Inactivation of Staphylococcus hyicus lipase by hexadecylsulfonyl fluoride: evidence for an active site serine

The Staphylococcus hyicus lipase is an acyl hydrolase with broadsubstrate specificity including neutral glycerides and phospholipids.To obtain further insight into the mechanism of action of thisenzyme, we tested several sulfonyl fluorides as active site-directedinhibitors. The enzyme is resistant to the well-known serineprotease/esterase inhibitor phenylmethanesulfonyl fluoride (PMSF),but is rapidly inactivated by hexadecylsulfonyl fluoride. Thekinetics of inactivation were studied in Triton X-100 micelles.Inactivation is fast and the rate of inactivation is constantover the pH range where this lipase is active. Metal ions likeCa²⁺ and Sr²⁺ do not appreciably influence the rate of inactivation,although the enzymatic activity is significantly increased,suggesting a structural role for these ions. The S.hyicus lipasecontains a consensus sequence G-H/Y-S-X-G. Substitution by site-directedmutagenesis of this serine (Ser369) by a cysteine resulted ina mutant with only 0.2% residual activity. The activity of thismutant could not be inhibited with water-soluble sulfhydrylreagents either in the presence or absence of Triton X-100 micelles.In the presence of Triton X-100 micelles, inactivation of themutant occurred with 4-nitrophenylhexadecyl disulfide (t_1/2= 125 min) while the wild-type enzyme does not react at all.We conclude that Ser369 is the active site residue and thatin water this residue is inaccessible. Only after interfacialactivation Ser369 (or Cys369) becomes exposed and reacts withirreversible inhibitors.     

Parasitic energy barriers in SiGe HBTs

Parasitic energy barriers can easily be introduced during processing. Measurements and calculations of experimental n-p-n HBTs (heterojunction bipolar transistors) are presented, showing that a parasitic conduction-band barrier at the base-collector junction reduces the collector current and the cutoff frequency. A simple analytical model explains the f_T degradation, caused by the reduction of the collector current and a pileup of minority carriers in the base. With the model the effective height and width of the barrier can also be derived from the measured collector current enhancement factor I_C(SiGe)/I_C(Si)     

Effect of the gap size on the SSI efficiency of split-gate memory cells

In this paper, new experimental results on the injection efficiency of split-gate memory cells programmed in the source-side-injection mode are reported. It is shown that the gap size has a negligible effect on the cell injection efficiency and, when the read current is not a limiting factor, it can be made large in order to increase the breakdown voltage of the oxide in the gap region, thus enhancing the cell reliability without detrimental effects on the performance. The experimental data is interpreted with the aid of fullband Monte Carlo simulations.     

Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K

The catalytic contribution of His48 in the active site of porcinepancreatic phospholipase A2 was examined using site-directedmutagenesis. Replacement of His48 by lysine (H48K) gives riseto a protein having a distorted lipid binding pocket. Activityof this variant drops below the detection limit which is 10⁷-foldlower than that of the wild-type enzyme. On the other hand,the presence of glutamine (H48Q) or asparagine (H48N) at thisposition does not affect the structural integrity of the enzymeas can be derived from the preserved lipid binding propertiesof these variants. However, the substitutions H48Q and H48Nstrongly reduce the turnover number, i.e. by a factor of 10⁵.Residual activity is totally lost after addition of a competitiveinhibitor. We conclude that proper lipid binding on its ownaccelerates ester bond hydrolysis by a factor of 10². With theselected variants, we were also able to dissect the contributionof the hydrogen bond between Asp99 and His48 on conformationalstability, being 5.2 kJ/mol. Another hydrogen bond with His48is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycolinteracts with the enzyme. Its contribution to binding of theinhibitor in the presence of an interface was found to be 5.7kJ/mol.     

Two-dimensional linear-combination model fitting of magnetic resonance spectra to define the macromolecule baseline using FiTAID,a Fitting Tool for Arrays of Interrelated Datasets

Object  

To propose the determination of the macromolecular baseline (MMBL) in clinical 1H MR spectra based on T₁ and T₂ differentiation using 2D fitting in FiTAID, a general Fitting Tool for Arrays of Interrelated Datasets.     

