The C-terminal domain of dimeric serine hydroxymethyltransferase plays a key role in stabilization of the quaternary structure and cooperative unfolding of protein: domain swapping studies with enzymes having high sequence identity

The serine hydroxymethyltransferase from Bacillus subtilis (bsSHMT) and B. stearothermophilus (bstSHMT) are both homodimers and share approximately 77% sequence identity; however, they show very different thermal stabilities and unfolding pathways. For investigating the role of N- and C-terminal domains in stability and unfolding of dimeric SHMTs, we have swapped the structural domains between bs- and bstSHMT and generated the two novel chimeric proteins bsbstc and bstbsc, respectively. The chimeras had secondary structure, tyrosine, and pyridoxal-5'-phosphate microenvironment similar to that of the wild-type proteins. The chimeras showed enzymatic activity slightly higher than that of the wild-type proteins. Interestingly, the guanidium chloride (GdmCl)-induced unfolding showed that unlike the wild-type bsSHMT, which undergoes dissociation of native dimer into monomers at low guanidium chloride (GdmCl) concentration, resulting in a non-cooperative unfolding of enzyme, its chimera bsbstc, having the C-terminal domain of bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding from native dimer to unfolded monomer. In contrast, the wild-type dimeric bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding, whereas its chimera bstbsc, having the C- terminal domain of bsSHMT, showed dissociation of native dimer into monomer at low GdmCl concentration and a GdmCl-induced non-cooperative unfolding. These results clearly demonstrate that the C-terminal domain of dimeric SHMT plays a vital role in stabilization of the oligomeric structure of the native enzyme hence modulating its unfolding pathway.     

Guanidine Hydrochloride Denaturation of Glycosylated and Deglycosylated Stem Bromelain

Glycosylation is one of the major naturally occurring covalent modifications of proteins. We have used stem bromelain, a thiol protease with a single, N-glycosylated polypeptide chain as a model to investigate the role of glycosylation of proteins. Periodate oxidation was used to obtain the deglycosylated form of the enzyme. Denaturation studies in the presence of guanidine hydrochloride (Gn·HCl) were performed using fluorescence and circular dichroism spectroscopy. The glycosylated stem bromelain was found to be stabilized by 1.9 kcal/mol as compared to the deglycosylated one. At a given concentration of denaturant, the fraction of denatured protein was higher in the case of deglycosylated stem bromelain. In short, deglycosylated bromelain showed more susceptibility towards guanidine hydrochloride denaturation, indicating the contribution of the carbohydrate part of the glycoprotein to the stability of the enzyme.     

Peroxidation of liposomal palmitoyllinoleoylphosphatidylcholine (PLPC), effects of surface charge on the oxidizability and on the potency of antioxidants

Peroxidation of membrane phospholipids is an important determinant of membrane function. Previously we studied the kinetics of peroxidation of the polyunsaturated fatty acid (PUFA) residues in model membranes (liposomes) made by sonication of palmitoyllinoleoylphosphatidylcholine (PLPC). Since most biomembranes are negatively-charged, we have now studied the effect of negative surface charge on the kinetics of peroxidation of liposomes made of PLPC and 9% of one of the negatively-charged phospholipids phosphatidylserine (PS) or phosphatidic acid (PA). Peroxidation was initiated by either CuCl2 or AAPH and continuously monitored spectrophotometrically. The following results were obtained: (i) The negative charge had only a slight effect on AAPH-induced peroxidation, but accelerated markedly copper-induced peroxidation of the liposomes, probably by increasing the binding of copper to the membrane surface. (ii) Ascorbic acid (AA) inhibited AAPH-induced but promoted copper-induced peroxidation in all the studied liposomes, probably by enhancing the production of free radicals upon reduction of Cu(II) to Cu(I). (iii) alpha-tocopherol (Toc) inhibited AAPH-induced peroxidation in all the studied liposomes, whereas the effect of tocopherol on copper-induced peroxidation varied from being pro-oxidative in PA-containing liposomes, to being extremely anti-oxidative in PS-containing liposomes, even at very low tocopherol concentrations. The significance of the latter unusual protective effect, which we attribute to recycling of tocopherol by a PS-Cu complex, requires further investigation.     

