High prevalence of diabetes mellitus in a cohort of Trinidadian patients with chronic obstructive pulmonary disease

OBJECTIVE: To define the prevalence of diabetes mellitus (DM) in a cohort of Trinidadian chronic obstructive pulmonary disease (COPD) patients, and investigate its relationship to lung function, quality of life, and depression. DESIGN AND METHODS: Anthropometric and spirometric data were obtained from 108 COPD (91 males) patients from Trinidad chest clinics, who also had HbA1c test results. Questionnaires on quality of life St. George’s Respiratory questionnaire ({SGRQ} and COPD Assessment Test {CAT}) and depression (Center for Epidemiologic Studies Depression Scale {CES-D & CESD-R}) were administered, and an interview conducted. RESULTS: Mean (SD) age: 67.4 (11.0) years. Median (IQR) HbA1c: 6.1 (5.7, 6.7) %. HbA1c values were obtained for 105 patients of whom 40% had diabetes, and 40% pre-diabetes. Diabetics had a greater (p=0.001) median (IQR) BMI [27.3 (24.1, 30.4)] than non-diabetics [24.2 (21.2, 27.2)]. Patients with at least one chest infection/exacerbation in the past year had increasing CAT & SGRQ Total (p<0.001), and CES-D & CESD-R (p≤0.013) scores. CAT and SGRQ total scores were negatively related to lung function and Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages (p<0.001). All lung function parameters, except Forced expiratory volume/Forced vital capacity (FEV1/FVC), decreased with increasing CES-D and CESD-R scores (p<0.05). CAT & SGRQ correlated well with CES-D & CESD-R scores (p<0.001). Intravenous corticosteroid use was positively associated with HbA1c (p=0.043). Dosage of inhaled corticosteroids was associated with lower FEV1 (p=0.034) and higher SGRQ & CAT (p≤0.048). FVC % predicted was negatively related to HbA1c (p=0.033). CONCLUSION: The prevalence of DM in the COPD patients was 40%; however no significant correlations of DM to outcome measures were observed. Patients with worse quality of life due poor lung function were more depressed.

Properties of bound inorganic phosphate on bovine mitochondrial F1F0-ATP synthase.

Beef-heart mitochondrial F1F0-ATP synthase contained six molecules of bound inorganic phosphate (Pi). This phosphate exchanged completely with exogenous 32Pi when the enzyme was exposed to 30% (v/v) dimethyl sulfoxide (DMSO) and then returned to a DMSO-free buffer (Beharry and Bragg 2001). Only two molecules were replaced by 32Pi when the enzyme was not pretreated with DMSO. These two molecules of 32Pi were not displaced from the enzyme by the treatment with 1 mM ATP. Similarly, two molecules of bound 32Pi remained on the DMSO-pretreated enzyme following addition of ATP, that is, four molecules of 32Pi were displaced by ATP. The ATP-resistant 32Pi was removed from the enzyme by pyrophosphate. It is proposed that these molecules of 32Pi are bound at an unfilled adenine nucleotide-binding noncatalytic site on the enzyme. Brief exposure of the enzyme loaded with two molecules of 32Pi to DMSO, followed by removal of the DMSO, resulted in the loss of the bound 32Pi and in the formation of two molecules of bound ATP from exogenous ADP. A third catalytic site on the enzyme was occupied by ATP, which could undergo a Pi <--> ATP exchange reaction with bound Pi. The presence of two catalytic sites containing bound Pi is consistent with the X-ray crystallographic structure of F1 (Bianchet, et al., 1998). Thus, five of the six molecules of bound Pi were accounted for. Three molecules of bound Pi were at catalytic sites and participated in ATP synthesis or Pi <--> ATP exchange. Two other molecules of bound Pi were present at a noncatalytic adenine nucleotide-binding site. The location and role of the remaining molecule of bound Pi remains to be established. We were unable to demonstrate, using chemical modification of sulfhydryl groups by iodoacetic acid, any gross difference in the conformation of F1F0 in DMSO-containing compared with DMSO-free buffers.