The lipase from Staphylococcus aureus. Expression in Escherichia coli, large-scale purification and comparison of substrate specificity to Staphylococcus hyicus lipase

The genes coding for the mature part of the lipases from Staphylococcus aureus NCTC8530 and Staphylococcus hyicus have been cloned and overexpressed in Escherichia coli as fusion proteins with an N-terminal hexa-histidine tag. The enzymes accumulated in the cytoplasm and were purified using sequential precipitation with protamine sulphate and ammonium sulphate, followed by metal-affinity and hydroxyapatite chromatography. The yield of pure lipase was 4.5 mg/g wet cells for S. aureus lipase and 13 mg/g for S. hyicus lipase. The purified enzymes need calcium for activity, albeit with different affinities, and a low residual activity was found in the absence of calcium. In contrast to S. hyicus lipase, not only strontium but also barium can replace calcium with full retention of activity of S. aureus lipase. Whereas S. hyicus lipase is optimally active at pH 8.5, the optimum pH for enzymatic activity for S. aureus lipase was found to be pH 6.5. The S. aureus lipase has a narrow substrate specificity: short-chain triacylglycerols and acyl esters of both p-nitrophenol and umbelliferone are readily degraded, whereas medium- and long-chain lipids, as well as phospholipids, are poor substrates. In contrast, S. hyicus lipase prefers phospholipids as substrate and hydrolyses neutral lipids irrespective of their chain length. The results are discussed in view of the large sequence similarity between both lipases.     

Measurements of bandgap narrowing in Si bipolar transistors

Theory predicts appreciable bandgap narrowing in silicon for impurity concentrations greater than about 10¹⁷ cm^?3. This effect influences strongly the electrical behaviour of silicon devices, particularly the minority carrier charge storage and the minority carrier current flow in heavily doped regions. The few experimental data known are from optical absorption measurements on uniformly doped silicon samples. New experiments in order to determine the bandgap in silicon are described here. The bipolar transistor itself is used as the vehicle for measuring the bandgap in the base. Results giving the bandgap narrowing (ΔV_g0) as a function of the impurity concentration (N) in the base (in the range of 4.10¹⁵–2.5 10¹⁹ cm^?3) are discussed. The experimental values of ΔV_g0 as a function of N can be fitted by:

$\text{δV}{}_{\mathrm{g0}}\text{= V}{}_1\text{ln}\text{N}\text{N}{}_0\text{+}\text{ln}{}^2\text{N}\text{N}{}_0\text{+C}$

where V₁, N₀ and C are constants.It is also shown how the effective intrinsic carrier concentration (n_ie) is related with the bandgap narrowing (ΔV_g0).     

Electrothermal characterization of silicon-on-glass VDMOSFETs

Electrothermal consequences of implementing bulk-silicon RF power MOS processes in the silicon-on-glass substrate transfer technology are investigated in this paper. Fabricated silicon-on-glass vertical double-diffused MOSFETs are measured on-wafer and very large thermal resistance values are extracted for each design. The influence of the thermal resistance on RF performance is analyzed, and it is shown that strong electrothermal feedback severely lowers the power capability and strongly increases the operating temperature. A combination of low-thermal contacting and surface mounting to thermally conducting printed circuit board is shown to be very efficient in reducing the large thermal resistance. Numerical thermal simulations demonstrate that surface-mounted silicon-on-glass transistors can have lower thermal resistance than the bulk-silicon device with a wafer thickness reduced down to 100 μm.     

Hydropathy profile alignment: a tool to search for structural homologues of membrane proteins

Hydropathy profile alignment is introduced as a tool in functional genomics. The architecture of membrane proteins is reflected in the hydropathy profile of the amino acid sequence. Both secondary and tertiary structural elements determine the profile which provides enough sensitivity to detect evolutionary links between membrane proteins that are based on structural rather than sequence similarities. Since structure is better conserved than amino acid sequence, the hydropathy profile can detect more distant evolutionary relationships than can be detected by the primary structure. The technique is demonstrated by two approaches in the analysis of a subset of membrane proteins coded on the Escherichia coli and Bacillus subtilis genomes. The subset includes secondary transporters of the 12 helix type. In the first approach, the hydropathy profiles of proteins for which no function is known are aligned with the profiles of all other proteins in the subset to search for structural paralogues with known function. In the second approach, family hydropathy profiles of 8 defined families of secondary transporters that fall into 4 different structural classes (SC-ST1-4) are used to screen the membrane protein set for members of the structural classes. The analysis reveals that over 100 membrane proteins on each genome fall in only two structural classes. The largest structural class, SC-ST1, correlates largely with the Major Facilitator Superfamily defined before, but the number of families within the class has increased up to 57. The second large structural class, SC-ST2 contains secondary transporters for amino acids and amines and consists of 12 families.