High calpain-1 expression predicts a poor clinical outcome and contributes to tumor progression in pancreatic cancer patients.

BACKGROUND: Pancreatic cancer (PC) is a highly aggressive and metastatic disease, with an elevated mortality rate. It is, therefore, crucial to assess factors affecting the prognosis of PC patients. Meanwhile, calpain-1 is associated with malignant tumor progression and metastasis. Thus, it is meaningful to evaluate the relationship between calpain-1 and PC. MATERIALS AND METHODS: Calpain-1 protein expression was assessed by immunohistochemistry in 96 pancreatic cancer samples and paired adjacent non-cancerous specimens. In addition, calpain-1 protein levels were assessed in six PC cell lines by western blot (WB). Next, PC cells were transfected with calpain-1 siRNA, and silencing was confirmed by WB. Finally, cell proliferation, colony formation, migration and invasion assays, and cell apoptosis analysis were performed to examine the effects of calpain-1 knockdown on proliferation, growth, apoptosis, migration, and invasion in PC cells. RESULTS: The results showed that calpain-1 was overexpressed in PC tissues and cells. Meanwhile, calpain-1 overexpression was associated with tumor site (P = 0.029), metastasis (P = 0.000), and TNM stage (P = 0.000), but showed no associations with histological grade (P = 0.396), age (P = 0.809), sex (P = 1.000), and lesion size (P = 0.679). The Kaplan-Meier method demonstrated that the low calpain-1 expression group had increased overall survival (OS) compared with patients expressing high calpain-1 levels (28.7 ± 4.1 vs. 17.0 ± 2.3 months) (P = 0.005). Besides, calpain-1 in PC cells was successfully silenced by liposome-mediated RNA interference, resulting in reduced cell growth, invasion, and metastasis in PC cells, with no effect on apoptosis. CONCLUSION: The above findings suggest that calpain-1 should be considered a potential biomarker for PC prognosis and therapy.

[A cross-sectional study of seroepidemiology of viral hepatitis among Uighurs in Shufu of Xinjiang].

Objective: To explore the status of seroepidemiology on hepatitis A, B and C among students and residents aged equal or greater than 18 years in south Xinjiang, and to provide scientific evidence for prevention and control of viral hepatitis. Methods: Uyghur students in four towns and villages were selected by cluster random sampling from Feb to May, 2015, and Uyghur residents aged 18 to 69 years were selected by stratified cluster sampling from May to September, 2016. 4 507 middle and primary Uygur students and 4 833 Uyghur resides equal or greater than 18 years attended this survey. Self-designed questionnaire was used to collect the demographic information. And Elisa test was adopted to detect HAV-IgG, HBsAg, HBsAb and HCV-IgG. Chi-square test was used to calculate the difference on antibody positive rate of three types of hepatitis among the participants. Results: The overall HAV-IgG positive rate was 99.45% (9 289/9 340). The positive rates were 99.70% (4 006/4 018) in male and 99.27% (5 283/5 322) in female (χ(2)=7.95, P=0.005). The HAV-IgG positive rate among people aged 10 to 14 years was the highest (99.91%, 2 233/2 235), and the difference between the age specific HAV-IgG positive rates was statistical significant (χ(2)=38.21, P<0.001). The overall HBsAg positive rate was 4.11% (384 cases), with 4.55% (183 cases) for male and 3.78% (201 cases) for female. The HBsAg positive rate among participants 19 to 24 years old was the highest (9.46%, 21/222) and the differences between the age specific HBsAg was statistically significant (χ(2)=116.22, P<0.001). The HBsAb positive rate was 35.03% (3 272 cases), with 35.59% (1 430 cases) for male and 34.61% (1 842 cases) for female, and the HBsAb positive rate among participants aged 15 to 18 years was the highest, which was 69.12% (696/1 007). The differences between the age specific HBsAb rates appeared statistically significant (χ(2)=671.80, P<0.001). The overall HCV-IgG positive rate was 0.65%(61 cases). The HCV-IgG positive rate in female was 0.86% (46 cases) and 0.37% (15 cases) in male (χ(2)=8.51, P=0.004).The HCV-IgG positive rate among participants aged over 70 years was the highest (3.78%,9/238) , and the difference between the age specific HCV-IgG positive rates had statistic significance (χ(2)=70.30, P<0.001). The HAV-IgG positive rate in hepatitis A vaccinees (100%, 876/876) was higher than that among the non-vaccinees (99.40%, 8 413/8 464) (χ(2)=4.26, P=0.039). The HBsAb positive rate in hepatitis B vaccinees was 39.32% (1 816/4 619) which was higher than that among the non-vaccinees (30.84%, 1 456/4 721) (χ(2)=73.68, P<0.001). Conclusion: The positive rate of overall HAV-IgG was high, male and participants aged between 10 to 14 had higher probability to be infected. But the infection rates of hepatitis B and C were low, with the low vaccination rate and the poor protective effectiveness of hepatitis B.

