Immunochemical studies on glutamic acid decarboxylase (EC 4.1.1.15) from mouse brain

Purified homogenous glutamic acid decarboxylase (GAD) from mouse brain and rabbit antiserum prepared to partially purified GAD gave only one sharp precipitin band in the Ouchterlony double diffusion test. GAD activity was inhibited partially by incubating with the antiserum. The maximal extent of inhibition was approximately 50 per cent. In the presence of antiserum all enzyme activity could be precipitated. The precipitates formed by GAD and antiserum had about 50 per cent of the enzyme activity and the K_m values for both glutamic acid and pyridoxal phosphate were significantly higher than those of the control system. Pyridoxal phosphate protected GAD from inhibition only slightly, even at very high concentrations. The results suggest that the antibodies may not react with the catalytic site, but rather that the inhibition of enzyme activity is attributable to indirect effects.     

Electrophoresis of proteins and DNA on horizontal sodium dodecyl sulfate polyacrylamide gels

An inexpensive Plexiglas apparatus which allows a simple and rapid preparation of horizontal polyacrylamide gels of different dimensions for different purposes, is described. Preparation of such gels is as easy and rapid as agarose gel preparation, and polymerized polyacrylamide gels are used to fractionate proteins or small DNA fragments using a common horizontal electrophoretic tank. This apparatus was used to electrophoretically fractionate proteins or DNA for immuno-blot analyses, particularirly in the study of the allergenic response to Parietaria judaica pollen in senescence, for Southern-blot hybridizations and in the study of DNA polymorphisms.     

Method for rapid removal of sodium dodecyl sulfate from polyacrylamide gels

A method is described for the isolation of hepatic microsomes by polyethylene glycol 6000 fractionation of the postmitochondrial fraction of liver homogenate. The procedure is simple and rapid requiring two centrifugation steps at 8000g for 10 min. The preparation has peptide patterns and levels of drug metabolic and other enzymatic activity similar to those of the microsomal fraction isolated by high-speed centrifugation and is referred to as polyethylene glycol 6000 microsomes. It is clarified with detergents and can serve as the starting material for the purification of microsomal proteins.     

Localization of cellular antigens in sodium dodecyl sulfate- polyacrylamide gels

A procedure is described for localizing antigen-antibody complexes in sodium dodecyl sulfate (SDS) polyacrylamide gels using 125I-labeled protein A from Staphylococcus aureus. We use the procedure to probe antigenic cross-reactivities between Strongylocentrotus and Chlamydomonas alpha- and beta-tubulins; we also domonstrate how the procedure can detect minor antibody species in an antiserum directed against a cell membrane.     

Chromatographic recovery of polypeptides from copper-stained sodium dodecyl sulfate polyacrylamide gels.

A procedure of preparative electrophoresis is described in which proteins separated on sodium dodecyl sulfate gels, stained with copper and eluted by simple diffusion, are highly concentrated on a fluorocarbon packing and freed of small molecular weight substances, including sodium dodecyl sulfate and buffer components and gel-related substances. This method can be used for microscale preparations or it can be scaled up to recover milligram amounts of protein. The purified polypeptides, however denatured, are suitable for amino acid sequencing.     

Distribution and tissue specificity of glutamate decarboxylase (EC 4.1.1.15).