Celiac sprue: another autoimmune syndrome associated with hepatitis C.

OBJECTIVE: Celiac sprue is being diagnosed with increasing frequency by screening individuals with epidemiologically associated autoimmune syndromes. We sought to test our hypothesis that hepatitis C also may predispose to celiac sprue because it can trigger autoimmune reactions. METHODS: Two hundred fifty-nine consecutively evaluated patients with chronic hepatitis C infection, 59 with autoimmune liver disease, 137 with other hepatic diseases, 356 with various GI syndromes, and 221 normal volunteers underwent serologic screening for celiac sprue. Patients with antigliadin, antiendomysial, and antitissue transglutaminase antibodies in serum underwent duodenoscopy and biopsy. RESULTS: There was a statistically significantly higher prevalence of antigliadin antibody in all groups of patients with liver disease compared with GI controls and normal controls. However, only patients with hepatitis C (n = 3; 1.2%) or autoimmune liver disease (n = 2; 3.4%) had antiendomysial/antitissue transglutaminase antibody in serum. One of 221 normal volunteers (0.4%) was antigliadin, antiendomysial, and antitissue transglutaminase positive; this individual also was found to have hepatitis C (previously undiagnosed). Each of these six individuals had mild intestinal symptoms, duodenal histopathology consistent with celiac sprue, and the celiac-associated HLA-DQ2 allele. Five of the six followed a prescribed gluten-free diet and experienced symptomatic improvement. CONCLUSION: Celiac sprue is epidemiologically associated with chronic hepatitis C infection and with autoimmune liver disease. Because hepatitis C is much more frequently encountered than autoimmune liver disease, hepatitis C appears to be the most common hepatic disease associated with the development of celiac sprue.

Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase.

Purified soluble bovine mitochondrial F(1)F(o)-ATP synthase contained 2 mol of ATP, 2 mol of ADP and 6 mol of P(i)/mol. Incubation of this enzyme with 1 mM [(32)P]P(i) caused the exchange of 2 mol of P(i)/mol of F(1)F(o)-ATP synthase. The labelled phosphates were not displaced by ATP. Transfer of F(1)F(o)-ATP synthase to a buffer containing 30% (v/v) DMSO and 1 mM [(32)P]P(i) resulted in the loss of bound nucleotides with the retention of 1 mol of ATP/mol of F(1)F(o)-ATP synthase. Six molecules of [(32)P]P(i) were incorporated by exchange with the existing bound phosphate. Removal of the DMSO by passage of the enzyme through a centrifuged column of Sephadex G-50 resulted in the exchange of one molecule of bound [(32)P]P(i) into the bound ATP. Azide did not prevent this [(32)P]P(i)<-->ATP exchange reaction. The bound labelled ATP could be displaced from the enzyme by exogenous ATP. Addition of ADP to the DMSO-pretreated F(1)F(o)-ATP synthase in the original DMSO-free buffer resulted in the formation of an additional molecule of bound ATP. It was concluded that following pretreatment with and subsequent removal of DMSO the F(1)F(o)-ATP synthase contained one molecule of ATP at a catalytic site which was competent to carry out a phosphate-ATP exchange reaction using enzyme-bound inorganic radiolabelled phosphate. In the presence of ADP an additional molecule of labelled ATP was formed from enzyme-bound P(i) at a second catalytic site. The bound phosphate-ATP exchange reaction is not readily accommodated by current mechanisms for the ATP synthase.

Surface hydrophobicity is increased in the ileum and proximal colon of cystic fibrosis mice.

