Effects of medium-chain fatty acids and tributyrin supplementation in milk replacers on growth performance, blood metabolites, and hormone concentrations in Holstein dairy calves.

This study aimed to evaluate the effects of triglycerides containing medium-chain fatty acids (MCT) and tributyrin (TB) supplementation in a milk replacer (MR) on growth performance, plasma metabolites, and hormone concentrations in dairy calves. Sixty-three Holstein heifer calves (body weight at 8 d of age, 41.1 ± 2.91 kg; mean ± SD) were randomly assigned to 1 of 4 experimental MR (28% crude protein and 18% fat): (1) containing 3.2% C8:0 and 2.8% C10:0 (in fat basis) without TB supplementation (CONT; n = 15), (2) containing 6.7% C8:0 and 6.4% C10:0 without TB supplementation (MCT; n = 16), (3) containing 3.2% C8:0 and 2.8% C10:0 with 0.6% (dry matter basis) TB supplementation (CONT+TB; n = 16), (4) containing 6.7% C8:0 and 6.4% C10:0 with 0.6% TB supplementation (MCT+TB; n = 16). The MR were offered at 600 g/d (powder basis) from 8 to 14 d, up to 1,300 g/d from 15 to 21 d, 1,400 g/d from 22 to 49 d, down to 700 g/d from 50 to 56 d, 600 g/d from 57 to 63 d, and weaned at 64 d of age. All calves were fed calf starter, chopped hay, and water ad libitum. The data were analyzed using a 2-way ANOVA via the fit model procedure of JMP Pro 16 (SAS Institute Inc.). Medium-chain fatty acid supplementation did not affect the total dry matter intake. However, calves that were fed MCT had greater feed efficiency (gain/feed) before weaning (0.74 ± 0.098 vs. 0.71 ± 0.010 kg/kg) compared with non-MCT calves. The MCT calves also had a lower incidence of diarrhea compared with non-MCT calves during 23 to 49 d of age and the weaning period (50 to 63 d of age; 9.2% vs. 18.5% and 10.5% vs. 17.2%, respectively). Calves fed with TB had a greater total dry matter intake during postweaning (3,465 vs. 3,232 g/d). Calves fed TB also had greater body weight during the weaning (90.7 ± 0.97 vs. 87.9 ± 1.01 kg) and postweaning period (116.5 ± 1.47 vs. 112.1 ± 1.50 kg) compared with that of non-TB calves. The plasma metabolites and hormone concentrations were not affected by MCT or TB. These results suggest that MCT and TB supplementation in the MR may improve the growth performance and gut health of dairy calves.

A randomized controlled trial of hypnosis compared with biofeedback for adults with chronic low back pain.

BACKGROUND: Chronic low back pain (CLBP) is common and results in significant costs to individuals, families and society. Although some research supports the efficacy of hypnosis for CLBP, we know little about the minimum dose needed to produce meaningful benefits, the roles of home practice and hypnotizability on outcome, or the maintenance of treatment benefits beyond 3 months. METHODS: One hundred veterans with CLBP participated in a randomized, four-group design study. The groups were (1) an eight-session self-hypnosis training intervention without audio recordings for home practice; (2) an eight-session self-hypnosis training intervention with recordings; (3) a two-session self-hypnosis training intervention with recordings and brief weekly reminder telephone calls; and (4) an eight-session active (biofeedback) control intervention. RESULTS: Participants in all four groups reported significant pre- to post-treatment improvements in pain intensity, pain interference and sleep quality. The hypnosis groups combined reported significantly more pain intensity reduction than the control group. There was no significant difference among the three hypnosis conditions. Over half of the participants who received hypnosis reported clinically meaningful (≥ 30%) reductions in pain intensity, and they maintained these benefits for at least 6 months after treatment. Neither hypnotizability nor amount of home practice was associated significantly with treatment outcome. CONCLUSIONS: The findings indicate that two sessions of self-hypnosis training with audio recordings for home practice may be as effective as eight sessions of hypnosis treatment. If replicated in other patient samples, the findings have important implications for the application of hypnosis treatment for chronic pain management.

Obsessive-compulsive behavior as a symptom of dementia in progressive supranuclear palsy.