Drug release from Kollicoat SR 30D-coated nonpareil beads: evaluation of coating level,plasticizer type,and curing condition

A newly available polyvinylacetate aqueous dispersion, Kollicoat SR 30D, was evaluated with respect to its ability to modulate the in vitro release of a highly water-soluble model compound (diphenhydramine hydrochloride) from nonpareil-based systems. Kollicoat SR 30D premixed with a selected plasticizer (10% wt/wt propylene glycol, 2.5% triethyl citrate, or 2.5% dibutyl sebacute), talc, and red #30 lake dye was coated onto the drug beads in an Aeromatic Strea I fluid-bed drier with a Wurster insert using bottom spray. With propylene glycol as the plasticizer, increases in polymer coating level retarded drug release from beads in a stepwise fashion along with apparent permeability, indicating a consistent release mechanisms. Stability studies at 40°C/75% RH revealed gradual decreases in dissolution rate, and additional curing studies further confirmed the dependence of release kinetics on curing condition. Furthermore, the type of plasticizer was found to play a key role. Unplasticized formulations exhibited the fastest dissolution, followed by formulations plasticized with triethyl citrate, propylene glycol, and dibutyl sebacate. All 4 formulations (unplasticized and plasticized), nevertheless, revealed a marked difference between uncured and cured dissolution profiles. Kollicoat SR 30D has, thereby, been demonstrated to effectively retard drug release from nonpareilbased systems. However, selected plasticizer type and subsequent curing condition play important roles in controlling drug release from such a system.     

Kinetics of protein aggregation. Quantitative estimation of the chaperone-like activity in test-systems based on suppression of protein aggregation

The experimental data on the kinetics of irreversible aggregation of proteins caused by exposure to elevated temperatures or the action of denaturing agents (guanidine hydrochloride, urea) have been analyzed. It was shown that the terminal phase of aggregation followed, as a rule, first order kinetics. For the kinetic curves registered by an increase in the apparent absorbance (A) in time (t) the methods of estimation of the corresponding kinetic parameters A _lim and k _I (A _lim is the limiting value of A at t and k _I is the rate constant of the first order) have been proposed. Cases are revealed when the reaction rate constant k _I calculated from the kinetic curve of aggregation of the enzymes coincides with the rate constant for enzyme inactivation. Such a situation is interpreted as a case when the rate of aggregation is limited by the stage of denaturation of the enzyme. A conclusion has been made that, in order to establish the mechanism of protein aggregation, the kinetic investigations of aggregation should be carried out over a wide range of protein concentrations. The refolding experiments after denaturation of proteins by guanidine hydrochloride or urea have been also analyzed. It was shown that aggregation accompanying refolding follows first order kinetics at the final phase of the process. The model of protein refolding explaining such a kinetic regularity has been proposed. When aggregation of protein substrate follows first order kinetics, parameters A _lim and k _I may be used for the quantitative characterization of the chaperone-like activity in the test-systems based on suppression of protein aggregation.     

Bupivacaine hydrochloride induces muscle fiber necrosis and hydroxyl radical formation-dimethyl sulphoxide reduces hydroxyl radical formation

We induced acute skeletal muscle necrosis in rats using bupivacaine hydrochloride and found that both 2,5- and 2,3-dihydroxybenzoic acid significantly increased in skeletal muscle. A single administration of dimethyl sulphoxide, a free radical scavenger, significantly lowered concentrations of 2,5- and 2,3-dihydroxybenzoic acid. These results suggest that dimethyl sulphoxide is an effective hydroxyl radical scavenger and may be useful in the treatment of myopathy.     

Differences in the denaturation behavior of ribonuclease A induced by temperature and guanidine hydrochloride

Moderate temperatures or low concentrations of denaturants diminish the catalytic activity of some enzymes before spectroscopic methods indicate protein unfolding. To discriminate between possible reasons for the inactivation of ribonuclease A, we investigated the influence of temperature and guanidine hydrochloride on its proteolytic susceptibility to proteinase K by determining the proteolytic rate constants and fragment patterns. The results were related to changes of activity and spectroscopic properties of ribonuclease A. With thermal denaturation, the changes in activity and in the rate constants of proteolytic degradation coincide and occur slightly before the spectroscopically observable transition. In the case of guanidine hydrochloride-induced denaturation, however, proteolytic resistance of ribonuclease A initially increases accompanied by a drastic activity decrease far before unfolding of the protein is detected by spectroscopy or proteolysis. In addition to ionic effects, a tightening of the protein structure at low guanidine hydrochloride concentrations is suggested to be responsible for ribonuclease A inactivation.     