Patients with cystic fibrosis (CF) have abnormal concentrations and composition of electrolytes and macromolecules in gastrointestinal secretions. Such alterations could change intestinal surface properties, such as surface hydrophobicity, and may influence the adhesion of macromolecules, bacteria, or microbial toxins to the intestinal surface. The objective of this study was to compare the surface hydrophobicity of the gastrointestinal tract in wild type and CF mice. We used axisymmetric drop shape analysis-contact diameter to determine surface hydrophobicity by measuring contact angles of sessile water droplets placed onto epithelial surfaces. In wild type mice, there were no differences in contact angles between the duodenum, upper jejunum, lower jejunum, and ileum. The contact angle of the gastric mucosa was lower than the rest of the gastrointestinal tract. Contact angles of the proximal colon and distal colon were both higher than that of the gastric mucosa and those of the small intestinal sections. In CF mice, contact angles along the gastrointestinal tract followed the same pattern as in wild type mice. However, contact angles in the ileum and proximal colon of CF mice were greater than those from wild type mice. This study of the murine intestine showed regional differences in surface hydrophobicity comparable to those observed in other mammalian species. In addition, we showed that the ileum and proximal colon of CF mice were more hydrophobic than the corresponding segments in wild type mice. These observations are of potential clinical relevance because patients with CF exhibit clinical manifestations of gastrointestinal disease primarily in the ileum and proximal colon.

Synergistic effects of cAMP- and calcium-mediated amylase secretion in isolated pancreatic acini from cystic fibrosis mice.

We evaluated pancreatic enzyme secretory response to secretagogues (cAMP- and Ca2+-mediating) involved in exocytosis and in chloride channel activation in an exon 10 knockout cystic fibrosis (CF) mouse model. Experiments were performed in isolated pancreatic acini from liquid-fed Cftr-/- mice (5-6 wk of age) and age-matched Cftr+/+ controls fed a solid or liquid diet. BrcAMP and forskolin alone induced higher amylase secretion (% initial amylase content) in the Cftr+/+ acini than carbachol (p < 0.05). Carbachol and BrcAMP or BrcAMP and forskolin, given in combination, produced additive effects on enzyme secretion in the Cftr+/+ acini. Ca2+- and cAMP-mediated amylase secretion in isolated pancreatic acini from the Cftr-/- mice was no different to that observed in the age- and diet-matched Cftr+/+ animals. However, Cftr-/- pancreatic acini showed a significantly greater amylase response to the combination of BrcAMP and carbachol than the sum of the individual responses in separate experiments (p < 0.05). The amylase response was not different in acini from solid-fed or liquid-fed Cftr+/+ controls. In summary, this study suggests that cystic fibrosis transmembrane conductance regulator is not essential for enzyme secretion as evidenced by no reduction in cAMP-mediated amylase secretion in Cftr-/- mice. The results in Cftr+/+ acini suggest pancreatic enzyme secretion is mediated via multiple intracellular pathways acting in parallel and probably converge at a distal step in the secretory process. However, Cftr-/- pancreatic acini exhibited a synergistic secretory response following stimulation by BrcAMP plus carbachol. The enhanced secretory response may partially contribute to the development of pancreatic dysfunction in CF patients by facilitating occlusion of digestive enzymes in the secretory canaliculus of the pancreatic acini.

Acute effect of glucose tablets on desire to smoke.

RATIONALE: Previous research suggests that glucose may reduce desire to smoke during periods of abstinence but a definitive test is needed. OBJECTIVE: The present study aimed to determine whether a single administration of oral glucose would reduce desire to smoke in abstaining smokers. METHODS: Thirty-eight smokers attended the laboratory in the afternoon having not smoked since the previous evening. They rated their desire to smoke immediately before and at 5-min intervals for 20 min after chewing four 3-g glucose tablets (experimental group) or four matched placebo tablets (control group). RESULTS: Ratings of desire to smoke decreased to a greater extent in the experimental than the control group. The effect was apparent after 10 min. There was no difference between the groups in terms of feeling "sick" or "satisfied". CONCLUSION: A single dose of glucose has a relatively rapid and detectable effect on desire to smoke and the effect is not mediated by feeling sick. Glucose tablets may be useful in helping to control desire to smoke during periods of abstinence.

Impact of protein deprivation on protein and DNA synthesis in the developing rat pancreas.