AIMS: To describe obsessive-compulsive symptoms (OCS) as under-recognized behavioral and psychological symptoms of dementia of progressive supranuclear palsy (PSP) and to discuss possible mechanisms based on MRI and SPECT findings. METHODS: We studied 74 PSP patients. OCS are defined as persistent and unreasonable, but non-delusional/hallucinatory, ideas and behaviors. Demography, cognition, the widths of middle cerebellar peduncles (MCP) and the inter-caudate distances (ICD), both corrected by the intracranial size (MCP and ICD ratios), and changes on voxel-based SPECT were compared between the subgroups with and without OCS. Finally, the predicative power of various factors to OCS was investigated. RESULTS: We observed OCS in 18 patients (24%). They were obsessed with daily trifles and physical symptoms among other things. OCS was not associated with demography or cognitive levels. OCS-positive patients had significantly smaller MCP and ICD ratios and showed marked uptake decreases in the orbitofrontal cortex, caudate and thalamus. Relative uptake increases in the cerebellum, specifically the tonsils, were milder in OCS-positive than -negative patients. A smaller right MCP, a smaller ICD ratio and lower uptake increases in the right cerebellar were the significant predictors of OCS. CONCLUSIONS: OCS are frequent but under-recognized behavioral and psychological symptoms of dementia in PSP. Dysfunction of the fronto-caudate-thalamus-cerebellum circuit may be involved.

Dysmenorrhea among Japanese women.

OBJECTIVE: To investigate the epidemiology of dysmenorrhea in Japanese women of menstrual age. METHODS: A prospective cohort study was conducted using a health diary in a sample representative of Japanese women. Information on health care use was also collected. RESULTS: Among 823 enrolled participants (age range, 18-51 years), dysmenorrhea (mean duration 1.75 days; range 1-5 days) was reported in 15.8% (95% CI, 13.3-18.3) during the 1-month study period. Common associated symptoms included headache (10.77%), back pain (6.92%), and fatigue (5.38%). No participant with dysmenorrhea visited a physician, while 51.5% of the women used self-medication, and 7.7% used complementary/alternative medicine. CONCLUSION: Dysmenorrhea is common in Japanese women. In our study, about half used self-medication, while some preferred complementary/alternative medicine. Dysmenorrhea is significantly associated with younger age and employment status.

Association between patient education and health-related quality of life in patients with Parkinson's disease.

BACKGROUND: Providing patients with disease- and treatment-related information is an important role of medical staff and is now reimbursed in Japan by the national health insurance system under the rubric 'patient education'. Evaluation of the effectiveness of patient education programs is necessary to ensure that limited health care resources are used efficiently. OBJECTIVE: The objective is to determine whether educating patients with Parkinson's disease (PD) is related to better health-related quality of life (HRQOL). DESIGN: A cross-sectional study was conducted. SETTING: Members of the Japan Association of Patients with Parkinson's disease were randomly selected. PARTICIPANTS: A total of 1200 patients with PD were asked to fill in written questionnaires and replies from 762 (63.5%) were analyzed. MEASUREMENTS: The questionnaire inquired about clinical characteristics, comorbidity, symptoms of PD, complications of therapy, HRQOL, and patient education. SF-36 was used to assess HRQOL. The section on patient education comprised one question each on patient-perceived satisfaction with information provided on (1) disease condition and pathophysiology, (2) effectiveness of drug therapy, (3) adverse drug reactions, (4) publicly available financial and social resources, and (5) rehabilitation and daily activities. Patient education score was defined as the sum of the individual scores for these five questions. The relationships between scores on the SF-36 subscales and the patient education score were examined. RESULTS: More satisfaction with patient education was associated with higher scores in all SF-36 subscales except physical functioning and bodily pain. The difference in score between the most satisfied and the least satisfied patients ranged from 8.4 points on the subscales of general health and 16.7 points on the subscale of role limitation due to emotional problems. CONCLUSION: The conclusion that patient education is associated with better HRQOL in patients with PD is drawn.

Normalization of low biotinidase activity in a child with biotin deficiency after biotin supplementation.

We report findings in a Japanese boy with severe skin rash attributable to biotin deficiency. The patient had an intracranial malformation and developed biotin deficiency due to tube feeding with a single formula for over one year. Results of urinary organic acid analysis were consistent with multiple carboxylase deficiency, and low biotinidase activity was also observed. After biotin supplementation, the skin rash improved and biotinidase activity normalized. We speculate that biotin is one regulating factor in the biosynthesis of biotinidase.

A new quantitative analytical method of serum biotinidase activity using biocytin as a substrate and its clinical significance in Japan.