[Prevalence of dyslipidemia and normal blood lipids level in Uygur population in Kashgar area of Xinjiang Uygur Autonomous Region].

Objective: To understand the prevalence of dyslipidemia and normal blood lipids level in Uygur diabetes patients in Kashgar prefecture in southern area of Xinjiang. Methods: A total of 5 078 local residents aged ≥18 years (42.56% were men) selected through cluster random sampling in Kashgar were surveyed by means of questionnaire survey, physical examination and laboratory test, and 521 diabetes patients were screened. Results: The overall prevalence of dyslipidemia in diabetes patients was 59.50% (310/521) with adjusted rate as 49.39%. Age ≥65 years, overweight, obesity and abdominal obesity increased the risk for dyslipidemia by 0.771 times (95% CI: 1.015-3.088), 1.132 times (95% CI: 1.290-3.523), 1.688 times (95% CI: 1.573-4.592) and 0.801 times (95% CI: 1.028-3.155) respectively. Compared with males, female was a protective factor for dyslipidemia (OR=0.507, 95%CI: 0.334-0.769). The overall normal rate of blood lipids level including total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) for type 2 diabetes patients was 11.13%. Female, higher BMI and abdominal obesity were the factors influencing the overall normal blood lipids level. The normal rate of LDL-C level decreased with increase of age, BMI and waist circumferences (trend test χ(2)=18.049, P<0.001; trend test χ(2)=10.582, P=0.001; χ(2)=19.081, P<0.001), but increased with educational level (trend test χ(2)=9.764, P=0.002). Conclusion: The prevalence of dyslipidemia in Uygur diabetes patients in Kashgar was high, however, the overall normal rate of blood lipid level was relatively low. Obesity was the most important risk factor for dyslipidemia in this area. More attention should be paid to dyslipidemia prevention in women.

[Prevalence of diabetes mellitus and associated risk factors in Uygur population in Kashgar area of Xinjiang Uygur Autonomous Region].

Objective: To investigate the epidemiologic characteristics of diabetes mellitus (DM) in Uygur residents in Kashgar of Xinjiang. Methods: The survey was conducted among the Uygur residents aged ≥18 years selected through stratified cluster sampling in Kashgar by means of questionnaire survey, physical examination and basic laboratory test. The prevalence of different groups were calculated and risk factors of DM was analyzed by logistic vegression model. Results: A total of 4 608 adults were surveyed. The prevalence of DM was 11.31% (standardized prevalence: 10.59%) and the prevalence was 13.65% (standardized prevalence: 12.34%) in males and 10.04% (standardized prevalence: 9.83%) in females. The prevalence increased with age. The prevalence of DM was higher than the prevalence of impaired fasting glucose (IFG) in people aged >60 years, especially in females. The rates of awareness, treatment and control of DM were 28.02%, 21.31% and 5.57%, respectively. Multivariate logistic regression analysis indicated that people aged 45-55, 55-65 and >65 years had higher risk of DM and the odds ratio were 2.08 (95%CI: 1.24-3.48), 2.73 (95%CI: 1.63-4.56) and 3.90 (95%CI: 2.24-6.78) for men and 2.63 (95% CI: 1.71-4.02), 3.14 (95%CI: 2.00-4.94) and 5.56 (95%CI: 3.47-8.92) for woman, respectively. Family history of DM (OR=2.88 for men, 95% CI: 1.45-5.72; OR=2.52 for women, 95% CI: 1.49-4.26) and BMI≥28.0 kg/m(2) (OR=1.77 for men, 95% CI: 1.19-2.64, OR=1.80 for women, 95% CI: 1.30-2.50) were also risk factors for DM. Conclusion: The prevalence of DM was high in Uygur residents in Kashgar, but the rate of awareness, treatment and control of DM were low. It is necessary to improve the detection rate of DM and conduct targeted prevention and control of DM.

