Expression and characterization of the tau subunit of phosphorylase kinase

A cDNA encoding the entire tau subunit of rabbit skeletal muscle phosphorylase kinase was reconstructed and inserted into a plasmid containing the Escherichia coli ptac promoter and a constructed plasmid containing the ptac promoter and bacterial chloramphenicol acetyl transferase (CAT) gene, respectively. A significant phosphorylase kinase activity was found, in the first case. In the second case, a fused protein containing 73 amino acids from the CAT protein was obtained. After renaturation, the CAT-tau subunit protein shows enzymatic activity similar to the HPLC-purified and renatured tau subunit.     

Expression of lipoprotein lipase gene in combined lipase deficiency

The expression of the gene for lipoprotein lipase (LPL) was studied in brown adipose tissue and the liver of combined lipase deficient (cld/cld) and unaffected mice. The mRNA specific for LPL was detected in both animals. Although the size of LPL mRNA in cld mice was similar to that of unaffected mice, the mRNA concentration in affected animals was higher than in unaffected animals. We also studied the LPL gene mutation in cld mice by Southern blot analysis. No restriction fragment length polymorphisms were observed after digestion with 16 endonucleases. These data indicate that there is no gene insertion or deletion, but do not exclude the possibility of point mutation in the LPL structural gene. However, the present results agree with the hypothesis that the genetic defect in cld is not due to a mutation in the LPL structural gene, but instead involves the defective post-translational processing of LPL or defective cellular function affecting transport and secretion of this enzyme group.     

The use of micropreparative electrophoresis of protein/peptide isolations for primary structure determinations

High-performance electrophoresis chromatography (HPEC) is a recent development that features continuous-elution gel electrophoresis for isolating proteins or peptides in range of 1 to 300 microgram quantities. Column gel electrophoresis is conducted under thermostated conditions, and the field voltage can be varied within a run with a programmable power supply. Applications of this apparatus in protein purification are presented to demonstrate the utility of the (Model 230A) HPEC. These examples include on-line detection with direct analyte recovery of highly purified sample, which mimics high-performance liquid chromatography, for subsequent structure-function characterization. A method to remove salts from sodium dodecyl sulfate electrophoresed samples for subsequent sequencing or amino acid analysis is described. This desalting procedure recovers from 90%-95% of the sample and employs a low molecular weight cut-off membrane during sample centrifugation onto a polyvinylidene difluoride membrane. Subsequent washings are performed to efficiently remove salts, free amino acids and detergents that are known to interfere with sequence analysis. Sequence information such as initial recovery, repetitive yields and chromatogram comparisons are presented to demonstrate the utility of this procedure when used following isolation of sample with HPEC.     

用免疫荧光技术研究细胞周期中核基质的分布变化

本文利用含有抗核基质自发抗体的硬皮人血清,以小鼠艾氏腹水癌细胞为材料,用间接免疫荧光染色的方法,追踪了对应核基质抗原在细胞周期中分布的变化。结果显示,在末期和间期之间存在一个核基质抗原从细胞质向细胞核内转移的过程。由于这一过程是通过核膜进行的,从而提示核基质结构可能有解聚和再聚合的行为。用酶化学结合间接免疫荧光染色的方法,初步研究了抗原的化学性质。染色形态的比较研究显示所用血清中可能含有不同于以前发现的、抗新的核基质抗原的自发抗体。     

白腹锦鸡机体营养成分的分析与探讨

本文报道笼养和野生白腹锦鸡机体营养成分及其差异。分析表明,笼养的比野生种营养成分含量高的有:腿肌蛋白质高11％,胸肌、腿肌、全血的氨基酸分别高2.64％,1.39％和4.68％,胸肌、腿肌和肝脏的碳水化合物分别高0.076％、0.092％和3.962％,胸肌和腿肌的维生素A分别高188.63和84.09 I.U.,胸肌和腿肌的维生素D分别高47.2和12.8 I.U.。但是胸肌蛋白质含量笼养的比野生的低26％。     

白腹锦鸡,红腹锦鸡,中国雉鸡SC组型的比较研究

以微铺展—硝酸银染色技术制备三种鸡的SC标本,进行电镜观察。结果表明:三种鸡的SC组非常相似,即2n=82,ZZ/ZW型性别决定,雄性为ZZ。除1号SC和Z-SC为中着丝粒外,其余均为端着丝粒。Z-SC的相对长度有明显的细胞间差异;平均相对长度度介于第3和第4号SC之间。三者SC组型上的差异主要表现在相应SC长度上的不同。并对其亲缘关系及在鸟类进化中的可能地位进行了讨论。此外,在微铺展法制备的锦鸡精母细胞SC标本中还发现了巨大中心粒,这在高等动物尚属首次。     

Identification of three novel missense PKU mutations among Chinese.

Three novel missense mutations have been identified in the phenylalanine hydroxylase (PAH) genes of Chinese individuals afflicted with various degrees of phenylketonuria (PKU). A T-to-C transition was observed in exon 5 of the gene, resulting in the substitution of Phe161 by Ser161. Two substitutions, G-to-T and T-to-G, were observed in exon 7, resulting in the substitution of Gly247 by Val247 and Leu255 by Val255, respectively. Expression analysis demonstrated that these mutant proteins produced between 0 and 15% of normal PAH enzyme activity. Population screening of a Chinese sample population indicates that these mutations are quite rare, together accounting for only about 4% of all PKU alleles among the Chinese. The P161S and G247V mutations were each present on a single PAH RFLP haplotype 4 chromosome in patients form Northern China, while the L255V mutation was present on chromosomes of both haplotypes 18 and 21 in patients from Southern China. These results suggest that the remaining 30% of uncharacterized PKU alleles in the Chinese population may bear a large number of relatively rare PAH mutations.     

Purification and characterization of pea chloroplastic phosphoriboisomerase

Pea (Pisum sativum L.) chloroplastic phosphoriboisomerase (EC 5.3.1.6) can be purified to apparent homogeneity in less than 2 days time with a 53% yield. Important steps in the purification include heat treatment and pseudoaffinity chromatography on Red H-3BN Sepharose. The purified isomerase has a subunit molecular mass of 26.4 kD. The N-terminal sequence has been determined through 34 residues. pH optima are 7.8 (ribose-5-phosphate) and 7.7 (ribulose-5-phosphate); K_m values are 0.9 millimolar (ribose-5-phosphate) and 0.6 millimolar (ribulose-5-phosphate). The enzyme is inhibited by erythrose-4-phosphate, sedoheptulosebisphosphate, glyceraldehyde-3-phosphate, and 3-phosphoglycerate at concentrations close to those found in photosynthesizing chloroplasts. Countercurrent phase partitioning experiments indicate that the pea chloroplastic phosphoriboisomerase interacts physically with phosphoribulokinase.