We have developed a new quantitative analytical method of serum biotinidase activity, which uses the native substrate biocytin, and to which can be applied the improved agar plate method of biotin bioassay. Assay characteristics were within acceptable ranges (intra-assay CVs, 4.44% and 1.95% at 1.82+/-0.08 and 3.08+/-0.06 pmol/min/ml; day-to-day CV, 5.92% at 2.68+/-0.16 pmol/min/ml). The enzyme activity with biocytin was stable at 4 degrees C for 90 days. The mean value of the serum biotinidase levels in 129 healthy adults was 2.71+/-0.93 pmol/min/ml. The method was clinically comparable with a colorimetric method for detection of biotinidase deficiency. Biotin supplementation treatment normalized our partial biotinidase deficiency patient's serum biotinidase activity. This normalized phenomenon has not yet been observed in a Caucasian patient. We also found that the distribution of the enzyme activities with biotinyl-p-aminobenzoate in 8 of 11 patients with suspected biotin metabolic disorders shifted to a higher level than that of the controls. Although, we have few opportunities to analyze the sera of biotin metabolic disorders in Japan, the new method are suitable for clinical research applications in combination with the colorimetric method.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant bacteria expressing the PHA synthase gene phaC1 from Pseudomonas sp. 61-3.

Pseudomonas sp. 61-3 accumulated a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer consisting of 3-hydroxyalkanoate (3HA) units of 4-12 carbon atoms. The genes encoding beta-ketothiolase (PhbA(Re)) and NADPH-dependent acetoacetyl-CoA reductase (PhbB(Re)) from Ralstonia eutropha were expressed under the control of promoters for Pseudomonas sp. 61-3 pha locus or R. eutropha phb operon together with phaC1(Ps) gene (PHA synthase 1 gene) from Pseudomonas sp. 61-3 in PHA-negative mutants P. putida GPp104 and R. eutropha PHB(-4) to produce copolyesters [P(3HB-co-3HA)] consisting of 3HB and medium-chain-length 3HA units of 6-12 carbon atoms. The introduction of the three genes into GPp104 strain conferred the ability to synthesize P(3HB-co-3HA) with relatively high 3HB compositions (up to 49 mol%) from gluconate and alkanoates, although 3HB units were not incorporated at all or at a very low fraction (3 mol%) into copolyesters by the strain carrying phaC1Ps gene only. In addition, recombinant strains of R. eutropha PHB(-4) produced P(3HB-co-3HA) with higher 3HB fractions from alkanoates and plant oils than those from recombinant GPp104 strains. One of the recombinant strains, R. eutropha PHB(-4)/ pJKSc46-pha, in which all the genes introduced were expressed under the control of the native promoter for Pseudomonas sp. 61-3 pha locus, accumulated P(3HB-co-3HA) copolyester with a very high 3HB fraction (85 mol%) from palm oil. The nuclear magnetic resonance analyses showed that the copolyesters obtained here were random copolymers of 3HB and 3HA units.

Bidi smoking and health.

Bidi, made of low-grade tobacco, is popular among rural folk and the urban poor, in the South Asian region. The objectives of this study were: (1) to assess the extent of bidi smoking in different countries; (2) to compare its smoke and health hazards with conventional cigarettes and; (3) to recommend preventive measures. English language articles were identified through a MEDLINE computerised search (1966-1998) using the term 'bidi', 'beedi', 'beedie', 'biri' and manually in order to collate all the available information and to present a descriptive epidemiological profile. The prevalence of bidi smoking was found to be 21-56% among men in South Asian countries with very little information available about women. Concentrations of nicotine, tar and other toxic agents in the smoke are higher for bidi than for other cigarettes. Bidi smoking is also considered to cause about 2-3 times greater nicotine and tar inhalation than do conventional cigarettes, due to the poor combustibility of the bidi wrapper and greater puff frequency needed to keep the bidi alight. It has also been shown to have greater odds ratios, although not statistically significant, for various cancerous and chronic conditions in comparison with other cigarettes. Since bidi is hand-rolled, workers employed in bidi factories are at risk of developing cancerous conditions due to exposure to tobacco dust and flakes. We suggest that anti-smoking campaigns should be directed to the bidi smokers of all South Asian countries. Most bidi smokers are illiterate and malnourished which makes them more vulnerable to smoking-related morbidity and mortality. At the same time, measures should be taken to make bidi less harmful and to produce bidi by machine to reduce exposure-related toxicity among bidi production workers.

Effect of freeze-dried soybean curd (tofu) on various bodily functions.