[Knowledge, attitude and practice to chronic diseases and associated influencing factors in Uygur population in Kashgar area of Xinjiang Uygur Autonomous Region].

Objective: To investigate the knowledge, attitude and practice (KAP) to chronic diseases and associated influencing factors in Uygur adults in Kashgar of southern Xinjiang, and provide basic information for developing ethnic specific prevention and control strategies for chronic diseases. Methods: With stratified cluster random sampling, investigations, including questionnaire survey, physical examination and laboratory tests were performed among local residents who were aged ≥18 years and selected in Shufu county in southern Xinjiang. KAP scores were calculated and unconditional logistic regression models were used to analyze influencing factors. Results: A total of 4 772 Uygur adults were surveyed. The awareness rate of chronic disease related knowledge ranged from 4.32% to 56.04%, while the awareness rate of preventive measures were from 1.76% to 85.18% and the participation rate of prevention program varied from 4.00% to 97.99%. The average KAP score was 15.90±4.20 and the rate of total KAP was 47.86%. Multi-factor analysis suggested the pass rate of total KAP score increased with educational level. Other factors positively associated with 'KAP score pass rate' were commercial insurance investment, hypertension, family history of common chronic diseases, female and abdominal obesity, while overweight was negatively associated with the KAP score pass rate. Conclusions: The KAP level on chronic diseases was low in Uygur adults in Kashgar. It is necessary to continuously implement ethnic targeted health education and health promotion campaigns, especially in low education level, low income level, overweight and male groups.

[Prevalence of hypertension and risk factors in Uygur population in Kashgar area of Xinjiang Uygur Autonomous Region].

Objective: To investigate the prevalence of hypertension and risk factors in Uygur adults in Kashgar of Xinjiang. Methods: A total of 4 748 Uygur adults aged ≥18 years selected through cluster random sampling in Shufu county of Kashgar prefecture were surveyed by means of questionnaire survey, physical examination and laboratory test. The prevalence, awareness, treatment and control rate of hypertension of different groups were calculated and risk factors of hypertension was analyzed by using non-conditional logistic regression model. Results: The overall prevalence of hypertension was 15.73% (age-adjusted prevalence was 13.75%). The prevalence rates of hypertension in men and women were 16.36% (age-adjusted prevalence was 12.96%), 15.39% (age-adjusted prevalence was 14.34%), respectively. The rates of awareness, treatment and control of hypertension were 59.57%, 52.74%, 21.29%, respectively. The prevalence of hypertension increased with age. Compared with age group 18-34 years, the age groups 55-64, ≥65 years had higher risk of hypertension and the OR values were 10.53, 20.96 for men and 16.27, 33.20 for women. The overweight (OR=1.47 for men, OR=1.82 for women, P<0.05) and obesity (OR=1.88 for men, OR=2.66 for women, P<0.05) also increased the risk of hypertension. The groups with family history of hypertension (OR=3.85 for men, OR=2.34 for women, P<0.05) also had higher risk of hypertension. Hypertriglyceridemia was positively correlated with the prevalence of hypertension in men (OR=1.62, 95%CI: 1.09-2.41). Conclusions: The prevalence of hypertension in Uygur adults in Kashgar area of Xinjiang was at relatively low level. The related risk factors were age, overweight, obesity, family history and hypertriglyceridemia in men and the risk factors were similar in women except hypertriglyceridemia.

[Status of seroepidemiology of hepatitis A, B and C in primary and middle school students in Shufu county, Xinjiang Uygur Autonomous Region of China].

