The identification of extrahepatic "glucokinase" as N-acetylglucosamine kinase

Several research groups have reported the presence of a high Km glucokinase (ATP:D-glucose 6-phosphotransferase, EC 2.7.1.2) in tissues other than adult liver. As shown in this report, protein fractions catalyzing glucose phosphorylation only at high substrate concentrations (100 mM) are indeed found in bovine spleen, rat kidney, human placenta, and newborn rat liver. However, the study of substrate specificities and Michaelis constant values showed that those fractions could be better described as N-acetylglucosamine kinase (ATP:acetamide-2-deoxy-D-glucose-6-phosphotransferase, EC 2.7.1.9) which, in addition to N-acetylglucosamine (Km = 0.066 mM), can also phosphorylate glucose although with very high Km values (370 mM). Furthermore, a homogeneous preparation from bovine spleen was able to phosphorylate both N-acetylglucosamine and glucose. An immune serum against bovine spleen N-acetylglucosamine kinase did not cross-react with purified hexokinases or with glucokinase from rat. However, it was able to remove the putative "glucokinases" from extracts of rat kidney, newborn rat liver, and one of two electrophoretic bands of liver "glucokinase." It is proposed that any report of extrahepatic glucokinase should explicity rule out N-acetylglucosamine kinase as the enzyme being described.     

The separation and identification of picomole amounts of intermediates of glucose metabolism by high performance liquid chromatography on pellicular resins.

A column (CarboPac PA1, Dionex) containing an anion-exchange pellicular resin was used for the separation of phosphoryl-hexoses derived from labeled glucose microinjected into individual frog oocytes or from cultures of Escherichia coli. Intermediates were identified by: a) comparison of retention times with those of authentic commercial compounds; b) the use of internal labeled standards; c) incubation of samples with specific enzymes and noting the disappearance of one radioactive peak and appearance of another at a new retention time.     

Assessing native and non-native conformational states of a protein by methylene carbene labeling: the case of Bacillus licheniformis beta-lactamase

Much knowledge of protein folding can be derived from the examination of the nature and size of solvent-exposed surfaces along conformational transitions. We exploit here a general photochemical modification with methylene carbene of the accessible surface area (ASA) of the polypeptide chain. Labeling of Bacillus licheniformis beta-lactamase (BL-betaL) with 1 mM 3H-diazirine yielded 8.3 x 10(-3) mol CH2/mol protein, in agreement with the prediction for an unspecific surface labeling phenomenon. The unfolded state U in 7 M urea was labeled 60% more than the native state N. This result lies well below the increment of ASA expected from theoretical estimates and points to the presence of residual organization in state U and/or of cavities or crevices favoring the partition of the reagent in state N. A partially folded state I was demonstrated from two sequential transitions occurring at 1.5-3.0 M and 3.5-6.5 M urea. This technique shows a close correlation with optical probes most sensitive to changes in tertiary structure, a statement supported by the fact that the largest change occurs along the N-I portion of the N-I-U transition and along the acid pH-induced N-A transition. In the latter case, state A is labeled 70% more than state N, an increment consistent with the loosening of tight interactions in the core of the protein. Fragmentation of labeled BL-betaL into peptides provides a sequential map of solvent accessibility. Thus, amino acid residues pertaining to the Omega-loop and to helices alpha5 and alpha6 line the major cavity of the protein, that is big enough to lodge the diazirine reagent. Methylene labeling, by introducing an original (and perhaps unique) experimental measurement of ASA, enlightens subtle aspects of complex transitions and makes possible a comparative structural characterization of the native as well as non-native states.     

gamma-aminobutyric acid increases the water accessibility of M3 membrane-spanning segment residues in gamma-aminobutyric acid type A receptors

gamma-Aminobutyric acid type A (GABA(A)) receptors are members of the ligand-gated ion channel gene superfamily. Using the substituted cysteine accessibility method, we investigated whether residues in the alpha(1)M3 membrane-spanning segment are water-accessible. Cysteine was substituted, one at a time, for each M3 residue from alpha(1)Ala(291) to alpha(1)Val(307). The ability of these mutants to react with the water-soluble, sulfhydryl-specific reagent pCMBS(-) was assayed electrophysiologically. Cysteines substituted for alpha(1)Ala(291) and alpha(1)Tyr(294) reacted with pCMBS(-) applied both in the presence and in the absence of GABA. Cysteines substituted for alpha(1)Phe(298), alpha(1)Ala(300), alpha(1)Leu(301), and alpha(1)Glu(303) only reacted with pCMBS(-) applied in the presence of GABA. We infer that the pCMBS(-) reactive residues are on the water-accessible surface of the protein and that GABA induces a conformational change that increases the water accessibility of the four M3 residues, possibly by inducing the formation of water-filled crevices that extend into the interior of the protein. Others have shown that mutations of alpha(1)Ala(291), a water-accessible residue, alter volatile anesthetic and ethanol potentiation of GABA-induced currents. Water-filled crevices penetrating into the interior of the membrane-spanning domain may allow anesthetics and alcohol to reach their binding sites and thus may have implications for the mechanisms of action of these agents.     

