastic cerebellar hemispheres and fluid in the posterior fossa. MRI, additionally visualized pontine hypoplasia. Fluid collection in the posterior fossa and translocation of cerebellar hemispheres were observed in the other 6 neonates by US with both approaches. MRI revealed arachnoid cysts (n = 2), mega cisterna magna (n = 3) and Blake's pouch (n = 1). CONCLUSIONS: US using posterolateral fontanelle is the method of choice for the diagnosis of cerebellar hemorrhage. These lesions are not visualized through anterior fontanelle. US visualization of the abnormal structures in some cerebellar malformations has similar effectiveness for both approaches. MRI plays the crucial role in identification and differential diagnosis of these malformations.

Plasminogen initiates and potentiates the healing of acute and chronic tympanic membrane perforations in mice.

BACKGROUND: Most tympanic membrane (TM) perforations heal spontaneously, but approximately 10-20% remain open as chronic TM perforations. Chronic perforations can lead to an impaired hearing ability and recurrent middle ear infections. Traditionally, these perforations must be surgically closed, which is costly and time consuming. Therefore, there is a need for simpler therapeutic strategies. Previous studies by us have shown that plasminogen (plg) is a potent pro-inflammatory regulator that accelerates cutaneous wound healing in mice. We have also shown that the healing of TM perforations is completely arrested in plg-deficient (plg(-/-)) mice and that these mice develop chronic TM perforations. In the present study, we investigated the therapeutic potential of local plg injection in acute and chronic TM perforation mice models. METHODS: Plg(-/-) mice and wild-type mice were subjected to standardized TM perforations followed by local injection of plg into the soft tissue surrounding the TM. TM perforations with chronic characteristics were induced by leaving TM perforations in plg(-/-) mice untreated for 9 days before treatment. The healing process was observed through otomicroscope and finally confirmed by immunostaining. The quality of TM healing was evaluated based on the morphology of the TM. RESULT: Daily local injections of plg into the soft tissue surrounding the TM restored the ability to heal TM perforations in plg-/- mice in a dose-dependent manner, and potentiated the healing rate and quality in wild-type mice. A single local injection of plg initiated the healing of the chronic-like TM perforations in these mice, resulting in a closed TM with a continuous but rather thick outer keratinocyte layer. However, three plg injections led to a completely healed TM with a thin keratinizing squamous epithelium covering a connective tissue layer. CONCLUSION: Our data suggests that plg is a promising drug candidate for the treatment of chronic TM perforations in humans.

[Ultrasound examination in diagnosis and monitoring of necrotizing enterocolitis in a group of newborns with birth weight below 2000 g--a preliminary report]. / Ultrasonografia w ocenie martwiczego zapalenia jelit u noworodków z urodzeniowa masa ciala ponizej 2000 g--donlesienie wstepne.

OBJECTIVES: Necrotizing enterocolitis (NEC) is a common cause of morbidity in the neonatal care units, especially in cases of preterm neonates with low and very low birth weight. Plain abdominal radiography remains to be the main diagnostic tool in the diagnosis and follow-up of NEC. However; it is sometimes impossible to depict all pathological findings in the radiographs. Furthermore, radiography exposes the youngest, most sensitive patients to consecutive episodes of radiation. Ultrasound examination seems to be an interesting alternative to current standard usage of radiography and its role is still underestimated. The aim of the paper was to assess the applicability of ultrasound examination in the diagnosis and monitoring of neonates suffering from NEC. MATERIAL AND METHODS: The study group consisted of 12 neonates (gestational age 25-36 weeks, weight 540-1900 g), suspected of NEC development. Abdominal radiographs obtained with the use of anterior-posterior and lateral projections, as well as ultrasound examination, were performed. During bowel sonography attention was paid to the presence of intraabdominal fluid, free intraperitoneal gas, bowel wall thickness and bowel wall perfusion. Intramural gas, free intraperitoneal gas and signs of bowel distension were evaluated on the radiographs. RESULTS: Bowel distension was found in all patients. The presence of intraluminal gas was detected in 3 neonates, whereas the signs of bowel perforation were present in only 2 patients. Ultrasound evaluation revealed bowel wall thickening together with increased bowel wall perfusion in 9 patients. Only one neonate presented thinning of the bowel wall, decreased bowel wall perfusion and presence of free intraperitoneal fluid. These findings were connected with a poor outcome of that patient. CONCLUSIONS: Ultrasound examination can be extremely helpful for the initial diagnosis as well as the follow-up of patients developing NEC. It allows to depict the majority of pathological findings for NEC, even those not visible on plain abdominal radiography It is important to emphasize that abdominal sonography (with special reference to the bowel sonography), together with plain abdominal radiography should be considered as standard imaging modalities for the assessment of necrotizing enterocolitis.

