Evidence for a bicarbonate "escort" site in Haemophilus influenzae beta-carbonic anhydrase .

The Haemophilus influenzae beta-carbonic anhydrase (HICA) allosteric site variants V47A and G41A were overexpressed and purified to homogeneity. These variants have k(cat)/K(m) values similar to that of the wild-type enzyme and exhibit a similar dramatic decrease in catalytic activity at pH <8.0. However, both HICA-G41A and -V47A were serendipitously found to bind sulfate ion or bicarbonate ion near pairs of Glu50 and Arg64 residues located on the dimerization interface. In the case of HICA-V47A, bicarbonate ions simultaneously bind to both the dimerization interface and the allosteric sites. For HICA-G41A, two of 12 chains in the asymmetric unit bind bicarbonate ion exclusively at the dimerization interface, while the remaining 10 chains bind bicarbonate ion exclusively at the allosteric site. We propose that the new anion binding site along the dimerization interface of HICA is an "escort" site that represents an intermediate along the ingress and egress route of bicarbonate ion to and from the allosteric binding site, respectively. The structural evidence for sulfate binding at the escort site suggests that the mechanism of sulfate activation of HICA is the result of sulfate ion competing for bicarbonate at the escort site, preventing passage of bicarbonate from the bulk solution to its allosteric site.

Antecedent hypoglycemia impairs autonomic cardiovascular function: implications for rigorous glycemic control.

OBJECTIVE: Glycemic control decreases the incidence and progression of diabetic complications but increases the incidence of hypoglycemia. Hypoglycemia can impair hormonal and autonomic responses to subsequent hypoglycemia. Intensive glycemic control may increase mortality in individuals with type 2 diabetes at high risk for cardiovascular complications. We tested the hypothesis that prior exposure to hypoglycemia leads to impaired cardiovascular autonomic function. RESEARCH DESIGN AND METHODS: Twenty healthy subjects (age 28 +/- 2 years; 10 men) participated in two 3-day inpatient visits, separated by 1-3 months. Autonomic testing was performed on days 1 and 3 to measure sympathetic, parasympathetic, and baroreflex function. A 2-h hyperinsulinemic [hypoglycemic (2.8 mmol/l) or euglycemic (5.0 mmol/l)] clamp was performed in the morning and in the afternoon of day 2. RESULTS: Comparison of the day 3 autonomic measurements demonstrated that antecedent hypoglycemia leads to 1) reduced baroreflex sensitivity (16.7 +/- 1.8 vs. 13.8 +/- 1.4 ms/mmHg, P = 0.03); 2) decreased muscle sympathetic nerve activity response to transient nitroprusside-induced hypotension (53.3 +/- 3.7 vs. 40.1 +/- 2.7 bursts/min, P < 0.01); and 3) reduced (P < 0.001) plasma norepinephrine response to lower body negative pressure (3.0 +/- 0.3 vs. 2.0 +/- 0.2 nmol/l at -40 mmHg). CONCLUSIONS: Baroreflex sensitivity and the sympathetic response to hypotensive stress are attenuated after antecedent hypoglycemia. Because impaired autonomic function, including decreased cardiac vagal baroreflex sensitivity, may contribute directly to mortality in diabetes and cardiovascular disease, our findings raise new concerns regarding the consequences of hypoglycemia.

Hypoglycemia increases serum interleukin-6 levels in healthy men and women.

OBJECTIVE: Inflammation may have a major role in the pathogenesis and prognosis of critical illness. Hyperglycemia increases levels of the inflammatory cytokine interleukin-6 (IL-6) and is associated with increased risks of morbidity and mortality. Because hypoglycemia is also associated with adverse outcomes, we tested the hypothesis that hypoglycemia increases IL-6. RESEARCH DESIGN AND METHODS: Seventeen healthy men and women participated in hypoglycemic and euglycemic-hyperinsulinemic clamp studies (target blood glucose levels 2.7 and 5.0 mmol/l, respectively), separated by 1-3 months. IL-6, ACTH, and cortisol were measured at baseline and at 45, 75, 105, and 135 min after initiation of the insulin infusion. RESULTS: IL-6, ACTH, and cortisol levels increased significantly (P < 0.0001) during hypoglycemia but not euglycemia. IL-6 increased from mean +/- SEM 1.0 +/- 0.2 pg/ml at baseline to 2.6 +/- 0.2 pg/ml after 135 min of hypoglycemia, whereas IL-6 levels were unchanged during euglycemia. CONCLUSIONS: Hypoglycemia increases IL-6 levels in healthy individuals.