The Common Bed Bug (Hemiptera: Cimicidae) Does Not Commonly Use Canines and Felines as a Host in Low-Income, High-Rise Apartments.

The common bed bug (Cimex lectularius L.) is a known pest and an obligate blood-feeding ectoparasite. Bed bugs can feed on warm-blooded animals including humans, bats, poultry, and rabbits, but no research has investigated the use of companion animals (canines and/or felines) as a blood source. This study investigates how long known host DNA could be detected in a bed bug and the prevalence of bed bugs feeding on companion animals. Laboratory-reared bed bugs were fed host blood to determine how long DNA of human, feline, canine, and rabbit blood could be detected up to 21 d postfeeding. Additionally, 228 bed bugs were collected from 12 apartments with pets (6: canine, 5: feline, and 1: canine and feline), characterized as engorged or unengorged, and then screened with host-specific primers to identify the bloodmeal. Host meals of human, rabbit, feline, and canine blood were detected up to 21 d after feeding laboratory strains. All bed bugs died after feeding on the canine blood, but DNA could be detected up to 21 d post feeding/death. Of the field-collected bed bugs analyzed, human DNA was amplified in 158 (69.3%) bed bugs, canine DNA amplified in 7 bed bugs (3.1%), and feline DNA amplified in 1 bed bug (0.4%). Results of this study suggest that bed bugs predominately feed on humans and rarely feed on companion animals when they cohabitate in low-income, high-rise apartments. Additionally, results from this study warrant future investigations into host use by bed bugs in different housing structures and socioeconomic environments.

Effects of pH on inactivation of maize phosphoenolpyruvate carboxylase.

Maize leaf phosphoenolpyruvate carboxylase (PEPC) is inactivated by incubation at pH's above neutrality. Both the amount and the rapidity of inactivation increase as the pH rises. The presence of phosphoenolpyruvate (PEP), malate, glucose 6-phosphate and dithiothreitol in the incubation medium give protection to the enzyme. While the presence of PEP during incubation at pH 8 prevents inactivation, the level of PEP in the assay after incubation has no effect on the relative inactivation. When the enzyme is incubated at pH 7 with 5 mM malate (a treatment known to cause dimerization) subsequent assay at saturating levels of MgPEP completely restores activity while assay at less than Km MgPEP produces greater than 99% inhibition of the same sample, showing that high PEP concentration has reconverted the PEPC to the malate-resistant tetramer. Thus the protective effect of PEP against inactivation at high pH probably is not related to its effect on the aggregation state of the enzyme but rather is due to the presence of PEP at the active site. Protection of PEPC at pH 8 by EDTA and its inactivation by low concentrations of Cu2- indicates that the loss of activity at high pH probably is in a sense an artifact resulting from the binding to a deprotinated cysteine of heavy metal ions contaminating the enzyme preparation or present in reagents. This suggests that caution should be used in the interpretation of experiments involving PEPC activity at alkaline pH's.

The influence of pH on substrate form specificity of phosphoenolpyruvate carboxylase purified from Crassula argentea.

Purified phosphoenolpyruvate carboxylase from both the crassulacean acid metabolism plant Crassula argentea and the C4 plant Zea mays was shown by kinetic studies at saturating fixed-varying concentrations of free mg2+ to selectively use the metal-complexed form of phosphoenolpyruvate when assayed at pH 8.0. A similar response to added magnesium at high free phosphoenolpyruvate concentrations was obtained for both enzymes, consistent with the use of the complex as the substrate. Kinetic studies at pH 7.0 indicated that at this pH the total concentration of phosphoenolpyruvate (including both free and metal-complexed forms) could be used by the enzyme from C.argentea while the C4 enzyme still utilized the complex. The loss of specificity induced by the decrease in the pH of the assay medium was accompanied by a decrease in the Km of this enzyme for phosphoenolpyruvate whatever the form considered and an increase in Vmax/Km. In contrast, a similar decrease of pH led to an increased Km of the C4 enzyme for phosphoenolpyruvate and a decrease of Vmax/Km. For the enzyme from C. argentea (previously shown to contain an essential arginine at the active site), protection of activity by the different forms of substrate against inactivation by the specific arginyl reagent 2,3-butanedione changes markedly with pH. At pH 8.1, the metal complex is the better protector while at pH 7.0 free phosphoenolpyruvate gives the best protection consistent with the observed kinetic changes in substrate form utilization. The relationship between the enzyme affinity for substrate, substrate specificity, and the requirement for magnesium for substrate turnover is discussed.

