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11.
A general method is described for the assay of glycosyltransferase activity, which makes use of synthetic glycoside acceptors attached to hydrophobic aglycones. The products formed by incubation of an enzyme with acceptor and radiolabelled sugarnucleotide can then be rapidly (one minute) separated from interfering radioactivity by adsorption on to reverse-phase C-18 cartridges. After aqueous washing, products are easily isolated by elution with methanol. The utility of the method for the assay of (1–4)galactosyltransferase, (1–2)fucosyltransferase andN-acetylglucosaminyltransferase I and V is demonstrated. 相似文献
12.
Endogenous opioid peptides (EOP) and prior exercise may modulate the stimulatory effect of glucose on insulin secretion. To gain insights into these relationships, we studied male Wistar rats (187-245 g) during sustained hyperglycemia by use of the glucose clamp technique. Four groups of sedentary fed rats (n = 8/group) either ran (Ex) at 24 m/min, 0% grade, or rested (R) for 40 min. Thirty minutes after Ex or R, arterial blood glucose was elevated to and maintained at 11 mM for 2 h by a variable glucose infusion. At the start of Ex or R rats had saline (Sal) or naloxone (Nal, an opioid antagonist) intravenous infusions for 160 min (40 min Ex + 30 min R + 90 min of a 120-min glucose clamp). Steady-state glucose infusion rates (SSGIR) were approximately 55 mg.kg-1.min-1 at the start of the clamp and declined significantly over the 2nd h to approximately 45 mg.kg-1.min-1. No significant differences existed in SSGIR between groups. R-Sal and Ex-Sal groups did not differ in their insulin response to hyperglycemia. In contrast, when all groups were compared at the end of the Nal or Sal infusion, Ex-Nal had the lowest insulin concentration (749 +/- 174 pmol/l), whereas the R-Nal group had the highest (1,581 +/- 216 pmol/l, P less than 0.05). These data suggest a stimulatory role for EOP on insulin secretion that is expressed after a prior stress (Ex). Thus one function of exercise-induced activation of EOP may be to regulate insulin secretion in the immediate postexercise period. 相似文献
13.
Lakhu M. Keshvara Elizabeth M. Newton A. Heather Good Ole Hindsgaul Monica M. Palcic 《Glycoconjugate journal》1992,9(1):16-20
ELISA assays have been developed for (1–3)N-acetylgalactosaminyltransferase (blood group A transferase) and (1–3)galactosyltransferase (blood group B transferase) activities. In these assays, microtitre plates coated with the bovine serum albumin conjugate of a synthetic Fuc1–2Gal-R acceptor substrate are incubated with the appropriate nucleotide donor (UDP-GalNAc or UDP-Gal) and human serum as the enzyme source. The resulting trisaccharide products Fuc1–2(GalNAc1–3)Gal-R-BSA or Fuc1–2(Gal1–3)Gal-R-BSA are detected and quantified with monoclonal antibodies selected not to cross-react with the substrate structure. With less than a microliter of human serum, product formation is proportional to enzyme concentration and to time of incubation of up to 90 min. 相似文献
14.
Ole Olsen Rainer Borriss Ortwin Simon Karl Kristian Thomsen 《Molecular & general genetics : MGG》1991,225(2):177-185
Summary Hybrid (1-3,1-4)--glucanase genes were constructed by extension of overlapping segments of the (1-3,1-4)--glucanase genes from Bacillus amyloliquefaciens and B. macerans generated by the polymerase chain reaction (PCR). Four hybrid genes were expressed in Escherichia coli cells. The mature hybrid enzymes contain a 16, 36, 78, or 152 amino acid N-terminal sequence derived from B. amyloliquefaciens (1-3,1-4)--glucanase followed by a C-terminal segment derived from B. macerans (1-3,1-4)--glucanase. Biochemical characterization of parental and hybrid enzymes shows a significant increase in thermostability of three of the hybrid enzymes when exposed to an acidic environment thus combining two important enzyme characteristics within the same molecule. At pH 4.1, 85%-95% of the initial activity was retained after 1 h at 65° C in contrast to 5% and 0% for the parental enzymes from B. amyloliquefaciens and B. macerans. After 60 min incubation at 70° C, pH 6.0, the parental enzymes retained 5% or less of the initial activity whilst one of the hybrids still exhibited 90% of the initial activity. Of the parental enzymes B. macerans (1-3,1-4)--glucanase had the lower specific activity while the hybrid enzymes exhibited specific activities that were 1.5- to 3-fold higher. These experimental results demonstrate that exchange of homologous gene segments from different species may be a useful technique for obtaining new and improved versions of biologically active proteins.Abbreviations AMY
mature form of Bacillus amyloliquefaciens (1-3,1-4)--glucanase;
- MAC
mature form of B. macerans (1-3,1-4)--glucanase
- SUB
mature form of B. subtilis (1-3,1-4)--glucanase
- H(A16-M), H(A36-M), H(A78-M), H(A107-M), H(A152-M)
mature forms of hybrid enzymes having 16, 36, 78, 107, 152 N-terminal amino acids, respectively, derived from AMY with the remaining amino acids derived from MAC 相似文献
15.
