Purification and characterization of a second form of acid lipase in human liver.

We describe here the purification and characterization of a form of acid lipase from human liver (designated ALII), which differed from the more abundant Mr-29000 form (ALI). ALII was solubilized from frozen human liver with Triton X-100 and purified 8500-fold by chromatography over concanavalin A-sepharose, CM-cellulose and finally h.p.l.c. over a Mono S column. ALII migrated as a single band on polyacrylamide-gel electrophoresis in both the presence and the absence of SDS. The Mr of ALII was estimated to be 58,500 by SDS/polyacrylamide-gel electrophoresis. Gel filtration on Sephacryl S-200 gave an apparent Mr of 69,000. 4-Methylumbelliferyl (4MU) palmitate, cholesterol oleate and triolein were substrates for ALII, with apparent Vmax values of 5000, 1100 and 2500 nmol/min per mg respectively and Km values of 1.0, 1.5 and 1.8 mM respectively. Cholesterol oleate and triolein were hydrolysed optimally by ALII at pH 4.5, whereas 4MU palmitate was hydrolysed optimally at pH 5.3. Antisera were raised against ALI and ALII and, on immunoblot analysis, no antigenic similarity was observed between ALI and ALII. Cellulose acetate electrophoresis followed by reaction with 4MU palmitate revealed two forms of lipase, corresponding to ALI and ALII. The two enzymes were also separated by hydrophobic chromatography. The activity of ALII was stimulated by several proteins and was partially inhibited by millimolar concentrations of NaCl, CaCl2 and MgSO4.     

Characterization of purified human liver acid beta-D-galactosidases A2 and A3.

1. Human liver acid beta-galactosidase A2 and A3 were isolated by chromatography on concanavalin A-Sepharose 4B, Sepharose 6B, and Sepharose 4B-6-aminohexyl 1-thio-beta-D-galactopyranoside. beta-Galactosidase A2 and A3 were purified to final specific activities of 45.5 and 20.6 mumol/min per mg respectively with 4-methylumbelliferyl beta-D-galactopyranoside as substrate. 2. Form A2 had a mol.wt. of 150000 +/- 15000 (gel filtration) and appeared as a single band of protein (mol.wt 65000 +/- 1000) on electrophoresis in the presence of sodium dodecyl sulphate. 3. Form A3 had a mol.wt. (gel filtration) of 660000 +/- 66000. On electrophoresis in the presence of sodium dodecyl sulphate, form A3 appeared as a major band of protein (72% of total) of mol.wt. 65000 +/- 1000 and minor protein bands of mol.wt. 44000 +/- 1000 and 26,000 +/- 1000 and 22000 +/- 1000. 4. Gel-filtration chromatography of purified beta-galactosidase A3 generated approximately equal amounts of forms A3 and A2. beta-Galactosidase A1 was not detected by gel-filtration chromatography of partially or highly purified preparations of forms A2 and A3. 5. Both forms A2 and A3 had identical isoelectric points of 4.42 +/- 0.02. The data suggest that forms A2 and A3 are dimeric and multimeric forms of beta-galactosidase A1. 6. Amino acid analysis of beta-galactosidase A2 gave a ratio of acidic to basic amino acids of 2.6:1. 7. beta-Galactosidase A2 contained 7.5% carbohydrate by weight and sialic acid, D-galactose, D-glucosamine and D-mannose were present in the molar proportions 1.1:1.0:1.7:2.7.     

Feline GM1 gangliosidosis: characterization of the residual liver acid beta-galactosidase.

The residual liver acid beta-galactosidase (beta-gal) activity from a case of feline GM1 gangliosidosis was partially purified and characterized with respect to its pH optimum, kinetic properties, thermostability, isoelectric point, molecular weight, and antigenicity. In comparison to the normal enzyme, the mutant enzyme had the same pH optima for the three substrates tested, a reduced Km for 4-methylumbelliferyl-beta-gal, elevated Km's for GM1 and asialofetuin (ASF), and increased thermolability. In addition, the mutant beta-gal had a higher isoelectric point, a reduced molecular weight, and appeared to be antigenically different from normal. The results suggest that the mutation in the Birmingham GM1 cat is structural and that the residual enzyme activity is a structurally altered acid beta-gal. The apparent lack of antigenic identity between the mutant and normal enzymes, in contrast to the situation in many human GM1 patients, is most unusual.     