Abstract— The activity of L–glutamate decarboxylase (EC 4.1.1.15) (GAD) in various mouse tissues was determined by five different methods, namely, the radiometric CO₂ method, column separation, electro–phoretic separation, the filtration method, and amino acid analysis. Results from the latter four methods agreed well, showing that brain had the highest activity, 4.27 nmol/min/mg protein (100%), followed by heart (7.4%), kidney (6.3%) and liver (1.5%). Measurement of brain GAD using the radiometric CO₂ assay method agreed with the other techniques. However, in heart, kidney, and liver, the GAD activities measured by the CO₂ method were about 3–4 times higher than those obtained by the GABA method, suggesting that the CO₂ method does not give a valid measurement of GAD activity in a crude non–neural tissue preparation. GAD activity also was detected in adrenal gland but not in pituitary, stomach, testis, muscle, uterus, lung, salivary gland, or spleen. GAD from brain, spinal cord, heart, kidney and liver were further compared by double immunodiffusion, enzyme inhibition by antibody, and microcomplement fixation using antibody against GAD purified from mouse brain. GAD from brain and spinal cord appear to be identical as judged from the following results: the immunoprecipitin bands fused together without a spur; the enzyme activity was inhibited by anti–GAD to the same extent; and the microcomplement fixation curves were similar in both the shape of the curve and the extent of fixation. No crossreactivity was observed between GAD from heart, kidney or liver and antibody against brain GAD in all the immunochemical tests described above, suggesting that GAD in non–neural tissues is different from that in brain and spinal cord.     

The identification of rat intestinal membrane enzymes after electrophoresis on polyacrylamide gels containing sodium dodecyl sulphate.

Brush-border membranes were isolated from the rat small intestine and then treated with sodium dodecyl sulphate under non-reducing conditions at room temperature. Analysis of the solubilized components by polyacrylamide-gel electrophoresis identified three major glycoproteins that co-migrate with glucoamylase-maltase-sucrase, lactase and isomaltase-maltase-sucrase activities. High activities of alkaline phosphatase and trehalase were detectable, but they could not be attributed to distinct co-migrating protein bands. Analysis of mucosa from the distal small intestine by the same methods showed a pattern of bands different from that obtained with the proximal intestine, which appeared to correlate with the relative deficiency of some of the enzymes in the distal region.     

Prestaining of glycoproteins in sodium dodecyl sulfate polyacrylamide gels by dansylhydrazine

A new fluorescent prestaining method for gel‐separated glycoproteins in 1D and 2D SDS‐PAGE was developed by using dansylhydrazine in this study. The prestained gels could be easily imaged after electrophoresis without any time‐consuming steps needed for poststains. As low as 4–8 ng glycoproteins (transferrin, α1‐acid glycoprotein) could be selectively detected, which is comparable to that of Pro‐Q Emerald 488, one of the most commonly used glycoprotein stain. In addition, a subsequent study of deglycosylation, glycoprotein affinity isolation, and LC‐MS/MS analysis was performed to confirm the specificity of the newly developed method.     

Identification of denatured enzyme proteins in sodium dodecyl sulfate polyacrylamide gels

A simple modification of the immunological sandwich method of Muilerman et al. for the identification of denatured enzyme proteins in sodium dodecyl sulfate-polyacrylamide gels is described, enabling the method to be used in principle for any enzyme whose activity is not inhibited by binding to antibodies. An immunological sandwich consisting of denatured enzyme, antibodies, and native enzyme is formed on a nitrocellulose filter blot of the gel, the filter is divided into strips, and each strip is tested for enzyme activity. The presence of enzyme activity serves to identify the region in the gel containing denatured enzyme protein. Experiments with human lysosomal alpha-glucosidase as a model system are described. The method was applied to identify a protein of Mr 125,000 as the main component with UDPgalactose pyrophosphatase activity in a partially purified preparation of the enzyme from rat liver.     

Cyanogen bromide cleavage of proteins in sodium dodecyl sulphate/polyacrylamide gels. Diagonal peptide mapping of proteins from epidermis.

A two-dimensional electrophoretic procedure employing CNBr has been devised for the analysis of proteins in sodium dodecyl sulphate/polyacrylamide gels. The technique allows the detection of an unusual class of epidermal proteins that lack methionine. The proteins have been identified by this method in newborn mouse, rat, and rabbit, because they are stable in the presence of CNBr and consequently lie on a diagonal. Adult human epidermis also contains CNBr-stable proteins, but in lesser amounts than in the newborn rabbit or newborn rodents. The methionine-containing proteins (i.e., the keratins) are degraded by CNBr into a series of unique and characteristics peptides which lie below the diagonal. Inter- and intra-species similarities and differences exist between the individual keratins, depending on the number and distribution of their methionine residues. The peptide-map patterns for the rodent and lagomorph proteins are more similar to each other than to that for the human proteins. The maps for rat and rabbit skin proteins are the most similar. We conclude that the epidermal keratins are a closely related, yet individually distinct, group of proteins that are found in conjunction with a class of proteins that lack methionine. The latter proteins are related to the histidine-rich basic protein, an epidermal structural protein that aggregates with keratin filaments.     

