Two methods for large-scale purification of recombinant human choline acetyltransferase

Choline acetyltransferase (ChAT) catalyzes the transfer of an acetyl group from acetyl-CoA to choline to produce the neurotransmitter acetylcholine (ACh). We have produced large quantities of pure human ChAT using two different bacterial expression systems. In the first, ChAT is fused to a chitin-binding domain via a self-cleavable linker allowing the release of ChAT without the use of proteases. In the second, ChAT is fused to a hexahistidine (His6) tag at the N-terminus with a linker incorporating a TEV protease cleavage site. In both cases, pure ChAT was produced that has a final specific activity of approximately 50 micromol ACh/min/mg and is suitable for structural characterization. Analysis of purified ChAT by Western blots and mass spectrometry revealed that the C-terminal 15 amino acids were slowly removed by endogenous proteolytic activity, to produce a stable 615 residue protein. Furthermore, we show that purified recombinant human ChAT is highly prone to oxidation, leading to the formation of covalent dimers and/or a loss of catalytic activity. Kinetic parameters of our purified proteins were obtained and, when compared to previously published constants for human placental ChAT, we found that recombinant human ChAT displays lower values for Michaelis and inhibition constants for ACh, which may be due to the complete absence of post-translational modifications.     

Synthesis of a naphthylvinylpyridine derivative and its use for affinity chromatography of choline acetyltransferase

N-(10-carboxy)decamethylene-4(1-naphthylvinyl)pyridinium chloride, a derivative of the choline acetyltransferase (CAT) inhibitor naphthylvinylpyridine (NVP) was synthesized and used as a ligand for affinity chromatography of choline acetyltransferase. The preparation of this inhibitor included the quaternization of naphthylvinylpyridine with 11-Br-undecanoic acid methyl ester to obtain N-(10-carbomethoxy)decamethylene-4-(1-naphthylvinyl)pyridinium bromide, followed by hydrolysis to free the carboxylic group. This inhibitor (C11-NVP+) had a potency comparable to that of N-methyl-4(1-naphthylvinyl) pyridinium iodide (C1-NVP+) which is the most potent derivative of NVP but which lacks a functional group for conjugation to Sepharose. The C11-NVP+ was then bound through the carboxylic group to aminoalkyl Sepharose by a carbodiimide promoted condensation reaction. Interaction of CAT with the inhibitor retarded its elution from a column of Sepharose-C11-NVP+ and permitted the purification of the enzyme to electrophoretic homogeneity starting from a preparation in which CAT represented about 20% of the total proteins. Conventional procedures of protein purification had previously been unsuccessful in isolating the enzyme in pure form. Inhibition studies showed that CAT could exhibit either a "high" or a "low" sensitivity to inhibition by naphthylvinylpyridine and its derivatives (I50 with C1-NVP+ = 0.57 microM or 5.2 microM). A direct relationship existed between the sensitivity of CAT to these inhibitors and the retention of the enzyme by the affinity column.     

Localization of the choline acetyltransferase (CHAT) gene to human chromosome 10

A cDNA clone encoding the complete sequence of porcine choline acetyltransferase (CHAT) isolated by S. Berrard et al. (1987, Proc. Natl. Acad. Sci. USA 84: 9280-9284) was hybridized to TaqI digests of a panel of 25 human-rodent somatic cell hybrids and to a complementary panel of 10 human-rodent hybrids in order to determine the chromosomal localization of human CHAT. To enhance the detection of the human signal, hybridization and washings were performed under low stringency conditions on membranes presaturated with sonicated DNA from parental rodent strains. All informative human fragments had the same distribution among the hybrids, mapping CHAT to a single human chromosome. CHAT was assigned to chromosome 10 because all other chromosomes were eliminated by exclusion based on the analysis of the signal segregation. This result indicates that mutation of the CHAT gene cannot be responsible for the primary defect in familial Alzheimer's disease.     