Objective: To explore the status of seroepidemiology on hepatitis A, B and C in primary and middle school students in Shufu county, Xinjiang Uygur Autonomous Region of China (Xinjiang) and to evaluate the effect of related immunization. Methods: Students in four towns and villages were selected by cluster random sampling method. HAV-IgG, HBsAg, HBsAb and HCV-IgG were detected in Feb to May, 2015. Results: The overall HAV-IgG positive rate was 99.75%, among 4 830 primary and middle school students. The positive rates were seen 99.92% in boys and 99.57% in girls, with difference statistically significant (χ2=5.798, P=0.016). The overall HBsAg positive rate appeared as 3.02%, with 3.55% for boys and 2.47% for girls, with difference statistically significant (χ2=4.782, P=0.029). The difference between age specific HBsAg positive rates also showed statistically significant (χ2=71.990,P=0.000). HBsAg positive rate in the students in rural area (3.28%) was higher than that in the students in urban area (1.61%, χ2=6.019, P=0.014). HBsAb positive rate was 38.84%, and the differences between the age specific HBsAb positive rates appeared statistically significant (χ2=837.699, P=0.000). HBsAg positive rate in students from the urban area (42.36%) was higher than those from the rural area (38.20%, χ2=4.598, P=0.032). 2 815 students, accounting for 58.28% of the total students, showed negative on both HBsAg and HBsAb. The overall HCV-IgG positive rate was 0.19%, and all appeared in students from the rural areas, with ethnicity solely as Uygur. Conclusions: The effect of hepatitis A vaccine was satisfactory in primary and middle school students in Shufu county but quiet a number of the students missed the vaccination. The infection rate of hepatitis C was low. Publicity and health education on hepatitis immunization and control should be revved up. Programs regarding primary and supplementary immunization on hepatitis, should be carried out timely for children of school age.

[Using Metronidazole and Hydroxyapatite for preventing dry socket after extraction of impacted mandibular 3rd molar]

Dry socket is one of the most frequent complications after teeth extraction,especially in impacted mandibular third molars.The etilogy and prevention is not clear.This study id based on principles of clinical epidemiology.Randomized double-blind method was carried out in 549 patients to test the value of the prophylactic use of Hydroxyapatite,to test the value of the prophylactic use of Hydroxyapatite and Metronidazole,placed in the sockets of extracted impacted mandibular third molars.The results of the incidence of DS was 7.1% of Metronidazole treated sockets,and 2.1% of Hydroxyapatite treated sockets,It is concluded that Hydroxyapatite is an effective preventive factor for dry socket,The possible mechanism of Hydroxyapatite and the dry socket etiology were discussed.

[Intraoperative transgallbladder duct endoscopy replaces conventional common duct exploration].

Twenty patients of cholelithiasis underwent intraoperative transcystic duct fiberendoscopy in whom choledochotomy was otherwise indicated. Among them choledocholithotomy was done endoscopically in three patients and auxiliary choledocho-cystic junction incision was obligatory for easy insertion of the fiberscope in six. No T-tube was needed after the procedure and the postoperative recovery was uneventful in all cases.

The NSR1 gene encodes a protein that specifically binds nuclear localization sequences and has two RNA recognition motifs.

We previously identified a protein (p67) in the yeast, Saccharomyces cerevisiae, that specifically recognizes nuclear localization sequences. We report here the partial purification of p67, and the isolation, sequencing, and disruption of the gene (NSR1) encoding this protein. p67 was purified using an affinity column conjugated with a peptide containing the histone H2B nuclear localization sequence from yeast. Using antibodies against p67 we have cloned the gene for this protein. The protein encoded by the NSR1 gene recognizes the wild-type H2B nuclear localization sequence, but does not recognize a mutant H2B sequence that is incompetent for nuclear localization in vivo. Interestingly, the NSR1 protein has two RNA recognition motifs, as well as an acidic NH2 terminus containing a series of serine clusters, and a basic COOH terminus containing arg-gly repeats. We have confirmed the nuclear localization of p67 by immunofluorescence and found that a restricted portion of the nucleus is highlighted. We have also shown that NSR1 (p67) is required for normal cell growth.

Active/inactive state transitions of the chloroplast F1 ATPase are induced by a slow binding and release of Mg2+. Relationship to catalysis and control of F1 ATPases.