Thiol-dependent proteases of African trypanosomes. Analysis by electrophoresis in sodium dodecyl sulphate/polyacrylamide gels co-polymerized with fibrinogen

The proteases of several species of African trypanosomes were analysed by electrophoresis in sodium dodecyl sulphate/polyacrylamide gels containing fibrinogen or collagen. After electrophoresis the gels were incubated in the presence of enzyme activators and/or inhibitors and then stained with Coomassie brilliant blue. The areas where the proteolytic activity had degraded the fibrinogen did not stain and so formed clear bands against a blue background. The proteases were found to have pH optima between 5 and 6, and required dithiothreitol or 2-mercaptoethanol for full expression of their activity. They were inhibited by amino acid chloromethanes, iodoacetamide, p-chloromercuribenzoate and other inhibitors of the thiol-dependent proteases, as well as by the trypanocidal drugs berenil (4,4'-diamidinodiazoaminobenzene-diacetamidoacetate) and pentamidine [1,5-di-(4-amidinophenoxy)pentane-di-(2- hydroxyethanesulphonate)]. Trypanosoma evansi, Trypanosoma brucei brucei and Trypanosoma brucei gambiense each have a protease with a relative molecular mass, Mr, of 28 000. In addition they occasionally exhibit activity at higher Mr values (up to 105000). Trypanosoma congolense has a low-Mr protease (31 000) and may exhibit higher-Mr proteases (up to 150000). The protease profiles of Trypanosoma vivax differ from the other species, T. brucei or T. congolense, and are present in lesser amounts. The proteases of the cultured procyclic forms are present in much smaller amounts than those of the metacyclic or mammalian blood stream forms of these parasites. The catalytic properties and inhibition characteristics of these thiol-dependent enzymes suggest that they resemble the mammalian lysosomal cathepsins B and L.     

Monoclonal antibodies against rat brain glutamic acid decarboxylase (GAD)

Monoclonal antibodies against rat brain GAD have been produced and immunochemically characterized in comparison with a traditional anti-GAD antiserum (Oertel et al., Neuroscience6, 2689–2700, 1981). An immunopurified fraction in which GAD represented an estimated 5% of the total protein was used as immunogen. Out of 10 mice injected with this fraction, 6 appeared to be immunized: their sera immunoprecipitated quantitatively GAD activity. Three cell fusions were performed between spleen cells of the best immunized mice and SP2/OAg14 myeloma cells. Around 500 hybridoma were generated in each hybridization experiment. The culture medium of 13 hybridoma significantly trapped GAD activity. All immunoprecipitation curves established with the ascitic fluid obtained from the positive hybridoma, showed a lower titer, at least 50-fold, than the titer of the conventional antiserum. None of these ascitic fluids was able to stain directly any protein from a rat high speed supernatant after western blotting. However, the electrophoretical analysis of the proteins immunotrapped by any of the monoclonal antibodies, followed by western blotting and immunolabelling with the anti-GAD antiserum (“cross-immunoblotting”) showed the same two stained monomers. They have the same molecular weight (respectively 59 and 62 kDa ± 2 kDa) as those stained directly by the anti-GAD antiserum from a rat brain supernatant. Although all monoclonal antibodies showed a lower affinity then the conventional antiserum, which prevents them from being used directly in immunoblotting they permit to definitively establish that the two monomers immunolabelled by the conventional antiserum are constitutive subunits of the rat brain GAD.