The present study was designed to examine the effect of freeze-dried soybean curd (tofu) on various bodily functions. The dietary experiment consisted of 4 days of a non-prescribed ordinary diet, 10 days of an experimental diet that contained 190 g of meat contributing about 38 g of protein (hereinafter referred to as the "meat period"), and 39 days of an experimental diet that contributed vegetable protein, i.e., freeze-dried tofu, corresponding to the protein that 190 g of meat would provide (hereinafter referred to as the "freeze-dried tofu period"). Three eggs (about 150 g) and 180 ml of cow's milk were prescribed for the daily diet, and staple and other foods were dispensed freely during both the meat and the freeze-dried tofu periods. The results showed that serum cholesterol levels and diastolic blood pressure significantly decreased in the freeze-dried tofu period as compared with the meat period. Freeze-dried tofu was found to be a valuable food for preventing lifestyle-related chronic diseases.

Low biotin content of infant formulas made in Japan.

Since minor vitamins such as biotin are not yet registered as food additives in Japan, these cannot be used to supplement infant formulas or foods, despite their nutritional value. Therefore, the biotin contents of infant formulas made in Japan were determined and compared with those from the United States (US). The Japanese products were obtained from five companies, while the US products were limited to those from one company. The average content of total biotin in 11 Japanese products for nursing of normal infants (standard formulas) was 1.04 micrograms/100 kcal (0.69 microgram/100 ml), which was significantly lower than that in two US products (2.56 micrograms/100 kcal, 1.71 micrograms/100 ml). The total biotin content in 26 Japanese infant formulas specially prepared for medical treatment and prevention of disease (special formulas) was 0.45 microgram/100 kcal (0.30 microgram/100 ml) on average. This was less than a fifth of the level in the three US products (1.82 micrograms/100 kcal, 1.22 micrograms/100 ml). There was no difference in the proportion of free biotin (the percent of active biotin to total biotin) between the Japanese and US standard formulas (67.7% and 77.2%, respectively). However, in special formulas the mean proportion of the free biotin in the Japanese products (29.4%) was lower than that in the US products (71.2%). The nutrient intake of infants is limited to the maternal milk and/or infant formulas. These findings suggest that Japanese infant formulas should be appropriately improved to maintain growth and good health. Biotin should be registered as a food additive in Japan so that it can be used to fortify infant formulas.

Efficient production of polyhydroxyalkanoates from plant oils by Alcaligenes eutrophus and its recombinant strain.

The ability of Alcaligenes eutrophus to grow and produce polyhydroxyalkanoates (PHA) on plant oils was evaluated. When olive oil, corn oil, or palm oil was fed as a sole carbon source, the wild-type strain of A. eutrophus grew well and accumulated poly(3-hydroxybutyrate) homopolymer up to approximately 80% (w/w) of the cell dry weight during its stationary growth phase. In addition, a recombinant strain of A. eutrophus PHB-4 (a PHA-negative mutant), harboring a PHA synthase gene from Aeromonas caviae, was revealed to produce a random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate from these plant oils with a high cellular content (approximately 80% w/w). The mole fraction of 3-hydroxyhexanoate units was 4-5 mol% whatever the structure of the triglycerides fed. The polyesters produced by the A. eutrophus strains from olive oil were 200-400 kDa (the number-average molecular mass). The results demonstrate that renewable and inexpensive plant oils are excellent carbon sources for efficient production of PHA using A. eutrophus strains.

p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells.

Superoxide anion formation is vital to the microbicidal activity of phagocytes. Recently, however, there is accumulating evidence that it is also involved in cell growth in vascular smooth muscle cells (VSMCs). We have shown that the hypertrophic agent angiotensin II stimulates superoxide production by activating the membrane-bound NADH/NADPH oxidase and that inhibition of this oxidase attenuates vascular hypertrophy. However, the molecular identity of this oxidase in VSMCs is unknown. We have recently cloned the cytochrome b558 alpha-subunit, p22(phox) (one of the key electron transfer elements of the NADPH oxidase in phagocytes), from a rat VSMC cDNA library, but its role in VSMC oxidase activity remains unclarified. Here we report that the complete inhibition of p22(phox) mRNA expression by stable transfection of antisense p22(phox) cDNA into VSMCs results in a decrease in cytochrome b content, which is accompanied by a significant inhibition of angiotensin II-stimulated NADH/NADPH-dependent superoxide production, subsequent hydrogen peroxide production, and [3H]leucine incorporation. We provide the first evidence that p22(phox) is a critical component of superoxide-generating vascular NADH/NADPH oxidase and suggest a central role for this oxidase system in vascular hypertrophy.