The results of genome analysis of five hybrids, viz.Elymus patagonicus ×Hordeum procerum, E. patagonicus ×H. tetraploidum, E. angulatus ×H. jubatum, E. angulatus ×H. lechleri, andE. angulatus ×H. parodii, are reported. The genomic constitution ofHordeum tetraploidum andH. jubatum is best given as H1H1H2H2, ofH. lechleri andH. parodii as H1H1H2H2H4H4, ofH. procerum as H1H1H2H2H3H3, and ofElymus patagonicus andE. angulatus as SSH1H1H2H2. 相似文献
16.
Else K. Hoffmann Ian H. Lambert Lars Ole Simonsen 《The Journal of membrane biology》1986,91(3):227-244
Summary The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K+ and Cl– transport pathways. RVD is accelerated when a parallel K+ transport pathway is provided by addition of gramicidin, indicating that the K+ conductance is rate limiting. Addition of ionophore A23187 plus Ca2+ also activates separate K+ and Cl– transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K+ and Cl– conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl– conductance is rate limiting. An A23187-induced activation of42K and36Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca2+-activated K+ channel. (ii) unaffected by substitution of NO
3
–
or SCN– for Cl–, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K+ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl– conductance. The Cl– conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl– transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca2+-free media (probably by release of Ca2+ from internal stores) is also transient, whereas the activation is persistent in Ca2+-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl– transport pathway. These findings suggest that a transient increase in free cytosolic Ca2+ may account for the transient activation of the Cl– transport pathway. The activated anion transport pathway is unselective, carrying both Cl–, Br–, NO
3
–
, and SCN–. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl– transport pathway and also blocks the activation of the K+ transport pathway. This is demonstrated directly by42K flux experiments and indirectly in media where the dominating anion (SCN–) has a high ground permeability. A comparison of the A23187-induced K+ conductance estimated from42K flux measurements at high external K+, and from net K– flux measurements suggests single-file behavior of the Ca2+-activated K+ channel. The number of Ca2+-activated K+ channels is estimated at about 100 per cell. 相似文献
17.
Summary The effect of cholecystokinin (CCK) and internal Ca2+ on outward K+ current in isolated pig pancreatic acinar cells has been investigated using the patch-clamp method for whole-cell current recording under voltage-clamp conditions. CCK (2 × 10–10
M) applied to the bath evoked a marked increase in the outward K+ current associated with depolarizing voltage steps, and this effect was fully reversible and acutely dependent on the presence of external Ca2+. When strongly buffered Ca2+-EGTA solutions were used inside the cells CCK failed to evoke an effect. Increasing the internal Ca2+ concentration ([Ca2+]
i
) from 5 × 10–10
M to 10–7 and 5 × 10–7
M mimicked the effect of CCK. It would appear therefore that CCK controls K+ conductance in the acinar cells via changes in the internal free ionized Ca2+ concentration. 相似文献
18.
The regional distribution of L-homocysteine (Hcy) was determined in brains from mouse, rat, guinea pig, and rabbit, using a sensitive radioenzymatic assay. Large interspecies variations in the Hcy content in various parts of the brain were observed, but cerebellum contained the highest amount in all species investigated. In the rat the amount of Hcy in cerebellum (6.4 nmol/g) was about sixfold higher than in most other parts of the brain, whereas in the mouse and guinea pig the amount in cerebellum (about 1 nmol/g) was only twofold higher than in the other brain regions. There was a remarkably high level of Hcy in all regions of the rabbit brain (4-10 nmol/g); the highest concentration was found in the cerebellar white matter. In this species the amount of Hcy in all brain regions examined exceeded that in the liver. 相似文献
19.
20.
Ole G. Mouritsen 《生物化学与生物物理学报:生物膜》1983,731(2):217-221
The gel-to-fluid first-order melting transition of lipid bilayers is simulated by the use of a microscopic interaction model which includes a variable number of lipid-chain conformational states. The results suggest that the experimental observation of ‘continuous melting’ in pure wet lipid bilayers, rather than being ascribed to the presence of impurities, may be explained as a result of kinetically caused metastability of intermediate lipid-chain conformations. 相似文献