Binding sites of cholinesterases. Alkylation by an aziridinium derivative

1. Alkylation of acetylcholinesterase and butyrylcholinesterase by 2-chloro-N-(chloroethyl)-N-methyl-2-phenylethylamine was observed. This alkylating agent was more potent than related compounds previously described, and less stable (half-life 8.5min. at 23 degrees ). 2. Alkylation had effects on hydrolysis of substrates varying from activation for indophenyl acetate to inhibition for acetylcholine, and intermediate effects with five other substrates. The effects were on V(max.) and not K(m). 3. Alkylation caused a variety of changes in sensitivity to inhibition by five carbamates, five organophosphates and four other inhibitors, varying from total protection against tetraethylammonium to mildly enhanced sensitivity to urea. 4. The findings suggested the existence of three binding sites, one of which was anionic and another hydrophobic, in addition to the esteratic site.     

Intra-specific heterogeneity of the rDNA internal transcribed spacer in the Simulium damnosum (Diptera: Simuliidae) complex

The internal transcribed spacer (ITS) of the rRNA gene cluster has been used as a model for the study of the action of concerted evolution and molecular drive on repeated sequence families. In contrast to this general finding, preliminary DNA sequence analysis of cloned representatives of the ITS from the West African black fly species complex Simulium damnosum s.1. demonstrated extensive intra-individual and intra-specific polymorphisms. Variability in the ITS was primarily confined to the ITS1 domain. The degree and type of intra-individual and intra-specific variability within the ITS was further characterized using gel electrophoresis, DNA hybridization, and heteroduplex analysis of the PCR products generated from the ITS1 domain. ITS1 copies from individual S. damnosum s.1. differed in length and sequence composition. These results, when taken together, demonstrate that a large degree of intra-individual and intra-specific heterogeneity exists in the ITS of S. damnosum s.1. The intra-individual heterogeneity was greater in the savanna-dwelling than forest-dwelling sibling species of S. damnosum s.1. This heterogeneity may be due in part to inter-breeding among sympatric sibling species, coupled with disturbance of S. damnosum s.1. populations resulting from intensive vector control efforts.      

ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo.

Hsp90 is an abundant molecular chaperone essential to the establishment of many cellular regulation and signal transduction systems, but remains one of the least well described chaperones. The biochemical mechanism of protein folding by Hsp90 is poorly understood, and the direct involvement of ATP has been particularly contentious. Here we demonstrate in vitro an inherent ATPase activity in both yeast Hsp90 and the Escherichia coli homologue HtpG, which is sensitive to inhibition by the Hsp90-specific antibiotic geldanamycin. Mutations of residues implicated in ATP binding and hydrolysis by structural studies abolish this ATPase activity in vitro and disrupt Hsp90 function in vivo. These results show that Hsp90 is directly ATP dependent in vivo, and suggest an ATP-coupled chaperone cycle for Hsp90-mediated protein folding.     

Strategies for imaging mitophagy in high-resolution and high-throughput

The selective autophagic removal of mitochondria called mitophagy is an essential physiological signaling for clearing damaged mitochondria and thus maintains the functional integrity of mitochondria and cells. Defective mitophagy is implicated in several diseases, placing mitophagy as a target for drug development. The identification of key regulators of mitophagy as well as chemical modulators of mitophagy requires sensitive and reliable quantitative approaches. Since mitophagy is a rapidly progressing event and sub-microscopic in nature, live cell image-based detection tools with high spatial and temporal resolution is preferred over end-stage assays. We describe two approaches for measuring mitophagy in mammalian cells using stable cells expressing EGFP-LC3 – Mito-DsRed to mark early phase of mitophagy and Mitochondria-EGFP – LAMP1-RFP stable cells for late events of mitophagy. Both the assays showed good spatial and temporal resolution in wide-field, confocal and super-resolution microscopy with high-throughput adaptable capability. A limited compound screening allowed us to identify a few new mitophagy inducers. Compared to the current mitophagy tools, mito-Keima or mito-QC, the assay described here determines the direct delivery of mitochondrial components to the lysosome in real time mode with accurate quantification if monoclonal cells expressing a homogenous level of both probes are established. Since the assay described here employs real-time imaging approach in a high-throughput mode, the platform can be used both for siRNA screening or compound screening to identify key regulators of mitophagy at decisive stages.     

Linkage relationships of the human methylmalonyl CoA mutase to the HLA and D6S4 loci on chromosome 6

The human methylmalonyl CoA mutase (MCM) cDNA has been used to localize the MUT locus on the short arm of chromosome 6 proximal to the glyoxalase locus in 6p deletion cell lines. A HindIII polymorphism identified by the MCM cDNA was used to study linkage relationships of MUT to HLA (A-B-DQ-DR) and D6S4 in the reference CEPH families. The maximum lod score for MUT versus HLA was 3.04 at a recombination fraction of 0.28. The maximum lod score for MUT versus D6S4 was 22.93 at a recombination fraction of 0.01. These data suggest that MUT and D6S4 loci are tightly linked and may be used as one locus in a haplotype form for linkage studies on proximal 6p and diagnostic analysis of pedigrees with mut methylmalonic acidemia.