The metabolic effects of protein malnutrition on growth and development of the exocrine pancreas are largely unknown. The aim of the present study was to evaluate the effects of protein malnutrition on pancreatic protein and DNA synthesis during postnatal development. Rat dams and their offspring were fed a protein-deficient diet (6% casein) or a control diet (25% casein) during gestation, lactation and after weaning. Pancreatic protein and DNA synthesis were measured in vitro at postnatal ages 1, 3, 10, 23, 36 and 60 days, by assessing [3H]leucine and [3H]thymidine incorporation in freshly isolated acini. Different patterns of protein synthesis were seen in the two groups. At birth, pancreatic protein synthesis was low in both control and malnourished animals. At day 3, protein synthesis in the control acini increased 10-fold while synthesis in acini of the malnourished animal group was only 50% of age-matched control values. No differences in protein synthesis were detected between the control and malnourished groups between 10 and 36 days of age. At 60 days (adulthood), acinar protein synthesis declined in the control-fed rats, but a significant increase was observed in the malnourished animals (p < 0. 0005). At birth, DNA synthesis was high in the acini from both control and malnourished animals. The low-protein diet induced a slight reduction in DNA synthesis at day 3, without altering the general pattern during later stages of development. In conclusion, protein deprivation has variable effects on pancreatic protein and DNA synthesis at different stages of postnatal development. Furthermore, the mechanisms of control within acini appear to be intrinsically regulated.

Postnatal development of the rat exocrine pancreas. I. Alterations in high- and low-affinity cholecystokinin receptors and enzyme secretion.

We tested the hypothesis that postnatal alterations in cholecystokinin (CCK) receptors are associated with developmental changes in enzyme secretory response. We used simultaneous measurements of CCK receptor binding and amylase release in pancreatic acini isolated from rat pups at various ages (1, 2, 5, 6, 18, and 36 days). CCK receptor binding was analyzed using the LIGAND program. The affinity of the high-affinity state increased postnatally at 18 and 36 days (p < 0.05); the capacity of the high-affinity state also increased at 2 days (p < 0.05), then declined sequentially up to 36 days. The affinity of the low-affinity state increased postnatally reaching statistical significance at 5 days; the capacity of the low-affinity state increased twofold at 2 days, reaching statistical significance at 5 days (p < 0.05); this was followed by a slight decrease at 36 days. At 1 day postnatally a small amylase response occurred (p < 0.05), but no dose-dependent response was observed. A significant CCK dose-dependent secretory response occurred at all other ages. Maximal amylase release was highest at 18 days (p < 0.05). CCK doses required to stimulate maximal amylase release were 20, 2, 1, 0.2 and 0.4 nM at 2, 5, 6, 18, and 36 days, respectively. The receptor occupancy rates for high- and low-affinity states decreased sequentially between 2 and 18 days of age, when maximal amylase release occurred. These data suggest that more spare receptors become available with increasing postnatal age. We conclude that postnatal alterations of both high- and low-affinity states of CCK receptors in pancreatic acini are associated with developmental changes in enzyme secretory response to CCK. An increase in the affinity of high-affinity state and the capacity of the low-affinity state may enhance acinar sensitivity to CCK.

Postnatal development of the rat exocrine pancreas. II. Effects of protein-calorie malnutrition on amylase secretion and CCK receptor binding.

Malnutrition induces pancreatic atrophy and intracellular derangement, but its effects on cholecystokinin (CCK) receptors and the CCK-induced secretory response remain unclear. We used a rodent model to study the developmental effects of protein-calorie malnutrition on exocrine pancreatic function. Simultaneous experiments evaluated postnatal alterations in CCK-induced amylase response and receptor binding of pancreatic acini. At all postnatal ages, somatic and pancreatic weight of the malnourished rats was significantly below age-matched controls (p < 0.01). The malnourished rats showed a higher secretory response to CCK at 1 day of age and increased acinar sensitivity at 2 days. Maximal amylase secretion was significantly higher at 5 and 18 days (p < 0.05), but remained similar to that of the age-matched controls at 36 days. CCK receptor binding showed no significant changes at 1 and 2 days postnatally in comparison with controls. At 5 and 18 days, the affinity of the high-affinity state showed a twofold increase, while the capacity of the high-affinity state decreased by 40-55%. At the same time, the affinity of the low-affinity state increased significantly (p < 0.05), but the capacity of the low-affinity state was essentially unchanged. The acinar sensitivity of malnourished rats was consistently reduced between 5 and 36 days, which coincided with a reduction in spare receptors in the malnourished rats. In conclusion, the increased amylase secretory response at 1 and 2 days of age may be due to an adaptive response of endocrine function to maternal metabolic stress. The increased affinities of CCK receptors at 5 and 18 days may be associated with a higher secretory responsiveness, while the decreased spare receptors may contribute to a reduction in the acinar sensitivity. These results demonstrate that malnutrition induces changes in CCK binding and its secretory response.

