Difference in the Pattern of Soluble Proteins from Rat Brain Regions

The electrophoretic pattern of soluble proteins from seven rat brain regions (amygdala, cerebellum, corpus striatum, cortex, hypothalamus, medulla, and midbrain) was examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Although the number of protein bands (36) was identical in all brain regions studied, there were differences in their relative densities, the greatest variation occurring in the low-molecular-weight region of the electrophoretogram. The bulk of the soluble proteins had molecular weights between 23,000 and 90,000 daltons. The medulla and amygdala showed the greatest range of protein band concentration. A large number of protein bands in the midbrain and corpus striatum showed a greater concentration of protein compared to the same bands in the other regions. A protein band that migrated with the same characteristic as albumin was found. It was consistently high in all regions, the midbrains showing a 1.5-fold greater concentration compared to other regions. Linear regression analysis of wet weight of regional brain tissue against protein concentration yielded a regression coefficient (r2) of 0.77. Midbrain and corpus striatum showed a relatively higher protein concentration: weight ratio than other regions.     

Detection of antimycin-binding subunits of Complex III by photoaffinity-labeling with an azido derivative of antimycin

Deformamidoazidoantimycin A (DAA), a photoactive derivative of antimycin A containing an azido group substituting for the formamido group attached to the phenyl ring, was synthesized. The ultraviolet spectrum of DAA was almost identical to that of antimycin A, indicating little alteration of the electronic structure of the substituted phenyl ring by the azido substitution. However, the inhibitory effectiveness of DAA toward ubiquinol-cytochromec reductase (Complex III) purified from bovine heart (K _i =ca. 0.5 µM) was considerably less than that of antimycin (K _i 3 pM), indicating a direct rather than a supporting role of the formamido group in the inhibitory activity of antimycin. Exposure of purified Complex III to [³H]DAA plus ultraviolet light caused a major labeling by tritium of SDS-PAGE band 7 (m=13 kDa by SDS-PAGE) and lesser but significant labeling of bands 3, 6, 8, and 9. Pretreatment of Complex III with antimycin greatly suppressed the labeling of bands 5, 6, and 7 but caused an apparent increased labeling of bands 8 and 9 by [³H]DAA, respectively. The labeling of band 7 by [³H]DAA also was strongly suppressed by reduction of Complex III by either sodium borohybride or ascorbate. Based on magnitude of labeling by [³H]DAA and the degree of suppression of labeling by antimycin, the protein of band 7 qualified as the principal component for specific binding of antimycin with the protein of band 6 (m=16 kDa) showing a lesser but significant amount of specific binding.     

Purification and characterization of thermostable leucine dehydrogenase from Bacillus stearothermophilus

Leucine dehydrogenase (l-leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) has been purified to homogeneity from a moderate thermophilic bacterium, Bacillus stearothermophilus. Am improved method of preparative slab gel electrophoresis was used effectively to purify it. The enzyme has a molecular mass of about 300,000 and consists of six subunits with identical molecular mass (Mr, 49,000). The enzyme does not lose its activity by heat treatment at 70° C for 20 min, and incubation in the pH range of 5.5–10.0 at 55° C for 5 min. It is stable in 10 mM phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol at over 1 month, and is resistant to detergent and ethanol treatment. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their keto analogs in the presence of NAD⁺ and NADH, respectively, as the coenzymes. The pH optima are 11 for the deamination of l-leucine, and 9.7 and 8.8 for the amination of -ketoisocaproate and -ketoisovalerate, respectively. The Michaelis constants were determined: 4.4 mM for l-leucine, 3.3 mM for l-valine, 1.4 mM for l-isoleucine and 0.49 mM for NAD⁺ in the oxidative deamination. The B. stearothermophilus enzyme shows similar catalytic properties, but higher activities than that from Bacillus sphaericus.Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday     

A method for efficient gene isolation from phage lambda gt11 libraries: use of antisera to denatured, acetone-precipitated proteins

Experience with cloning pseudorabies virus (PRV) DNA in the lambda gt11 phage vector has shown that there are special requirements for the antisera used in screening the libraries, in addition to the requirement that the antisera recognize proteins on a Western blot. Initial screening of a lambda gt11 library of sheared PRV DNA fragments in Escherichia coli for expression of PRV antigens using PRV hyperimmune antisera was unsuccessful. It was only after screening the library with antisera raised against PRV proteins eluted from sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels that positive results were obtained. These "gel-slice" antisera (GSA) were equivalent in potency to hyperimmune antisera in standard immunoassays (including ELISA, immunoprecipitation, Western blots, and neutralization of virus), but only the GSA could recognize PRV fusion proteins expressed by recombinant lambda gt11 phage. This difference was seen despite the fact that hyperimmune antisera performed satisfactorily on Western blots of denatured PRV-infected cell extracts. These results show that the efficiency of screening expression libraries in E. coli can be improved if antibodies are raised against denatured proteins.     

Analysis of the inducible MEL1 gene of Saccharomyces carlsbergensis and its secreted product, alpha-galactosidase (melibiase)

We have determined both the nucleotide sequence of the MEL1 gene of Saccharomyces carlsbergensis and the N-terminal amino acid (aa) sequence of its extracellular gene product, alpha-galactosidase (melibiase) (alpha-Gal). The predicted translation product of MEL1 is a pre-alpha-Gal protein containing an 18 aa N-terminal signal sequence for secretion. The purified enzyme is a dimer consisting of two 50-kDal polypeptides, each of which is glycosylated with no more than eight side chains. The 5'-flank of the MEL1 gene contains a region (UASm) having certain areas of sequence homology to similar sites found upstream of the structural genes GAL1, GAL7 and GAL10, which are also regulated by the action of the products of genes GAL4 and GAL80. There are three TATA boxes between UASm and the initiation codon of pre-alpha-Gal, as well as a typical yeast cleavage/polyadenylation sequence in the 3'-flank of the gene.     

流行性乙型脑炎病毒减毒株和野毒株在聚丙烯酰胺凝胶电泳上的表现

乙型脑炎病毒减毒株(2-8株)和野毒株(SA14株)在生物学性质上有明显的不同,特别是嗜神经毒力方面,2-8株已丧失了对小白鼠、马,猴等敏感动物的脑内致病力。为了探讨这些生物学性质变化的物质基础,我们开展了乙脑强、弱毒株聚丙烯酰胺凝胶     

Phosphoglucose isomerase isozymes in teleostean fishes from the Arabian Gulf

Two phosphoglucose isomerases (PGI) with different electrophoretic mobilities have been found in all groups of teleostean fishes studied, with the exception of the Clupeomorpha. PGI proved to be a good taxonomic criterion to differentiate members of the Nemipteridae, Sciaenidae, Platycephalidae and Stromateidae from the other teleost families.     

Genetic variation and divergence of the sibling pair of cyprinid fishes, Notropis cornutus and N. chrysocephalus

Horizontal starch gel electrophoresis was used to estimate the amount of genetic divergence between Notropis cornutus and N. chrysocephalus. Measures of genetic identity (1) and distance (D) were 0.881 and 0.127 ± 0.055 (s.e.), respectively. These estimates correspond more closely to the sibling species status of these taxa than other previously reported estimates. Notropis cornutus was found to be significantly more variable than N. chrysocephalus electrophoretically and morphologically. Assuming the existence of an electrophoretic clock, the time of divergence was calculated to be roughly 1.9–2.5 million years. This estimate corresponds very closely to a previously hypothesized late Pliocene divergence.