Mice deficient in urokinase-type plasminogen activator have delayed healing of tympanic membrane perforations.

Mice deficient in plasminogen, the precursor of plasmin, show completely arrested healing of tympanic membrane (TM) perforations, indicating that plasmin plays an essential role in TM healing. The activation of plasminogen to plasmin is performed by two plasminogen activators (PAs), urokinase-type PA (uPA) and tissue-type PA (tPA). To elucidate the functional roles of PAs in the healing of TM perforations, we investigated the phenotypes of single gene-deficient mice lacking uPA (uPA(-/-)) or tPA (tPA(-/-)) after TM perforation. Delayed healing of TM perforations was observed in uPA(-/-) mice but not tPA(-/-) mice. The migration of keratinocytes was clearly delayed and seemed to be misoriented in uPA(-/-) mice. Furthermore, fibrin deposition and the inflammatory response were persistent in these mice. Our findings demonstrate that uPA plays a role in the healing of TM perforations. The observed phenotypes in uPA(-/-) mice are most likely due to the reduced generation of plasmin.

Plasminogen is a key proinflammatory regulator that accelerates the healing of acute and diabetic wounds.

Despite decades of research on wound healing, effective biologic agents for the treatment of chronic wounds, especially diabetic wounds, are still lacking. In the present study, we report that the inert plasma protein plasminogen (plg) acts as a key regulatory molecule that potentiates wound healing in mice. Early in the healing process, plg bound to inflammatory cells is transported to the wound area, where the level of plg is increased locally, leading to the induction of cytokines and intracellular signaling events and to a potentiation of the early inflammatory response. Systemic administration of additional plg not only accelerates the healing of acute burn wounds in wild-type mice, but also improves the healing of chronic diabetic wounds in a mouse model of diabetes. Our results suggest that the administration of plg may be a novel therapeutic strategy to treat many different types of wounds, especially chronic wounds such as those caused by diabetes.

Substrate kinetics and substrate effects on the quaternary structure of barley UDP-glucose pyrophosphorylase.

UDP-Glc pyrophosphorylase (UGPase) is an essential enzyme responsible for production of UDP-Glc, which is used in hundreds of glycosylation reactions involving addition of Glc to a variety of compounds. In this study, barley UGPase was characterized with respect to effects of its substrates on activity and quaternary structure of the protein. Its K(m) values with Glc-1-P and UTP were 0.33 and 0.25 mM, respectively. Besides using Glc-1-P as a substrate, the enzyme had also considerable activity with Gal-1-P; however, the K(m) for Gal-1-P was very high (>10 mM), rendering this reaction unlikely under physiological conditions. UGPase had a relatively broad pH optimum of 6.5-8.5, regardless of the direction of reaction. The enzyme equilibrium constant was 0.4, suggesting slight preference for the Glc-1-P synthesis direction of the reaction. The quaternary structure of the enzyme, studied by Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA), was affected by addition of either single or both substrates in either direction of the reaction, resulting in a shift from UGPase dimers toward monomers, the active form of the enzyme. The substrate-induced changes in quaternary structure of the enzyme may have a regulatory role to assure maximal activity. Kinetics and factors affecting the oligomerization status of UGPase are discussed.

The relationship of carotid arterial stiffness to left ventricular diastolic dysfunction in untreated hypertension.