Mg2+ is known to be a potent inhibitor of F1 ATPases from various sources. Such inhibition requires the presence of a tightly bound ADP at a catalytic site. Results with the spinach chloroplast F1 ATPase (CF1) show that the time delays of up to 1 min or more in the induction or the relief of the inhibition are best explained by a slow binding and slow release of Mg2+ rather than by slow enzyme conformational changes. CF1 is known to have multiple Mg2+ binding sites with Kd values in the micromolar range. The inhibitory Mg2+ and ADP can bind independently to CF1. When Mg2+ and ATP are added to the uninhibited enzyme, a relatively fast rate of hydrolysis attained soon after the addition is followed by a much slower steady-state rate. The inhibited steady-state rate results from a slowly attained equilibrium of binding of medium Mg2+. The Kd for the binding of the inhibitory Mg2+ is in the range of 1-8 microM, in the presence or absence of added ATP, as based on the extent of rate inhibition induced by Mg2+. Assessments from 18O exchange experiments show that the binding of Mg2+ is accompanied by a relatively rapid change to an enzyme form that is incapable of hydrolyzing MgATP. When ATP is added to the Mg2+- and ADP-inhibited enzyme, the resulting reactivation can be explained by MgATP binding to an alternate catalytic site which results in a displacement of the tightly bound ADP after a slow release of Mg2+. Both an increase in temperature (to 50 degrees C) and the presence of activating anions such as bicarbonate or sulfite reduce the extent of the Mg2+ inhibition markedly. The activating anions may bind to CF1 in place of Pi near the ADP. Whether the inhibitory Mg2+ binds at catalytic or noncatalytic nucleotide binding sites or at another location is not known. The Mg2(+)- and ADP-induced inhibition appears to be a general property of F1 ATPases, which show considerable differences in affinity for ADP, Mg2+, and Pi. These differences may reflect physiological control functions.

Properties of chloroplast F1-ATPase partially modified by 2-azido adenine nucleotides, including demonstration of three catalytic pathways.

Previous investigations on the distribution of [18O]Pi isotopomers formed by hydrolysis of [gamma-18O]ATP by the chloroplast F1-ATPase (CF1) showed that a single reaction pathway is used by all participating sites and that the pathway is modulated by ATP concentration as expected for cooperative interactions between catalytic sites. Such oxygen exchange measurements have been applied to CF1 modified at a single catalytic or noncatalytic site by 2-azido adenine nucleotides. When less than one catalytic or one noncatalytic site per enzyme is modified, hydrolysis occurs in part by the pathway of the unmodified enzyme plus at least one additional pathway at 200 microM and two additional pathways at 4 microM [gamma-18O]ATP. Thus, three sites are potentially catalytically active. The two new pathways shown by the derivatized enzyme logically can arise from nonidentical interactions of the remaining two underivatized beta subunits with the derivatized beta subunit. Reversals of bound ATP cleavage before Pi is released are increased, and the amount of product formed by the new pathways is changed when the ATP concentration is lowered. These modulations must result from the behavior of two remaining active catalytic sites rather than of one catalytic and one regulatory site. When the CF1 is derivatized more extensively, the original catalytic pathway is lost, and two catalytic pathways that do not show modulation by ATP concentration are found. The remaining beta subunits now have weak but independent catalytic capacity. In addition, the enzyme is no longer activated by Ca2+, loses MgGTPase activity, and is much less sensitive to azide.

Relationship of tightly bound ADP and ATP to control and catalysis by chloroplast ATP synthase.

Whether the tightly bound ADP that can cause a pronounced inhibition of ATP hydrolysis by the chloroplast ATP synthase and F1 ATPase (CF1) is bound at catalytic sites or at noncatalytic regulatory sites or both has been uncertain. We have used photolabeling by 2-azido-ATP and 2-azido-ADP to ascertain the location, with Mg2+ activation, of tightly bound ADP (a) that inhibits the hydrolysis of ATP by chloroplast ATP synthase, (b) that can result in an inhibited form of CF1 that slowly regains activity during ATP hydrolysis, and (c) that arises when low concentrations of ADP markedly inhibit the hydrolysis of GTP by CF1. The data show that in all instances the inhibition is associated with ADP binding without inorganic phosphate (Pi) at catalytic sites. After photophosphorylation of ADP or 2-azido-ADP with [32P]Pi, similar amounts of the corresponding triphosphates are present on washed thylakoid membranes. Trials with appropriately labeled substrates show that a small portion of the tightly bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling by an ADP moiety at a catalytic site. We also report the occurrence of a 1-2-min delay in the onset of the Mg2+-induced inhibition after addition of CF1 to solutions containing Mg2+ and ATP, and that this delay is not associated with the filling of noncatalytic sites. A rapid burst of Pi formation is followed by a much lower, constant steady-state rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalytic properties of chloroplast F1-ATPase modified at catalytic or noncatalytic sites by 2-azido adenine nucleotides.