Antigen Binding Lymphocytes in Congenitally Athymic (Nude) Mice

THE autoradiographic detection of the binding of various radiolabelled antigens to a proportion of lymphocytes from animals not exposed to those antigens (“nonprimed” lymphocytes) is well documented^1–4. Such lymphocytes are thought to have patches of surface immunoglobulin, primarily IgM, which act as specific receptors for antigen^5,6. A proportion at least of these unprimed lymphocytes are immunologically competent as shown in vivo^7,8 and hence are true antigen reactive cells. Most assays have used peripheral lymphocyte suspensions from tissues of man, mouse, rat and chicken, not enriched or fractionated in any way for the two distinct lines of lymphocytes, thymic derived (T) and non-thymic derived (B)⁹. It is not clear whether antigen-binding cells (ABC), detected in routine assays where autoradiographs are exposed for 1–2 weeks, are of both T and B cell type or are predominantly of only one type. Experiments using unlabelled and radiolabelled immunoglobulin antisera with isolated T and B cells have inferred specific antigen binding on both populations although T cells seem to have far fewer antigen binding receptors than B cells¹⁰.     

Properties of choline acetyltransferase from the bulb miteRhizoglyphus echinopus (Acari: Acaridae)

In the presence of exogenous acetyl coenzyme A¹⁴-C and choline, the 20 000 g supernatant fraction of whole bulb-mite homogenates synthesized a radioactive product that chromatographed with authentic acetylcholine (ACh). ApparentK _m values were 0.12 and 1.14 mM for acetyl coenzyme A and choline, respectively, and the average rate of ACh synthesis was 2.06 mol h^–1 g^–1 equivalent of mite tissue (wet weight). Choline acetyltransferase (ChAT) activity was inhibited by 5,5-dithiobis(2-nitrobenzoic acid), suggesting the presence of functionally important sulfhydryl groups. However, ChAT activity was not inhibited by 27 acaricides from several different chemical classes, except for the thiazolidine flubenzimine which gave a pI₅₀ of 3.5 mol l^–1.Contribution from the Missouri Agricultural Experiment Station, Columbia, Missouri, Journal Series No. 11 157.     

Developmental changes of choline acetyltransferase (ChAc) activity in chick embryo spinal and sympathetic ganglia

The ChAc activity of spinal and sympathetic ganglia was measured throughout the embryonic life of the chick. In spinal ganglia, the ChAc activity reached a first peak when the maximal proliferation of neuroblasts occurred. Then, the relative ChAc activity decreased. After the 12th day of incubation, the enzyme activity increased again and reached a second peak on the 16th day. In sympathetic ganglia, the general course of the development of ChAc activity was similar to spinal ganglia. However, higher enzymic activity was found. Furthermore, the earlier peak of ChAc activity occurred 48 hr later than the corresponding peak in spinal ganglia. The behaviour of ChAc activity in these two areas of the developing nervous system is interpreted as a function of their histogenesis.     

Calotropis procera (Sodom apple)--a potential material for enzyme purification

A simple method based on precipitation with Calotropis procera latex was developed for the purification of crude enzyme from fermentation broth. C. procera latex (10(-2) dilution) clarified and concentrated the crude amylase of Aspergillus oryzae 4-fold with 97% recovery of the initial amylase activity in the filtrate in a single step operation. The latex was stable at pH < or = 4.5 and there was no significant difference (P < or = 0.05) in the purification potential of the latex at 4 and 28 degrees C. This method could prove useful to developing economies of the tropics where ambient temperature is around 28 degrees C.     

A simple method for monitoring chromatography column eluates for laccase activity during enzyme purification

A simple and economical method is described that allows rapid detection of laccase activity in chromatography column fractions during enzyme purification. Aliquots of column eluants are applied to filter paper coated with 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) containing a numbered grid, and incubated at ambient temperature for 20 min. Indications of enzyme activity are simply observed by a colour change. This method avoids having to manually assay each fraction of a chromatographic run for enzyme activity.     