Prevalence of risk factors for cardiovascular disease in Canadians 55 to 74 years of age: results from the Canadian Heart Health Surveys, 1986-1992

BACKGROUND: By 2016, the proportion of Canadians older than 65 years of age will increase to 16%, and there will be an increase in the absolute number of cases of cardiovascular disease in older Canadians. The Canadian Heart Health Surveys database provides information about this population upon which health policy related to cardiovascular disease can be based. This paper presents for the first time population-based data on the risk factors for cardiovascular disease in older Canadians. METHODS: Canadians from all 10 provinces participated in surveys of cardiovascular risk factors; health insurance registries were used as sampling frames. In each province, probability samples of 2200 adults 18 to 74 years old not living in institutions, on reserves or in military camps were asked to participate in interviews and to undergo testing at clinics for major risk factors for cardiovascular disease. RESULTS: A total of 2739 men (response rate 70%) and 2617 women (response rate 66%) aged 55 to 74 years participated in the survey and also provided follow-up clinical measurements at the clinic. Overall, 52% of participants were hypertensive, 26% had isolated systolic hypertension, and 30% had a total blood cholesterol level of 6.2 mmol/L or greater. Rates of current smoking were lower in women than men (17% v. 22%). Overall, 87% of men and 78% of women who were current smokers smoked at least 10 cigarettes per day. Only slightly more than half of participants exercised at least once a week for at least 15 minutes, and almost half had a body mass index of 27 or greater. In only 4% was no major risk factor for cardiovascular disease detected. INTERPRETATION: Significant numbers of older Canadians have one or more major risk factors for cardiovascular disease. Many of these risk factors are amenable to modification.     

Comparative studies on glucose phosphorylating isoenzymes of vertebrates. Identification and characterization of amphibian liver hexokinases

Glucose phosphorylating activities were measured in liver extracts from two urodeles and twenty-six anurans. Fractionation on diethylaminoethyl-cellulose columns of liver extracts from these amphibians permitted the recognition of four hexokinases which are called A, B, C, and D. However, any given amphibian displays only three liver hexokinases and the profiles so far observed are either of the type A-B-D or C-B-D. The distribution of the amphibians in either type of pattern does not show any simple taxonomic relationship. A wide generic and specific, but not individual, variation of the relative proportion of each isoenzyme was observed. Hexokinases A and B were shown to be low K_{m glucose} isoenzymes (0.06 and 0.15 mm glucose, respectively) with normal hyperbolic kinetics. Hexokinase C, also a low K_m isoenzyme (0.05 mm) was found to be inhibited by excess substrate at physiological levels of glucose. Hexokinases A, B, and C were able to phosphorylate fructose, mannose, and 2-deoxyglucose at equal or higher rates than glucose when assayed at saturating sugar levels. Hexokinase D was found to be a high K_m isoenzyme ($\text{K}{}_{0.5}\text{? 2}\text{mM}$) with sigmoidal saturation curves for glucose (Hill coefficient ? 1.6). Fructose and mannose were also phosphorylated by this isoenzyme at about 70% of the glucose rate when studied at saturating sugar concentrations. The properties of the amphibian hexokinases are thus similar, although not identical, to those of mammalian hexokinases.     

Sigmoidal kinetics of glucokinase.

Glucokinases obtained from the liver of several species of mammals and amphibians exhibit sigmoidal saturation functions for glucose. Hill coefficients (nH) are about 1.5, and half-saturation values (K0.5) lie between 1.5 and 8.5 mmol/l. The nH and K0.5 values are constant throughout the purification steps of rat glucokinase. A dimeric form of rat glucokinase appearing in aged preparations exhibits michaelian kinetics. Sigmoidal kinetics is considered as an adaptive feature of glucokinases to increase the efficiency of the liver uptake of glucose at the changeable concentrations in the blood resulting from variations in the amount of dietary glucose.