Apoflavodoxin (un)folding followed at the residue level by NMR

The denaturant-induced (un)folding of apoflavodoxin from Azotobacter vinelandii has been followed at the residue level by NMR spectroscopy. NH groups of 21 residues of the protein could be followed in a series of 1H-15N heteronuclear single-quantum coherence spectra recorded at increasing concentrations of guanidinium hydrochloride despite the formation of protein aggregate. These NH groups are distributed throughout the whole apoflavodoxin structure. The midpoints of unfolding determined by NMR coincide with the one obtained by fluorescence emission spectroscopy. Both techniques give rise to unfolding curves with transition zones at significantly lower denaturant concentrations than the one obtained by circular dichroism spectroscopy. The NMR (un)folding data support a mechanism for apoflavodoxin folding in which a relatively stable intermediate is involved. Native apoflavodoxin is shown to cooperatively unfold to a molten globule-like state with extremely broadened NMR resonances. This initial unfolding step is slow on the NMR chemical shift timescale. The subsequent unfolding of the molten globule is faster on the NMR chemical shift timescale and the limited appearance of 1H-15N HSQC cross peaks of unfolded apoflavodoxin in the denaturant range studied indicates that it is noncooperative.     

Regulation of G Protein-Coupled Receptor Kinase 2 in Brains of Opiate-Treated Rats and Human Opiate Addicts

Abstract: The effects of opiate drugs (heroin, morphine, and methadone) on the levels of G protein-coupled receptor kinase 2 (GRK2) were studied in rat and human brain frontal cortices. The density of brain GRK2 was measured by immunoblot assays in acute and chronic opiate-treated rats as well as in opiate-dependent rats after spontaneous or naloxone-precipitated withdrawal and in human opiate addicts who had died of an opiate overdose. In postmortem brains from human addicts, total GRK2 immunoreactivity was not changed significantly, but the level of the membrane-associated kinase was modestly but significantly increased (12%) compared with matched controls. In rats treated chronically with morphine or methadone modest increases of the enzyme levels (only significant after methadone) were observed. Acute treatments with morphine and methadone induced dose- and time-dependent increases (8–22%) in total GRK2 concentrations [higher increases were observed for the membrane-associated enzyme (46%)]. Spontaneous and naloxone-precipitated withdrawal after chronic morphine or methadone induced a marked up-regulation in the levels of total GRK2 in the rat frontal cortex (18–25%). These results suggest that GRK2 is involved in the short-term regulation of μ-opioid receptors in vivo and that the activity of this regulatory kinase in brain could have a relevant role in opiate tolerance, dependence, and withdrawal.     

复方黄连素片中盐酸小檗碱含量测定

目的：建立测试黄连素片中盐酸小檗碱含量的高效液相方法,并对此方法进行系统的方法学验证,以确保应用该方法测试的结果准确、可靠。方法：采用色谱柱：Agilent TC-C18柱（4．6 mm ×150 mm,5μm,Agilent,美国）,流动相：乙腈－0．05 mol／L磷酸二氢钾溶液（45∶55,含5％的0．05 mol／L庚烷磺酸钠溶液）,检测波长为265 nm。结果：盐酸小檗碱在2．5～80μg／mL范围内线性关系良好,得到线性回归方程为Y＝421 ．5X＋17．32,相关系数为0．9997（n＝6）;低、中、高浓度批内精密度的RSD值分别为0．30％、0．58％、0．30％,批间精密度的RSD为0．80％;重复性RSD为0．27％;低、中、高浓度的加样回收率分别为99．66％、99．88％、99．98％;供试品溶液室温放置8h稳定,RSD为0．41％。结论：本方法测定黄连素片中盐酸小檗碱的含量准确、可靠,操作简单、用时短,可用于黄连素片中盐酸小檗碱的含量测定。