Affinity chromatography of peptidylarginine deiminase from rabbit skeletal muscle on a column of soybean trypsin inhibitor (Kunitz)-Sepharose

We designed a simple procedure for the purification of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle using substrate affinity chromatography. Of the immobilized substrate ligands tested, i.e. protamine and soybean trypsin inhibitor (Kunitz) (STI), STI-Sepharose was found to be an effective affinity adsorbent for purification of the enzyme. The specific binding of peptidylarginine deiminase to STI-Sepharose was observed in the presence of calcium ion, and the enzyme could be selectively eluted from the affinity adsorbent by washing with chelator. A 1,800-fold purification with a 50% yield was achieved in the three-step procedure, which involved DEAE-Sephacel ion-exchange and STI-Sepharose affinity chromatography. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity and the recovery were considerably higher than have been obtained by any procedures previously reported. The specific interaction of peptidylarginine deiminase with STI immobilized on Sepharose was also investigated quantitatively by frontal affinity chromatography. In this method, a peptidylarginine deiminase solution was applied continuously to an STI-Sepharose column and the retardation of the elution front was measured as a parameter of the strength of the interaction. The dissociation constant for the enzyme with STI was found to be 2.3 X 10(-7)M. This value was in good agreement with that obtained by kinetic analysis in our previous studies. Peptidylarginine deiminase required millimolar Ca2+ for the binding to STI-Sepharose. The Ca2+ dependence of the enzyme binding was quite similar to that of the enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coexistence of GABA-and choline acetyltransferase (ChAT)-like immunoreactivity in the hypoglossal nucleus of the rat

Summary Single and sequential double immunocytochemical techniques were applied to localize gamma-aminobutyric acid (GABA)-and choline acetyltransferase (ChAT)-like immunoreactivity (-LI) in the hypoglossal nucleus of the rat. After subsequential double staining a relatively high number of hypoglossal motor neurons showed the coexistence of both ChAT-and GABA-LI. Coexistence of both substances was also revealed in the axons of the hypoglossal nerve situated within the medulla oblongata. Cells showing only ChAT-or GABA-LI were also observed. Differences in immunostaining between the different cell groups of the hypoglossal nucleus were established.Following axotomy of the right hypoglossal nerve, a decrease or loss of the immunoreactivity for both ChAT and GABA in the motor neurons was established until the 3rd week after the operation. The results obtained do not give evidence on the origin of the GABA-like immunoreactive material and its functional significance in the cholinergie neurons. It can be only speculated that the GABA-like material is either taken up from the intercellular space or is synthesized by the ChAT-LI nerve cells. Functionally, the importance of GABA for the synthesis of gamma-hydroxybutyrate (a novel neurotransmitter candidate) and its postsynaptic transmitter action or presynaptic regulatory action (through autoreceptors in the membrane of the nerve endings) on the release of acetylcholine (ACh) should be taken into consideration.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

抗人CD19单链抗体基因的构建、表达及功能测定

采用RT-PCR方法从分泌抗人类白细胞表面分化抗原CD19单克隆抗体的杂交瘤细胞中克隆出V_H和V_L可变区基因,再通过重叠延伸拼接（spliceoverlap extension）PCR方法在V_H和V_L可变区基因之间引入连接肽(Gly4Ser)3,体外构建抗人CD19单链抗体（抗CD19-ScFv）基因。将其克隆至表达载体PET28a并在大肠杆菌中表达。SDS-PAGE和Westernblot分析结果表明,抗CD19-ScFv在BL21（DE3）菌中获得表达,重组蛋白的相对分子量为27kD,表达产物以不溶性包涵体形式存在,经过溶解包涵体,镍柱亲和层析纯化和体外复性过程,获得了高纯度的单链抗体片段。流式细胞分析结果证实抗CD19ScFv可与人类白细胞表面的分化抗原CD19结合,保留了鼠源性单抗与CD19结合活性。抗人CD19-ScFv的构建与表达,为下一步针对B淋巴系统恶性肿瘤的靶向治疗奠定了基础。     

Coexistence of GABA- and choline acetyltransferase (ChAT)-like immunoreactivity in the hypoglossal nucleus of the rat

Single and sequential double immunocytochemical techniques were applied to localize gamma-aminobutyric acid (GABA)- and choline acetyltransferase (ChAT)- like immunoreactivity (-LI) in the hypoglossal nucleus of the rat. After subsequential double staining a relatively high number of hypoglossal motor neurons showed the coexistence of both ChAT- and GABA-LI. Coexistence of both substances was also revealed in the axons of the hypoglossal nerve situated within the medulla oblongata. Cells showing only ChAT- or GABA-LI were also observed. Differences in immunostaining between the different cell groups of the hypoglossal nucleus were established. Following axotomy of the right hypoglossal nerve, a decrease or loss of the immunoreactivity for both ChAT and GABA in the motor neurons was established until the 3rd week after the operation. The results obtained do not give evidence on the origin of the GABA-like immunoreactive material and its functional significance in the cholinergic neurons. It can be only speculated that the GABA-like material is either taken up from the intercellular space or is synthesized by the ChAT-LI nerve cells. Functionally, the importance of GABA for the synthesis of gamma-hydroxybutyrate (a novel neurotransmitter candidate) and its postsynaptic transmitter action or presynaptic regulatory action (through autoreceptors in the membrane of the nerve endings) on the release of acetylcholine (ACh) should be taken into consideration.     