The bound adenine nucleotides of purified bovine mitochondrial ATP synthase.

The experiments in this study were directed towards defining the nucleotide content of purified beef-heart mitochondrial F1F0 ATP synthase during binding and hydrolysis of ATP. The purified, soluble synthase as prepared contained 2 mol ATP and 2 mol ADP/mol enzyme. Three of these four nucleotides were exchangeable on incubation with radiolabelled MgATP. Passage of the ATP synthase through a column of Sephadex G-50 readily removed 1 mol ADP/mol. The remaining bound nucleotides were not displaced by incubation with 1 mM GTP or 5 mM sodium sulfite, the latter an activator of the ATPase activity of the synthase. Incubation of the synthase with 250 microM MgATP in the presence of 3 mM sodium azide, an inhibitor of the ATPase, resulted in the transitory formation of a form of the enzyme in which 5-6 nucleotide-binding sites were loaded with ATP and/or ADP, thus showing that the ATP synthase, like the soluble F1 ATPase, contained a minimum of six nucleotide-binding sites. The presence of an ATP-regenerating system during incubation with MgATP resulted in the loading of 5-6 sites to yield a form of the enzyme containing 3-4 mol ATP and 2 mol ADP/mol synthase even after passage through a centrifuged column. Following hydrolysis of the medium MgATP, the enzyme reached a stable form containing 2 mol ATP and 2 mol ADP/mol synthase. Like the form of the enzyme originally prepared, 1 mol ADP/mol synthase was readily released. However, this ADP remained bound to the synthase in the presence of GTP if azide was present. These results are discussed in the context of current ideas about nucleotide-binding sites on the F1 ATPase portion of the F1F0 ATP synthase. It is concluded that the properties of the sites on the F1F0 synthase show some differences from those on the F1 ATPase.

In vivo measurements of ion transport in long-living CF mice.

The Cftr (Cystic Fibrosis Transmembrane Conductance Regulator) gene codes for an epithelial chloride (C1) channel essential for fluid secretion into the respiratory and gastrointestinal tract and from exocrine glands. Mice lacking CFTR function due to a disruption of Cftr exon 10 or exon 1 (Cftr (m1UNC/m1UNC) or Cftr(m1HSC/m1HFC) mice, respectively) generally suffer from severe gastrointestinal disease resulting in death shortly after birth or at the time of weaning. However, a subgroup of the Cftr(m1HSC/m1HSC) mice have been characterized which exhibit relatively mild intestinal pathology resulting in a noncompromised lifespan compared to the more severely affected Cftr(m1UNC/m1UNC) mice. We compared the ion transport capacity of the intestinal mucosa of the mildly and severely affected CF mice using the in vivo technique of rectal potential difference (PD) measurement and found that the net calcium-activated chloride conductance toward the lumen was much greater in the rectum of mildly affected mice than in the severely affected mice. Hence, the milder phenotype may be related to the expression of a factor which enhances the net calcium-activated chloride conductance into the lumen of the intestinal tract.

The effect of depletion of nucleotide and of delta and epsilon subunits on ATP synthesis in dimethyl sulfoxide by F1-ATPase of Escherichia coli.