BACKGROUND: Interest in the role of arterial stiffness in the pathomechanism of left ventricular (LV) diastolic dysfunction has grown in recent years. AIM: To examine the relationship between local carotid arterial stiffness parameters assessed by the ultrasonic high-resolution echo-tracking (eT) method and LV diastolic function indices in patients with untreated hypertension (H). METHODS: The study group consisted of 173 subjects, 78 male and 95 female, 113 of them with untreated H, mean age 55.7 ± 10.4 years, and 60 age-matched controls. Using 2D echo, conventional and tissue Doppler echocardiography, LV systolic and diastolic function and left ventricular hypertrophy (LVH) indices were assessed. Hypertensives were divided into two groups: those with diastolic dysfunction (HDD+: with relaxation abnormalities, n = 55 and with pseudonormalisation pattern, n = 12); and those without diastolic dysfunction (HDD-, n = 46). Using carotid arteries ultrasound, intima media thickness (IMT) and eT arterial stiffness parameters were evaluated, as also were ß - beta, Ep - epsilon, AC - arterial compliance, PWVß - one-point pulse wave velocity and AI - augmentation index. RESULTS: Linear regression analysis revealed significant correlations between arterial stiffness indices and diastolic function parameters in the study groups: the ratio of early to late transmitral pulse Doppler velocities - E/A - correlated to Ep,ß, AC and PWVß (r = -0.30, r = -0.25, r = 0.26, r = -0.30, respectively, p < 0.05); early diastolic mitral annular velocity - e' - correlated to Ep, ß and PWVß (r = -0.22, r = -0.26, r = -0.25, respectively, p < 0.05); the ratio of early to late diastolic mitral annular velocities - e'/a' - was correlated with ß and PWVß (r = -0.28, r = -0.28, respectively, p < 0.05). HDD+ did not present echocardiographic LVH. Using ROC curve analysis, we identified optimal cut-off values of different parameters in the determination of diastolic dysfunction occurrence. Univariable analysis revealed the following significant variables in determining LV diastolic dysfunction: ß > 9.2 (OR 2.65, p = 0.026), Ep > 118 kPa (OR 3.53, p = 0.040), PWVß > 6.2 m/s (OR 3.92, p = 0.002), AI > 7.8 (OR 2.62, p = 0.049), age > 54 (OR 4.76, p < 0.001), diabetes presence (OR 2.78, p = 0.013), IMT > 0.51 mm (OR 4.49, p < 0.001), diastolic blood pressure < 70 mm Hg (OR 3.38, p = 0.047), pulse pressure > 64 (OR 2,90, p = 0.031) and ejection fraction < 76 (OR 3.38, p = 0.019). However, at multivariate analysis, only age (OR = 2.43, p = 0.073), IMT (OR = 4.56, p = 0.002) and PWVß (OR = 2.18; p = 0.091) were independently associated with diastolic dysfunction occurrence. CONCLUSIONS: Carotid IMT as a marker of subclinical atherosclerosis and PWVß as an index of carotid arterial stiffness are, besides age, independently associated with LV early diastolic dysfunction occurrence in untreated middle-aged hypertensives.

A common structural blueprint for plant UDP-sugar-producing pyrophosphorylases.

Plant pyrophosphorylases that are capable of producing UDP-sugars, key precursors for glycosylation reactions, include UDP-glucose pyrophosphorylases (A- and B-type), UDP-sugar pyrophosphorylase and UDP-N-acetylglucosamine pyrophosphorylase. Although not sharing significant homology at the amino acid sequence level, the proteins share a common structural blueprint. Their structures are characterized by the presence of the Rossmann fold in the central (catalytic) domain linked to enzyme-specific N-terminal and C-terminal domains, which may play regulatory functions. Molecular mobility between these domains plays an important role in substrate binding and catalysis. Evolutionary relationships and the role of (de)oligomerization as a regulatory mechanism are discussed.

Plasmin is essential in preventing periodontitis in mice.

Periodontitis involves bacterial infection, inflammation of the periodontium, degradation of gum tissue, and alveolar bone resorption, which eventually leads to loss of teeth. To study the role of the broad-spectrum protease plasmin in periodontitis, we examined the oral health of plasminogen (Plg)-deficient mice. In wild-type mice, the periodontium was unaffected at all time points studied; in Plg-deficient mice, periodontitis progressed rapidly, within 20 weeks. Morphological study results of Plg-deficient mice revealed detachment of gingival tissue, resorption of the cementum layer, formation of necrotic tissue, and severe alveolar bone degradation. IHC staining showed massive infiltration of neutrophils in the periodontal tissues. Interestingly, doubly deficient mice, lacking both tissue- and urokinase-type plasminogen activators, developed periodontal disease similar to that in Plg-deficient mice; however, mice lacking only tissue- or urokinase-type plasminogen activator remained healthy. Supplementation by injection of Plg-deficient mice with human plasminogen for 10 days led to necrotic tissue absorption, inflammation subsidence, and full regeneration of gum tissues. Notably, there was also partial regrowth of degraded alveolar bone. Taken together, our results show that plasminogen is essential for the maintenance of a healthy periodontium and plays an important role in combating the spontaneous development of chronic periodontitis. Moreover, reversal to healthy status after supplementation of Plg-deficient mice with plasminogen suggests the possibility of using plasminogen for therapy of periodontal diseases.

Bomapin is a redox-sensitive nuclear serpin that affects responsiveness of myeloid progenitor cells to growth environment.