Inhibition of phosphoenolpyruvate carboxylase by malate.

Malate has been noted to be a ;mixed' inhibitor of phosphoenolpyruvate (PEP) carboxylase. The competitive portion of this inhibition appears to be fairly constant regardless of the condition of the enzyme being measured, but the noncompetitive (V-type) inhibition is subject to variation depending on the source of the enzyme, its storage condition, the presence or absence of various ligands, and differences in pH. In the case of the maize (Zea mays L.) phosphoenolpyruvate carboxylase (PEPC), the V-type inhibition by malate is much less pronounced at pH 8 than at pH 7. Examination of the response of the maize PEPC to PEP concentration reveals a pronounced cooperativity at pH 8 which is not present at pH 7, and which results in the disappearance of the V-type inhibition at pH 8. The ability of high concentrations of PEP to convert PEPC from a form readily inhibited by malate to one resistant to malate inhibition has been previously demonstrated and we attribute the cooperativity shown at pH 8 to this response to high levels of PEP. Support for this proposal is provided by studies of the enzyme at pH 7 and pH 8 run in 20% glycerol. In this case there was no V-type inhibition of PEPC at either pH. Treatment with 20% glycerol has been shown to result in the aggregation of maize PEPC.

Activation of higher plant phosphoenolpyruvate carboxylases by glucose-6-phosphate.

Studies of the response of phosphoenolpyruvate carboxylase from C(3) (wheat [Triticum aestivum L.]), C(4) (maize [Zea mays L.]), and Crassulacean acid metabolism (CAM) (Crassula) leaves to the activator glucose-6-phosphate as a function of pH showed that the binding of the activator and the response path to activation were essentially identical for all three enzymes. The level of affinity for the activator differed, with the CAM enzyme having the highest affinity and the maize enzyme the lowest. The observed pK values suggest that histidine and cysteine groups may be involved in activation by glucose-6-phosphate. The presence of glucose-6-phosphate protected the enzyme against inactivation of the activation response by p-chloromercuribenzoate. The maximal activation response to glucose-6-phosphate showed differences among the three enzymes including different pH optima and different pH profiles. Here the maize leaf enzyme showed a potential response about twice as great as that of the C(3) and CAM enzymes.

Kinetic studies of the form of substrate bound by phosphoenolpyruvate carboxylase.

Phosphoenolpyruvate carboxylase isolated from maize (Zea mays L.) leaves was assayed with varying concentrations of free phosphoenolpyruvate at several fixed-varying concentrations of free magnesium higher than required to saturate the enzyme reaction. These assays produced velocity data which were found to form a family of individual lines when plotted against free phosphoenolpyruvate or against total phosphoenolpyruvate, but not when plotted against the concentration of the complex of phosphoenolpyruvate with magnesium. In this latter case, the points from all the fixed-varying concentrations fell on the same line, which can be fitted to a modified Michaelis-Menten equation with a multiple correlation coefficient R(2) = 0.995. Similar results were obtained when the enzyme from the C(4) plant maize was assayed with manganese rather than magnesium and when phosphoenolpyruvate carboxylase from leaves of the C(3) plant wheat (Triticum vulgare Vill.) was assayed with magnesium. However, at pH 7.0 the enzyme from the Crassulacean acid metabolism plant Crassula argentea did not produce a satisfactory single line when plotted against the complex of metal ion and substrate, but did so when the assay pH was raised to 8.0. It is concluded that in general the preferred form of substrate for phosphoenolpyruvate carboxylase is the complex of phosphoenolpyruvate with the metal ion.

Role of Magnesium in the Binding of Substrate and Effectors to Phosphoenolpyruvate Carboxylase from a CAM Plant.