The F1-ATPase of Escherichia coli contains 3 mol bound adenine nucleotide/mol F1. It is of the F1[2,1] type based on the ability of GTP to displace 1 mol adenine nucleotide/mol F1 (Kironde, F.A.S. and Cross, R.L. (1986) J. Biol. Chem. 261, 12544-12549). A portion of the adenine nucleotide (2 mol/mol F1) is not displaceable by GTP. F1[2,1] was converted to F1[1,0]. This form of the enzyme synthesized ATP from endogenous ADP and inorganic phosphate in a medium containing 30% (v/v) dimethyl sulfoxide (Me2SO). A delta,epsilon subunit-depleted form of the F1-ATPase was shown to be predominantly in the F1[0,1] form. ATP was not synthesized from endogenous ADP by the subunit-depleted enzyme in Me2SO unless additional molecules of ADP were bound. It is concluded that ATP synthesis from endogenous ADP in Me2SO occurs at GTP-nondisplaceable adenine nucleotide binding sites.

Escherichia coli F1-ATPase can use GTP-nonchaseable bound adenine nucleotide to synthesize ATP in dimethyl sulfoxide.

Escherichia coli F1-ATPase contained 3 mol of tightly-bound adenine nucleotide/mol enzyme. A further 3 mol could be loaded by incubation of the enzyme with ATP. The unloaded enzyme was designated as a F1[2,1] type on the basis of the ability of GTP to displace 1 mol of adenine nucleotide/mol of F1 [Kironde, F.A.S., & Cross, R.L. (1986) J. Biol. Chem. 261, 12544-12549]. The loaded enzyme was designated F1[3,3] since GTP could displace 3 of the 6 mol of bound adenine nucleotide/mol of F1. Incubation of F1[2,1], F1[2,0], and F1[3,0] with phosphate in the presence of 30% (v/v) dimethyl sulfoxide led to the synthesis of ATP from endogenous bound ADP. Hydrolysis of newly synthesized ATP occurred on transfer of the F1 from 30% (v/v) dimethyl sulfoxide to an entirely aqueous medium. Thus, synthesis and hydrolysis of ATP can occur at GTP-nonchaseable adenine nucleotide binding sites, and these sites in dimethyl sulfoxide are not necessarily equivalent to noncatalytic sites.

Interaction of beef-heart mitochondrial F1-ATPase with immobilized ATP in the presence of dimethylsulfoxide.

Dimethylsulfoxide [Me2SO, 30% (v/v)] promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F1-ATPase. The effects of this solvent on the interaction of beef-heart mitochondrial F1 with the immobilized ATP of Agarose-hexane-ATP were studied. In the presence of Me2SO, F1 bound less readily to the immobilized ATP, but once bound was more difficult to elute with exogenous ATP. This suggests that not only was the binding affinity for adenine nucleotide at the first binding site affected but that adenine nucleotide binding affinity at the second and/or third sites, which interact cooperatively with the first site to release bound nucleotide, was also affected. A reduction in the binding of [3H]ADP to these sites was shown. A change in the conformation of F1 in 30% (v/v) Me2SO was demonstrated by crosslinking and by the increased resistance of the enzyme to cold denaturation.

Changes in the adenine nucleotide and inorganic phosphate content of Escherichia coli F1-ATPase during ATP synthesis in dimethyl sulphoxide.

Escherichia coli F1-ATPase contained 2.9 +/- 0.1 mol of adenine nucleotide and 3.1 +/- 0.3 mol of Pi/mol of enzyme. After preincubation with ATP, the nucleotide and phosphate contents were 5.6 and 6.0 +/- 0.5 mol/mol of enzyme respectively. The F1-ATPase was induced to synthesize ATP in the presence of 30% (v/v) dimethyl sulphoxide (Me2SO). The ATP originated from endogenous bound ADP. The bound adenine nucleotide and Pi contents of the enzyme during the time course of ATP synthesis were investigated by using F1-ATPase which had been preincubated with ATP. We show that the process of ATP synthesis in Me2SO involves (i) an initial rapid loss of nucleotide from the enzyme, the process being facilitated by exogenous Pi, (ii) a rapid loss of Pi from the enzyme, at least in the absence of exogenous Pi, (iii) re-binding of a portion of the lost nucleotide, and (iv) synthesis of ATP from bound ADP and exogenous Pi. It is proposed that transfer of the F1-ATPase to the Me2SO medium induces a change in the conformation of the enzyme to a form favouring ATP synthesis.