Doctor-shopping patients and users of alternative medicine among Japanese primary care patients.

To describe the clinical characteristics of patients with doctor-shopping behavior (doctor-shopping patients) and users of alternative medicine among Japanese primary care patients, 1088 patients from the general medicine outpatient clinic of a medical school hospital answered an original questionnaire and 30-item General Health Questionnaire (GHQ). A random sample of patients was questioned in accordance with the Diagnostic Interview Schedule modified for use in Japan (DIS-JM). Twenty-three percent of these patients met our criteria for doctor-shopping patients, and 7.9% had used alternative medicine. Multivariate analysis showed no significant difference between the two groups of patients with regard to sex, age, residence, occupation, education, or marital status. The most striking characteristics of doctor-shopping patients were chronicity of illness (p < 0.005), inability to understand doctors' explanations (p < 0.005), disbelief of the doctor's diagnosis and treatment (p < 0.005), and high GHQ scores (p < 0.05). The major factor for alternative medicine users was high expectations of medical school hospitals (p < 0.01). DIS-JM interviews showed that doctor-shopping patients had a high lifetime prevalence of mental disorders. The lifetime prevalence of DSM-III somatization disorders was significantly higher in the two study groups. These results suggest that the risk factors for doctor shopping are strongly associated with chronic conditions and the doctor-patient relationship. Also, users of alternative medicine had high expectations of medical school hospitals as the symbol of modern medicine. Therefore, we emphasize the importance of accurate explanations and maintenance of good doctor-patient relationships by physicians providing care. We also suggest that when obtaining the patient's medical history, doctors ask how many prior visits the patient has made to medical facilities with the same complaint, and how many times the patient has used alternative medicine.

Abnormal fatty acid composition of lymphocytes of biotin-deficient rats.

The relative proportions (% of total fatty acids) of odd-chain (15:0-29:0) and long-chain (22:0-30:0) saturated fatty acids in phospholipids of biotin-deficient rat lymphocytes were significantly increased as compared with biotin-supplemented rats, and the ratio of unsaturated fatty acids to saturated fatty acids in the former was significantly decreased mainly due to the reduced composition of polyunsaturated fatty acids in the omega-3, omega-6, and omega-9 pathway. The ratio of cis-vaccenic acid to palmitoleic acid in biotin-deficient rats was significantly lower than that in control rats, and was thought to be another important, but previously unreported indicator of biotin deficiency. These changes imply that the elongation and desaturation of unsaturated fatty acids are depressed in lymphocytes of biotin-deficient rats, and may contribute to the associated immunological dysfunction in biotin deficiency through abnormal prostaglandin metabolism and/or cell membrane functions.

Site-directed random mutagenesis of AMP-binding residues in adenylate kinase.

Two highly conservative residues, Val67 and Gln101, in adenylate kinase are located in the hydrophobic region putatively involved in binding of the adenine ring of AMP. We have performed polymerase chain reaction-based random mutagenesis of the two residues using recombinant chicken muscle adenylate kinase cDNA as a template. The synthetic oligonucleotide primers contained A,G,C,T-mixed bases in the codons corresponding to those for Val67 and Gln101. The amplified fragments were ligated with the expression plasmid pKK223-3, and the mutant proteins expressed were identified by immunoblotting. Enzymatically active mutant proteins were selected on the basis of the growth at 45 degrees C of the temperature sensitive Escherichia coli mutant for adenylate kinase. At position 67, various amino acid residues other than Val have been found to restore the growth at 45 degrees C of the ts mutant. In contrast, only Gln, His, and Met could be present at position 101. These results are compatible with the proposal that Val67 contributes to the AMP binding through hydrophobic interactions and Gln101 by forming a hydrogen bond with the adenine ring. Indeed, several purified Val67 and Gln101 mutant enzymes exhibited markedly high Km values for AMP, whereas the Km values for MgATP were comparable to those of the wild-type enzyme. Substrate specificity for nucleoside monophosphates was changed significantly by the mutagenesis of the two residues.

Biotinidase activity in patients with liver disease.