Overexpression of (His)6-tagged human arginase I in Saccharomyces cerevisiae and enzyme purification using metal affinity chromatography

Arginase (EC 3.5.3.1; L-arginine amidinohydrolase) is a key enzyme of the urea cycle that catalyses the conversion of arginine to ornithine and urea, which is the final cytosolic reaction of urea formation in the mammalian liver. The recombinant strain of the yeast Saccharomyces cerevisiae that is capable of overproducing arginase I (rhARG1) from human liver under the control of the efficient copper-inducible promoter CUP1, was constructed. The (His)(6)-tagged rhARG1 was purified in one step from the cell-free extract of the recombinant strain by metal-affinity chromatography with Ni-NTA agarose. The maximal specific activity of the 40-fold purified enzyme was 1600 μmol min(-1) mg(-1) protein.     

Coexpression of glutamate vesicular transporter (VGLUT1) and choline acetyltransferase (ChAT) proteins in fetal rat hippocampal neurons in culture

A very small population of choline acetyltransferase (ChAT) immunoreactive cells is observed in all layers of the adult hippocampus. This is the intrinsic source of the hippocampal cholinergic innervation, in addition to the well-established septo-hippocampal cholinergic projection. This study aimed at quantifying and identifying the origin of this small population of ChAT-immunoreactive cells in the hippocampus at early developmental stages, by culturing the fetal hippocampal neurons in serum-free culture and on a patternable, synthetic silane substrate N-1 [3-(trimethoxysilyl) propyl] diethylenetriamine. Using this method, a large proportion of glutamatergic (glutamate vesicular transporter, VGLUT1-immunoreactive) neurons, a small fraction of GABAergic (GABA-immunoreactive) neurons, and a large proportion of cholinergic (ChAT-immunoreactive) neurons were observed in the culture. Interestingly, most of the glutamatergic neurons that expressed glutamate vesicular transporter (VGLUT1) also co-expressed ChAT proteins. On the contrary, when the cultures were double-stained with GABA and ChAT, colocalization was not observed. Neonatal and adult rat hippocampal neurons were also cultured to verify whether these more mature neurons also co-express VGLUT1 and ChAT proteins in culture. Colocalization of VGLUT1 and ChAT in these relatively more mature neurons was not observed. One possible explanation for this observation is that the neurons have the ability to synthesize multiple neurotransmitters at a very early stage of development and then with time follows a complex, combinatorial strategy of electrochemical coding to determine their final fate.     

Leishmanial excreted factors (EFs): purification by affinity chromatography

Leishmania species grown in culture excrete a polyanionic, carbohydrate-rich factor (EF) which binds to antibodies produced in rabbits against the parent Leishmania species. EF, previously purified by physical and chemical methods, was purified by affinity chromatography on a Ricinus lectin column. The purified samples were characterised and analysed. The results show a notable proportion of galactose in EF and clarify the reasons for its polyanionic properties. Heterogenicity of EF is demonstrated and discussed.     

Modified microassay for the rapid and sensitive determination of choline acetyltransferase activity using (3H)-acetyl-CoA and Fonnum's extraction.

A modified procedure for the quantitative estimation of choline acetyltransferase activity in brain tissue based upon the formation of [3H]-ACh from [3H]-acetyl-CoA is described. The labelled ACh is isolated by a modification of Fonnum's procedure using sodium tetraphenyl borate in ketonic solution. The ChAc-activity is independent on the specific activity of the [3H]-acetyl-CoA used. The substrate blank is higher than with [14C]-labelled substrate but highly stable and reproducible. The method permits the determination of ChAc activity in less than 5 mug of brain tissue. 30-40 samples may be handled by one person per hour easily.