Changes in the adenine nucleotide content of beef-heart mitochondrial F1 ATPase during ATP synthesis in dimethyl sulfoxide.

Beef-heart mitochondrial F1 ATPase can be induced to synthesize ATP from ADP and inorganic phosphate in 30% Me2SO. We have analyzed the adenine nucleotide content of the F1 ATPase during the time-course of ATP synthesis, in the absence of added medium nucleotide, and in the absence and presence of 10 mM inorganic phosphate. The enzyme used in these investigations was either pretreated or not pretreated with ATP to produce F1 with a defined nucleotide content and catalytic or noncatalytic nucleotide-binding site occupancy. We show that the mechanism of ATP synthesis in Me2SO involves (i) an initial rapid loss of bound nucleotide(s), this process being strongly influenced by inorganic phosphate; (ii) a rebinding of lost nucleotide; and (iii) synthesis of ATP from bound ADP and inorganic phosphate.

Beef-heart mitochondrial F1-ATPase can use endogenous bound phosphate to synthesize ATP in dimethyl sulfoxide.

Beef-heart mitochondrial F1-ATPase contained 5 mol of inorganic phosphate bound per mol of F1, following pretreatment with ATP. A portion of the phosphate, bound most likely at a catalytic site, reacted in dimethylsulfoxide with endogenous adenine nucleotide to form ATP.

The effect of dimethylsulfoxide on adenine nucleotide binding and ATP synthesis by beef-heart mitochondrial F1 ATPase.

Dimethylsulfoxide (Me2SO; 30%, v/v) promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F1 ATPase. The effects of this solvent on the adenine nucleotide binding properties of beef-heart mitochondrial F1 ATPase were examined. The ATP analog adenylyl-5'-imidodiphosphate bound to F1 at 1.9 and 1.0 sites in aqueous and Me2SO systems, respectively, with a KD value of 2.2 microM. Lower affinity sites were present also. Binding of ATP or adenylyl-5'-imidodiphosphate at levels near equimolar with the enzyme occurred to a greater extent in the absence of Me2SO. Addition of ATP to the nucleotide-loaded enzyme resulted in exchange of about one-half of the bound ATP. This occurred only in an entirely aqueous medium. ATP bound in Me2SO medium was not released by exogenous ATP. Comparison of the effect of different concentrations of Me2SO on ADP binding to F1 and ATP synthesis by the enzyme showed that binding of ADP was diminished by concentrations of Me2SO lower than those required to support ATP synthesis. However, one site could still be filled by ADP at concentrations of Me2SO optimal for ATP synthesis. This site is probably a noncatalytic site, since the nucleotide bound there was not converted to ATP in 30% Me2SO. The ATP synthesized by F1 in Me2SO originated from endogenous bound ADP. We conclude that 30% Me2SO affects the adenine nucleotide binding properties of the enzyme. The role of this in the promotion of the formation of ATP from ADP and phosphate is discussed.

Interaction of ox heart mitochondrial F1-ATPase with immobilized ADP and ATP.

The reaction of mitochondrial F1-ATPase with immobilized substrate was studied by using columns of agarose-hexane-ATP. Mg2+ was required for binding of the enzyme to the column matrix. The column-bound enzyme could be eluted fully by ATP and other nucleoside triphosphates. Nucleoside di- and mono-phosphates were less effective. At a fixed concentration of nucleotide the effectiveness of elution was proportional to the charge on the eluting molecule. The ATP of the column matrix was hydrolysed by the bound F1-ATPase to release phosphate, probably by a uni-site reaction mechanism. Thus the F1-ATPase was bound to the immobilized ATP by a catalytic site. Treatment of the bound F1-ATPase with 4-chloro-7-nitrobenzofurazan prevented complete release of the enzyme by ATP. Only one-third of the bound enzyme was now eluted by the nucleotide. The inhibition of release could be due either to the inhibitor blocking co-operative interactions between sites or to its increasing the tightness of binding of immobilized ADP at the catalytic site.