There are two forms of androgen binding protein in human testes. Comparison of their protomeric variants with serum testosterone-estradiol binding globulin

To determine how the androgen binding protein in human testes (hABP) is related to the serum protein, testosterone-estradiol binding globulin (hTeBG), both proteins were isolated and compared. The hABP in extracts of human testes was composed of two molecular species based on concanavalin A (ConA)-Sepharose chromatography. Form I hABP did not interact with ConA while Form II hABP bound to ConA and eluted with alpha-methylmannoside. Form I and Form II hABP from five batches of testes were then purified approximately 30,500- and 30,000-fold to apparent homogeneity by high-performance liquid chromatography and compared with hTeBG isolated from human pregnancy serum. Fractionation of both forms of hABP and hTeBG by polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate suggested that the native forms of these proteins were indistinguishable. However, analysis of the purified proteins on sodium dodecyl sulfate-containing polyacrylamide gels indicated that all three were dimers and that each was composed of monomers of at least two sizes which were not present in equimolar concentrations. Two distinctive monomers or protomers of each protein were designated as heavy (H) and light (L) according to their electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels. The H and L protomers of Form I hABP showed apparent molecular weights of 55,000 and 52,000, respectively, in all preparations and were usually present in a 4:5 ratio (H:L). The two components of Form II hABP had apparent molecular weights of 53,000 and 48,000, respectively, and existed in a ratio of approximately 20:1. These two components could not be distinguished in some preparations where Form II hABP migrated as a broad band rather than as distinct protomers. By contrast, hTeBG, which was similar to Form II hABP with respect to ConA binding, always exhibited discrete H and L protomers in a 10:1 ratio. Photolysis of these highly purified proteins with delta 6-[3H]testosterone resulted in specific covalent labeling of their binding sites, confirming that the products identified by silver staining and immunoblotting were indeed steroid binding proteins. The H and L protomers of Form I hABP and hTeBG were separated and examined by peptide mapping using Staphylococcus aureus protease V8 and chymotrypsin. The comparison of the respective fragmentation patterns of protomers indicated that Form I hABP and hTeBG contained distinctive peptides.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adenosine 5'-diphosphate as an allosteric effector of phosphorylase kinase from rabbit skeletal muscle

Equilibrium binding and activity studies indicate that adenosine 5'-diphosphate binds to phosphorylase kinase with high affinity at a site, or sites, distinct from the catalytic site. Equilibrium dialysis at pH 6.8 and 8.2, with and without Mg2+, and with phosphorylated and nonphosphorylated enzyme preparations revealed approximately 8 ADP binding sites per alpha 4 beta 4 gamma 4 delta 4 hexadecamer, with Kd values ranging from 0.26 to 17 microM. Decreasing the pH from 8.2 to 6.8 or removing the Mg2+ enhanced the affinity for ADP. At pH 6.8, ADP stimulated the phosphorylase conversion and autophosphorylation activities of the nonactivated enzyme. Analogs of ADP with modifications at the 2'-, 3'-, and 5'-positions allowed determination of structural requirements for the stimulation of activity. ADP seems to alter the conformation of the beta subunit because addition of the nucleotide inhibits its dephosphorylation by phosphoprotein phosphatase and its chemical cross-linking by 1,5-difluoro-2,4-dinitrobenzene. The binding affinities and effects of ADP suggest that it may function physiologically as an allosteric effector of phosphorylase kinase.     

Fluorescence resonance energy transfer study of the associative state of membrane-bound complexes of complement proteins C5b-8

Human complement protein C8 was labeled with the fluorescent chromophores fluorescein-5-isothiocyanate (FITC), 3-(4-isothiocyanatophenyl)-7-diethylamine-4-methyl coumarin (IPM), eosin-5-isothiocyanate (EOS), or Texas Red (sulforhodamine-101-sulfonyl chloride; TR) with only minor reduction in the specific hemolytic activity of the protein. The distribution of C5b-8 complexes bound to sheep erythrocyte membranes was investigated by monitoring fluorescence resonance energy transfer (RET) between the following RET donor/acceptor pairs of labeled C8: FITC-C8/EOS-C8, IPM-C8/EOS-C8, and FITC-C8/TR-C8. On binding to membranes containing pre-formed C5b67 complexes, specific RET was detected for each of the donor/acceptor pairs of labeled C8 investigated. In contrast, no energy transfer was observed for these RET donor/acceptor pairs of labeled C8 incubated in the presence of control membranes or in membrane-free solution. On the basis of a consideration of the transfer efficiency that would be expected for donor/acceptor pairs of labeled C8 that were uniformly dispersed on the membrane surface, these results suggest that C5b-8 complexes are aggregated into polymeric clusters when membrane-bound. The efficiency of donor-C8 to acceptor-C8 RET--and the hemolytic activity of membrane-bound C5b-8 (in the absence of C9)--are both related to the surface density of membrane-bound C5b67, suggesting that the physical clustering of the membrane-inserted C5b-8 complex may be related to the expression of its cytolytic activity.     

The role of the carbohydrate moiety on the size heterogeneity and immunologic determinants of human testosterone-estradiol-binding globulin

Human testosterone-estradiol-binding globulin (hTeBG) has been purified to apparent homogeneity by several laboratories using procedures which, in most instances, were labor intensive. In this report, hTeBG was purified from pregnancy serum by a newly developed two step procedure involving sequential affinity chromatography and ion-exchange high performance liquid chromatography (ion-exchange HPLC). The purity of the final product was confirmed by silver stained SDS-polyacrylamide gel and reverse phase HPLC monitored at 206 nm. hTeBG purified by ion-exchange-HPLC maintained binding activity by Dextran coated charcoal (DCC) assay and size heterogeneity on SDS-polyacrylamide gels which were indistinguishable from those of the proteins purified by conventional chromatography. Removal of the carbohydrate moiety from the molecule by both enzymatic and chemical treatment reduced the apparent molecular size and eliminated lectin binding of hTeBG subunits. Deglycosylation did not, however, abolish or alter the distribution of the protomeric forms of this subunit. We conclude that hTeBG is a dimer whose monomer exhibits two protomeric forms which is not a result of carbohydrate heterogeneity. In addition, disialylated and deglycosylated hTeBG exhibited antigenic determinants identical to the native protein.     