The binding of phosphoenolpyruvate, malate, and glucose 6-phosphate to phosphoenolpyruvate carboxylase purified from Crassula argentea Thunb. was measured using both the intrinsic tryptophan fluorescence of the enzyme and the extrinsic fluorescence of the complex of 8-anilino-1-napthalenesulfonate with the enzyme. It was found that the substrate phosphoenolpyruvate can bind in the absence of magnesium but is bound in greater quantities and more tightly when magnesium is present. Malate reduces the binding of phosphoenolpyruvate, while glucose 6-phosphate increases the binding of the substrate. Glucose 6-phosphate requires magnesium to bind to the enzyme, while malate does not. The general trends from the binding experiments using fluorescence methods were confirmed by activity determinations using assays performed in the absence of magnesium.

Oligomerization and the sensitivity of phosphoenolpyruvate carboxylase to inactivation by proteinases.

Phosphenolpyruvate (PEP) carboxylase from leaves of Crassula argentea displays varying levels of sensitivity to inactivation by various proteolytic enzymes. In general, the native enzyme is sensitive to proteinases known to attack at the carbonyl end of lysine or arginine (trypsin, papain, or bromelain). The ineffective proteolytic enzymes are those which have low specificity or which attack at the N-terminal end of hydrophobic amino acids, or which cannot attack lysine. The lack of an effect of endoproteinase arginine C, which is specific for arginine, probably indicates that lysine is the critical residue. When the native enzyme, which is comprised of an equilibrium of dimers with tetramers in approximately equal quantities, is treated by preincubation with 5 millimolar PEP, the enzyme becomes much more resistant to proteolytic inactivation. When the preincubation is with 5 millimolar malate rather than buffer alone, the effect is to slightly increase (ca. 15%) the sensitivity of the enzyme to inactivation by trypsin as measured by estimates of the pseudo-first order rate constant for inactivation. PEP carboxylase from corn leaves appears to be relatively susceptible to inactivation by trypsin, but is unaffected by preincubation with malate or PEP. The sensitivity of this C(4) enzyme to inhibition by malate is also unaffected by preincubation with these ligands.

Malate inhibition of phosphoenolpyruvate carboxylase from crassula.

Phosphoenolpyruvate carboxylase partially purified from leaves of Crassula and rendered insensitive to malate by storage without adjuvants can be altered to the form sensitive to malate inhibition by brief, 5-minute preincubation with 5 millimolar malate. The induction of malate sensitivity is reversible by lowering the malate(2-) concentration. Of the reaction components only HCO(3) (-) increases the sensitivity to malate in subsequent assay. Phosphoenolpyruvate (PEP), which itself tends to lower sensitivity to subsequent malate inhibition, also reduces the effect of malate in the assay, as does glucose-6-phosphate. PEP isotherms showed that the insensitive or unpreincubated enzyme, responds to the presence of 5 millimolar malate during assay with a 3-fold increase in K(m), but no effect on V(max). Enzyme preincubated with malate shows the same effect of malate on K(m), but in addition V(max) is inhibited 72%. It thus appears that both sensitive and insensitive forms of PEP carboxylase are subject to K-type inhibition by malate, but only the sensitive form also shows V-type inhibition. Preincubation with malate at different pH values showed that at pH 6.15, the inhibition by malate in subsequent assay at pH 7 was much lower than at pH 7 or 8. When the reaction is prerun for 30 minutes with increasing concentrations of PEP, subsequent assay with malate shows progressively less inhibition due to malate. When 0.3 millimolar PEP either alone or with 0.1 millimolar ATP and 0.3 millimolar NaF is present during preincubation, the effect of malate in a following assay is to activate the reaction. These results may indicate an effect of phosphorylation of the enzyme on sensitivity to malate.

Physical and Kinetic Properties and Regulation of the NAD Malic Enzyme Purified from Leaves of Crassula argentea.