When heat-activated F1-ATPase from chloroplasts was repeatedly exposed to Mg2+ and 2-azido-ATP, followed by separation from medium nucleotides and photolysis, a total of two sites per enzyme, both catalytic and noncatalytic, were labeled. In a coupled assay with pyruvate kinase about half the activity was lost when one site per enzyme was modified. However, increased modification resulted in no further loss of activity. In contrast, methanol-sulfite activation of the enzyme showed a loss of most of the catalytic capacity when one site per enzyme was modified. Predominant labeling of either one catalytic or one noncatalytic site caused a loss of most of the activity in either assay. An indication that the enzyme modified at one site retained some catalytic activity was verified by measurement of the [18O]Pi species formed when [gamma-18O]ATP was hydrolyzed by partially derivatized enzyme. With either catalytic or noncatalytic site modification, the distributions of [18O]Pi species formed showed that the modified enzyme had different catalytic characteristics. An interpretation is that with modification by azido nucleotides at either catalytic or noncatalytic sites, capacity for rapid catalysis is largely lost but the remaining sites retain weak modified catalytic properties.

Catalytic and noncatalytic nucleotide binding sites of chloroplast F1 ATPase. Photoaffinity labeling and peptide sequencing.

Exposure of chloroplast F1 ATPase to 2-azido-ATP results in the noncovalent tight binding of 2-azido-ATP or 2-azido-ADP to noncatalytic or to catalytic sites. Subsequent photolysis results in covalent labeling of adjacent tryptic peptides of the beta-subunit. Binding at noncatalytic sites results in labeling of tyrosine 385 by an ATP or an ADP moiety. Binding at catalytic sites results in labeling of tyrosine 362 by only an ADP moiety. Similar labeling patterns are observed for the heat-activated or the membrane-bound enzymes.

Adenine nucleotide binding sites on beef heart F1 ATPase: photoaffinity labeling of beta-subunit Tyr-368 at a noncatalytic site and beta Tyr-345 at a catalytic site.

2-Azidoadenine [32P]nucleotide was bound specifically at catalytic or noncatalytic nucleotide binding sites on beef heart mitochondrial F1 ATPase. In both cases, photolysis resulted in nearly exclusive labeling of the beta subunit. The modified enzyme was digested with trypsin, and labeled peptides were purified by reversed-phase high-pressure liquid chromatography. Amino acid sequence analysis of the major 32P-labeled tryptic fragments showed beta-subunit Tyr-368 to be present at noncatalytic sites and beta Tyr-345 to be present at catalytic sites. From the relationship between the degree of inhibition and extent of modification, it is estimated that one-third of the catalytic sites or two-thirds of the noncatalytic sites must be modified to give near-complete inhibition of catalytic activity.

Chloroplast F1 ATPase has more than three nucleotide binding sites, and 2-azido-ADP or 2-azido-ATP at both catalytic and noncatalytic sites labels the beta subunit.

The photolabeling of chloroplast F1 ATPase, following exposure to Mg2+ and 2-azido-ATP and separation from medium nucleotides, results in derivatization of two separate peptide regions of the beta subunit. Up to 3 mol of the analogue can be incorporated per mole of CF1, with covalent binding of one moiety or two moieties per beta subunit that can be either AMP, ADP, or ATP derivatives. These results, the demonstration of noncovalent tight binding of at least four [3H]adenine nucleotides to the enzyme and the presence of three beta subunits per enzyme, point to six potential adenine nucleotide binding sites per molecule. The tightly bound 2-azido nucleotides on CF1, found after exposure of the heat-activated and EDTA-treated enzyme to Mg2+ and 2-azido-ATP, differ in their ease of replacement during subsequent hydrolysis of ATP. Some of the bound nucleotides are not readily replaced during catalytic turnover and covalently label one peptide region of the beta subunit. They are on noncatalytic sites. Other tightly bound nucleotides are readily replaced during catalytic turnover and label another peptide region of the beta subunit. They are at catalytic sites. No alpha-subunit labeling is detected upon photolysis of the bound 2-azido nucleotides. However, one or both of the sites could be at an alpha-beta-subunit interface with the 2-azido region close to the beta subunit, or both binding sites may be largely or entirely on the beta subunit.