马立克氏病毒单克隆抗体的研究

获得了4株分泌马立克氏病毒(MDV)特异性单克隆抗体(McAb)的杂交瘤细胞:4BS10对MDV所有毒株呈阳性反应;4CN8 对MDV血清1,3型毒株发生反应;2BN90和4CN24只对MDV血清1型毒株有阳性反应。3个McAb属IgG1,1个为IgG2b,均不中和MDV,免疫扩散试验也无沉淀线。对禽白血病毒(ALV)无交叉反应。 以2BN90和辣根过氧化物酶、异硫氰酸荧光素的结合物进行直接酶联免疫吸附试验和直接荧光抗体试验,均获得成功。抗体滴度前者为1/51,200,后者为1/640。对ALV无交叉反应。     

肝素修饰尿激酶某些性质的研究

本文对比研究了溴化氰活化及高碘酸活化肝素修饰的两种修饰尿激酶的性质。结果表明尿激酶在溴化氰活化肝素(肝素CN),高碘酸钠活化肝素(肝素I_4)的共价修饰后,其残余自由氨基分别是64％和52％;酶活性分别保留94％和90％;抗胃蛋白酶水解以及抗冻融变性的能力均高于天然酶;在离体血浆中的失活速变低于天然酶。本文还对修饰酶进行了萤光及紫外差光谱的分析,讨论了修饰过程对构象的影响。     

The rabbit C family of short, interspersed repeats. Nucleotide sequence determination and transcriptional analysis

When the entire adeno-associated virus (AAV) genome is inserted into a bacterial plasmid, infectious AAV genomes can be rescued and replicated when the recombinant AAV-plasmid DNA is transfected into human 293 cells together with helper adenovirus particles. We have taken advantage of this experimental system to analyze the effects of several classes of mutations on replication of AAV DNA. We obtained AAV mutants by molecular cloning in bacterial plasmids of naturally occurring AAV variant or defective-interfering genomes. Each of these mutants contains a single internal deletion of AAV coding sequences. Also, some of these mutant-AAV plasmids have additional deletions of one or both AAV terminal palindromes introduced during constructions in vitro. We show here that AAV mutants containing internal deletions were defective for replicative form DNA replication (rep-) but could be complemented by intact wild-type AAV. This indicates that an AAV replication function, Rep, is required for normal AAV replication. Mutants in which both terminal palindromes were deleted (ori-) were also replication defective but were not complementable by wild-type AAV. The cis-dominance of the ori- mutation shows that the replication origin is comprised in part of the terminal palindrome. Deletion of only one terminal palindrome was phenotypically wild-type and allowed rescue and replication of AAV genomes in which the deleted region was regenerated apparently by an intramolecular correction mechanism. One model for this correction mechanism is proposed. An AAV ori- mutant also complemented replication of AAV rep- mutants as efficiently as did wild-type AAV. These studies also revealed an unexpected additional property of the deletion mutants in that monomeric single-stranded single-stranded DNA accumulated very inefficiently even though monomeric single-stranded DNA from the complementing wild-type AAV did accumulate.     

Comparison of rabbit androgen binding protein with testosterone estradiol binding globulin--I. Physical and chemical properties

Rabbit epididymal androgen binding protein (rbABP) and serum testosterone estradiol binding globulin (rbTeBG) were purified and their physicochemical properties compared. Both proteins bound dihydrotestosterone (DHT) with high affinity. Both contained two components, Heavy (H) and Light (L), and their molecular weights and pI values were comparable. rbABP and rbTeBG were different with regard to their ConA-Sepharose binding property. rbABP was not bound by ConA-Sepharose while rbTeBG was found and retained by this lectin; thus, rbABP and rbTeBG differed in their carbohydrate structure. Peptide mapping on SDS-PAGE indicated that the H components of rbABP and rbTeBG were distinct even though they showed a high degree of homology. By contrast, the L components of these two proteins appeared to be identical. The structure of the steroid binding sites of these two proteins was analyzed by peptide mapping of [1,2(3)H]17 beta hydroxy-androsta-4,6-dien-3-one photoaffinity labeled protein. The size distribution of radioactive peptide fragments generated appeared to be identical for these two proteins. However, the distribution of labeled peptides was slightly different when examined by high pressure liquid chromatography (HPLC). The observations suggest that the differences between rbABP and rbTeBG might reside not only in carbohydrate moieties but also in their amino acid sequences.     

在甜椒花药培养中马来酰胼对小孢子发育的抑制作用

选用甜椒的小孢子单核期花药,用100、300、500、2000ppm的马来酰肼溶液浸泡处理,并设对照,进行无激素MS固体培养基培养。分别取样用各种组化方法对花药内部多糖、蛋白质、核酸及ATP酶进行组化反应和形态学上的观察。与对照组相比,处理组花药外部形态和内部结构出现许多变异。小孢子内多糖,蛋白质含量减少;绒毡层无明显变化;两组中,核酸的含量均无明显变化;ATP酶的活性低于对照组。可能,马来酰肼对于花药中ATP酶等产生抑制作用,致使花药败育。