The NAD malic enzyme has been purified to near homogeneity from the leaves of Crassula argentea Thunb. The enzyme has two subunits, one of 59,000 daltons, and one of 62,000 daltons. In native gels stained for activity, the enzyme appears to exist in the dimeric, tetrameric, and predominantly the octameric forms.The enzyme uses either Mg(2+) or Mn(2+) as the required divalent cation, and utilizes NADP at a rate less than 20% of that with NAD. With Mn(2+) the K(m) for malate(2-) is lower than with Mg(2+), but V(max) is lower than with Mg(2+). In the forward (malate-decarboxylating) direction with NAD, the kinetic parameters are essentially like those observed for the enzyme from C(3) plants. In the reverse reaction, run with Mn(2+), the activity is 1.5% of that in the forward reaction. The equilibrium constant is 1.1 x 10(-3) molar.The kinetic mechanism of the reaction, at least in the forward direction, is sequential, with apparently random binding of all reaction components. Product inhibition patterns confirm this.The enzyme displays a strong hysteretic lag, which is shortened by high enzyme concentrations, high substrate concentrations, and the presence of the product NADH.The enzyme is activated by coenzyme A with K(a) = 4 micromolar. AMP also shows competitive activation, with K(a) = 24 micromolar. The activation by coenzyme A and AMP is additive, implying separate sites for their binding. Phosphoenolpyruvate activates the reaction at low (micromolar) concentrations, but higher concentrations of phosphoenolpyruvate cause deactivation. Fumarate(2-) is a strong activator, with K(a) = 0.3 millimolar. Fructose-1,6-bisphosphate activates the enzyme, but its most pronounced effect is in shortening the lag. Citrate is a competitive inhibitor of malate, with K(i) = 4.9 millimolar.

Slow Transients in the Activity of the NAD Malic Enzyme from Crassula.

The NAD malic enzyme from Crassula argentea shows a slow reaction transient in the form of a lag before reaching a steady-state rate in assays. This lag, which has a half-time or tau ranging from seconds to many minutes under various conditions, poses problems in the interpretation of kinetic data with this enzyme. The NAD malic enzyme from Kalanchoë daigremontiana has a similar lag.The lag is greatest in freshly prepared enzyme and diminishes with storage at -70 degrees C, but the activity of the enzyme also diminishes with storage.The lag is inversely proportional to the concentration of enzyme, both in the assay and in storage prior to assay. The lag is also inversely proportional to the concentration of malate used in the assay, which poses particular problems because the lag with low malate concentrations may be so long that activity begins to be lost before the steady-state rate is reached.Various buffer ions produce different lags, but the lag with all buffers is longer than in the absence of buffer. The effectors CoA and SO(4) (2-) in the assay substantially decrease the lag. The lag is shorter with Mn(2+) as the required divalent cation than when Mg(2+) is used.The response of enzyme activity to pH shows that the intrinsic activity is greater with magnesium than with manganese, although the rate actually attained is lower with Mg(2+) because the pK values for the response to pH are closer together when that cation is used. The enzyme has a higher optimum pH and a broader response to pH when Mn(2+) is used. The change in lag with pH follows the general pattern of activity with longer lags at intermediate pH values.Preincubation of the enzyme with various reaction components and effectors reduces the lag, with NADH being the most effective. The presence of NADH in the assay is much more effective, but none of the treatments tried will completely eliminate the lag of freshly prepared enzyme.

Malate Dehydrogenase and NAD Malic Enzyme in the Oxidation of Malate by Sweet Potato Mitochondria.

Over a range of concentrations from less than 0.1 mm to more than 70 mm, sweet potato root mitochondria display a bimodal substrate saturation isotherm for malate. The high affinity portion of the isotherm has an apparent Km for malate of 0.85 mm and fits a rectangular hyperbolic function. The low affinity portion of the isotherm is sigmoid in character and gives an apparent S(0.5) of 40.6 mm and a Hill number of 3.7.Extracts of sweet potato mitochondria contain both malate dehydrogenase and NAD malic enzyme. The malate dehydrogenase, assayed in the forward direction at pH 7.2, shows typical Michaelis-Menten kinetics with a Km for malate of 0.38 mm. The NAD malic enzyme shows pronounced sigmoidicity in response to malate with a Hill number of 3.5 and an S(0.5) of 41.6 mm.On the basis of the normal kinetics, the Km, and the fact that oxaloacetate production from malate by mitochondria appears most active at low malate concentrations, the high affinity portion of the malate isotherm with mitochondria is attributed to malate dehydrogenase. The low affinity portion of the malate isotherm with mitochondria is thought, on the basis of the similarity of S(0.5) values, the Hill numbers, and the greater production of pyruvate from malate at high malate concentrations, to represent the activity of the NAD malic enzyme.