To investigate whether biotinidase deficiency may occur in liver disease, we determined biotinidase activity, biotin levels, and organic acids in patients with liver disease. Serum biotinidase activity in patients with liver disease (2.63 +/- 1.40 nmol/min/ml) was significantly lower than in the control group (5.43 +/- 1.06 nmol/min/ml). Serum biotinidase activity in decompensated liver cirrhosis (LC) and hepatoma was significantly lower than in acute viral hepatitis (AVH), chronic viral hepatitis (CVH), and compensated LC. The mean serum level of biotin in decompensated LC (1.8 +/- 0.6 microgram/ml) and hepatoma (1.7 +/- 0.8 microgram/ml) was significantly lower than in the control group (2.5 +/- 1.0 microgram/ml), and urinary excretion of biotin was increased in patients with liver disease, particularly in decompensated LC. Biotinidase activity correlated positively with serum biotin level and correlated negatively with urinary biotin level. Moreover, in four of five patients with severe liver disease the excretion of propionate, lactate, and 3-hydroxybutyrate decreased after biotin supplementation. The data for patients with severe liver disease so resembled those for late-onset multiple carboxylase deficiency that biotinidase deficiency is likely in patients with severe liver disease.

Molecular cloning, nucleotide sequencing, and affinity labeling of bovine liver UDP-glucose pyrophosphorylase.

A bovine liver cDNA encoding UDP-glucose pyrophosphorylase [EC 2.7.7.9], which catalyzes the reversible uridylyl transfer between glucose 1-phosphate and MgUTP, has been cloned by the use of oligonucleotide probes synthesized on the basis of partial amino acid sequences of the enzyme. The cDNA clone contained a 1,689 base-pair insert including the complete message for the subunit polypeptide (508 amino acid residues) of the octameric enzyme. The bovine liver enzyme shows significant sequence similarities with the enzymes from potato tuber and a slime mold, Dictyostelium discoideum, but not with the enzyme from Escherichia coli, or ADP-glucose pyrophosphorylases from rice seed and E. coli. To probe the substrate-binding site in the bovine liver enzyme, the purified enzyme was incubated with an affinity labeling reagent, uridine triphosphopyridoxal, and then reduced with sodium borohydride. The enzyme was inactivated rapidly and irreversibly by the reagent at low concentrations. The inactivation was almost completely retarded by UDP-glucose and MgUTP. Structural analysis of the labeled enzyme revealed that three lysyl residues, Lys291, Lys357, and Lys396, were modified by the reagent. The three lysyl residues are conserved at the corresponding positions in the sequence of the potato tuber enzyme, in which they have catalytically important functions. These results show that the active-site structure of bovine liver UDP-glucose pyrophosphorylase is very similar to that of the potato tuber enzyme.

Utilization behavior and concomitant motor neglect by bilateral frontal lobe damage.

We reported on a patient who had bilateral mesial frontal lesions, presumably of a primary cerebral malignant lymphoma. The patient presented with a combination of unusual behavioral disorders of the upper extremities, which has not yet been documented in the literature: bilateral utilization and imitation behaviors and motor neglect of the left arm. Utilization behavior was closely associated with bilateral manual grasping behavior and was caused by the bifrontal lesions. Damage to the right supplementary motor area resulted most likely in concomitant motor neglect of the left hand. Discussion includes differentiation from other related behavioral disorders.

Probing the pyrophosphate-binding site in potato tuber UDP-glucose pyrophosphorylase with pyridoxal diphosphate.

Potato tuber UDP-glucose pyrophosphorylase (EC 2.7.7.9) catalyzes the reversible uridylyl transfer from UDP-glucose to MgPPi forming glucose 1-phosphate and MgUTP, according to an ordered bi-bi mechanism in which UDP-glucose and MgPPi bind in this order. To probe the active site of this enzyme, we have applied pyridoxal 5'-diphosphate, a reactive PPi analogue. The enzyme was rapidly inactivated when incubated with the reagent in the presence of Mg2+ followed by sodium borohydride reduction. The degree of the inactivation was decreased by MgUTP, MgPPi, and glucose 1-phosphate, but enhanced by UDP-glucose. The enhancement was prevented by co-addition of Pi, the competitive inhibitor with respect to PPi. The complete inactivation corresponded to the incorporation of 0.9-1.1 mol of reagent/mol of enzyme monomer. In the presence of UDP-glucose, labels were almost exclusively incorporated into Lys-329. Thus, this residue may be located near the bound MgPPi and its modification is promoted, probably through conformational changes, by the binding of UDP-glucose to the enzyme. The results of the modification by the same reagent of the mutant enzymes in which Lys-329 and Lys-263 are individually replaced by Gln suggest the roles of these lysyl residues in the binding of MgPPi and in the UDP-glucose-induced conformational changes, respectively.