BACKGROUND: Haematopoiesis is a process of formation of mature blood cells from hematopoietic progenitors in bone marrow. Haematopoietic progenitors are stimulated by growth factors and cytokines to proliferate and differentiate, and they die via apoptosis when these factors are depleted. An aberrant response to growth environment may lead to haematological disorders. Bomapin (serpinb10) is a hematopoietic- and myeloid leukaemia-specific protease inhibitor with unknown function. RESULTS: We found that the majority of naturally expressed bomapin was located in the nucleus. Both the natural and recombinant bomapin had a disulfide bond which linked the only two bomapin cysteines: one located in the CD-loop and the other near the C-terminus. Computer modelling showed that the cysteines are distant in the reduced bomapin, but can easily be disulfide-linked without distortion of the overall bomapin structure. Low-level ectopic expression of bomapin in bomapin-deficient K562 cells resulted in about 90% increased cell proliferation under normal growth conditions. On the other hand, antisense-downregulation of natural bomapin in U937 cells resulted in a decreased cell proliferation. Bomapin C395S mutant, representing the reduced form of the serpin, had no effect on cell proliferation, suggesting that the disulfide bond-linked conformation of bomapin is biologically important. The bomapin-dependent effect was specific for myeloid cells, since ectopic expression of the serpin in HT1080 cells did not change cell proliferation. In contrast to the survival-promoting activity of bomapin in cells cultured under optimal growth conditions, bomapin enhanced cell apoptosis following growth factor withdrawal. CONCLUSIONS: We propose that bomapin is a redox-sensitive nuclear serpin that augments proliferation or apoptosis of leukaemia cells, depending on growth factors availability.

Domain-specific determinants of catalysis/substrate binding and the oligomerization status of barley UDP-glucose pyrophosphorylase.

UDP-glucose (UDPG) pyrophosphorylase (UGPase) produces UDPG for sucrose and polysaccharide synthesis and glycosylation reactions. In this study, several barley UGPase mutants were produced, either single amino acid mutants or involving deletions of N- and C-terminal domains (Ncut and Ccut mutants, respectively) and of active site region ("NB loop"). The Del-NB mutant yielded no activity, whereas Ncut deletions and most of Ccut mutants, including short deletions at the so called "I-loop" region of C-terminal domain, as well as a single K260A mutant resulted in very low activity. For wt and the mutants, kinetics with UDPG were linear on reciprocal plots, whereas PPi at concentrations above 1 mM exerted strong substrate inhibition. Both K260A and most of the Ccut mutants had very high Km with PPi (up to 33 mM), whereas Ncut deletions had greatly increased Km with UDPG (up to 57 mM). Surprisingly, an 8 amino acid deletion from end of the C-terminus resulted in an enzyme (Ccut-8 mutant) with 44% higher activity when compared to wt, but with similar Km values. Whereas Ccut-8 existed solely as a monomer, other deletion mutants had a more oligomerized status, e.g. Ncut mutants existing primarily as dimers. Overall, the data confirmed the essential role of NB loop in catalysis, but also pointed out to the role of both N- and C-termini for activity, substrate binding and oligomerization. The importance of oligomerization status for enzymatic activity of UGPase is discussed.

Molecular and kinetic characterization of two UDP-glucose pyrophosphorylases, products of distinct genes, from Arabidopsis.

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme in the production (and conversions) of UDP-glucose, a key precursor for carbohydrate biosynthesis. cDNAs corresponding to two UGPase isozymes in Arabidopsis were overexpressed in Escherichia coli and, subsequently, the recombinant proteins were purified and characterized. Both proteins were highly conserved, sharing 93% identity. Based on crystal structure-derived images, the main amino acid differences mapped to N- and C-termini domains, but not to central active site region. The two proteins existed mainly as monomers, and they had similar molecular masses of ca. 53 kDa. However, comparison of molecular masses of UGPases from Arabidopsis root and leaf extracts revealed that the root protein was slightly larger, suggesting a post-translational modification. Specific activity of the purified UGPase-1 was ca. 10-30% lower than that of UGPase-2, depending on direction of the reaction, whereas its K(m) values with all substrates in both directions of the reaction were consistently ca. twice lower than those of UGPase-2 (0.03-0.14 mM vs. 0.07-0.36 mM, respectively). Both proteins were "true" UGPases, and had no activity with ADP-glucose/ATP or galactose-1-P. Equilibrium constant for both proteins was ca. 0.3, suggesting preference for the pyrophosphorolysis direction of the reaction. The data are discussed with respect to potential roles of UGPase in carbohydrate synthesis/metabolism in Arabidopsis.