Synthesis of two different molecular forms of ribulose-bisphosphate carboxylase/oxygenase in Rhodopseudomonas blastica grown in batch culture

The expression of the two different molecular forms (form I and form II) of ribulose-bisphosphate carboxylase/oxygenase (RuBisCO) in Rhodopseudomonas blastica during growth in batch on pyruvate–malate medium was investigated. During the early exponential phase of growth, only form I enzyme was synthesized. At the mid-exponential phase of growth, both forms were expressed, although form I enzyme was predominant. At the late exponential phase, form I and form II enzymes were synthesized but form II enzyme predominated. It is concluded that form I and form II RuBisCO enzymes in R. blastica are differentially expressed and this may be mediated by the level of CO2 in the growth medium.     

Synthesis of two different molecular forms of ribulose-bisphosphate carboxylase/oxygenase in Rhodopseudomonas blastica grown in batch culture

The expression of the two different molecular forms (form I and form II) of ribulose-bisphosphate carboxylase/oxygenase (RuBisCO) in Rhodopseudomonas blastica during growth in batch on pyruvate–malate medium was investigated. During the early exponential phase of growth, only form I enzyme was synthesized. At the mid-exponential phase of growth, both forms were expressed, although form I enzyme was predominant. At the late exponential phase, form I and form II enzymes were synthesized but form II enzyme predominated. It is concluded that form I and form II RuBisCO enzymes in R. blastica are differentially expressed and this may be mediated by the level of CO2 in the growth medium.     

Agarose gel electrophoretic analysis of damage to supercoiled DNA by adriamycin in the presence of beta-NADH dehydrogenase

In a cell-free system, the anticancer anthracycline antibiotic adriamycin was able to convert purified covalently closed circular, superhelical, form I bacteriophage PM2 DNA to relaxed circular form II DNA in the presence of either sodium borohydride (NaBH4), NADPH cytochrome P-450 reductase or beta-NADH dehydrogenase isolated from myocardial cells. There was no detectable increase in the amount of form III linear duplex DNA formed during the reaction even at high drug concentrations. Less drug was required for the conversion of form I to form II DNA in the presence of the enzymic reducing agents than in the presence of NaBH4. Form II DNA, prepared by irradiation using a Cs-137 source, was not degraded to form III linear duplex DNA. However, form I0 DNA, covalently closed circular DNA without superhelical turns, freshly prepared using topoisomerase I, was converted to form II DNA similar to the conversion of superhelical form I to form II DNA. Again, no increase in the amount of form III linear duplex DNA could be detected.     

Isoenzymic forms of NAD-linked glycerol-3-phosphate dehydrogenase from rabbit brain.

Two major enzyme forms of cytosolic NAD-linked glycerol-3-phosphate dehydrogenase in rabbit brain have been purified to apparent homogeneity. One major enzyme form designated I6.5 exhibits an iso-electric point at pH 6.5, and is indistinguishable from the major form I6.5 found in other tissues. The other major form, designated I5.9, has an isolectric point at pH 5.9, and by amino acid analysis is shown to be a true isoenzyme distinct from form I6.5. Form I5.9 appears to be closely related to or identical with the major enzyme characteristic of heart. Neither the brain enzyme form I5.9 nor the major heart isoenzyme are inhibited by antiserum to the muscle enzyme. Because of the high apparent Km for NADH, it is postulated that the brain isoenzyme I5.9 serves to maintain glycolysis when NADH levels rise under relatively anaerobic conditions especially during fetal and neonatal development.     

角蒿属6个种的核形态学研究

对紫葳科角蒿属（Incarvillea)6种植物（其中两头毛Incarvillen arguta包括红花和白花2个类型）进行了核形态学研究,它们的间期核均为简单染色中心型,前期染色体为中国型,体细胞中期染色体数目均为2n=22,核型公式分别为：（1）两头毛（红花类型）Incanillea ar-guta(Red-flower form)2n=22=14m (2SAT) 8sm(lSAT),着丝点端化值（T.C.%)为62.71%,臂指数（N.F.值）为44;（la)两头毛（白花类型I.arguta(White-flower form)2n=22=16sm(lSAT) 6st,T.C.%值为70.62%,N.F值为38;（2）鸡肉参I.mairei 2n=22=6m 8sm(lSAT) 8st,T.C.%值为70.07%,N.F.值为36;（3）红波罗花I.delavayi 2n=22=10m 6sm 6st,T.C.%值为61.33%,N.F.值为38;（4）单叶波罗花I.forrestii2n=22\4m 8sm 10st(lSAT).T.C.%值为73.10%,N.F.值为34;（5）中甸角蒿I.zhongdianensis2n=22=4m 8sm 10st,T.C.%值为72.31%,N.F.值为34;（6）黄波罗花I.lutea2n=22=4m 8sm(2SAT) 10st,T.C.%值为69.47%,N.F.值为34。上述几种植物中,除两头毛（红花类型）的核型不对称性为2A型外,其余几种的核型不对称性都属于3A型,本文观察的6种植物的核形态结构均为首次报道。     

Accuracy of Deoxynucleotide Incorporation by Soybean Chloroplast DNA Polymerases Is Independent of the Presence of a 3[prime] to 5[prime] Exonuclease

DNA polymerase was purified from soybean (Glycine max) chloroplasts that were actively replicating DNA. The main form (form I) of the enzyme was associated with a low level of 3[prime] to 5[prime] exonuclease activity throughout purification, although the ratio of exonuclease to polymerase activity decreased with each successive purification step. A second form (form II) of DNA polymerase, which elutes from DEAE-cellulose at a higher salt concentration than form I, was devoid of any exonuclease activity. To assess the potential function of the 3[prime] to 5[prime] exonuclease in proofreading, the fidelity of deoxynucleotide incorporation was measured for form I DNA polymerase throughout purification. Despite the steadily decreasing ratio of 3[prime] to 5[prime] exonuclease to polymerase activity, the extent of misincorporation by form I enzyme remained unchanged during the final purification steps, suggesting that the exonuclease did not contribute to the accuracy of DNA synthesis by this polymerase. Fidelity of form I DNA polymerase, when compared with that of form II, revealed a higher level of misincorporation for form I enzyme, a finding that is consistent with the exonuclease playing little or no role in exonucleolytic proofreading.     

质料拷贝数对宋内氏菌I相O—抗原表达的影响

试图通过提高质粒拷贝数来提高宋内Ｉ相Ｏ－抗原的表达水平,先将宋内Ｉ相Ｏ－抗原基因亚克隆至高拷贝的ｐＵＣ１８和ｐＧＥＭ３Ｚ,但没能找到相应的阳性转化子,可能Ｏ－抗原基因在高拷贝质粒上表达水平太高对宿主产生毒性效应。再将其亚克隆至中等拷贝的ｐＡＴ１５３,虽能找到阳性转化子,但表达水平仍不能满足以后实验的需要,表明质粒拷贝数与Ｉ相Ｏ－抗原的表达水平密切相关,要有效地表达宋内Ｉ相Ｏ－抗原,还需要寻找拷贝数     

Morphometric relationship of length–weight and chelae length–width of eastern white river crayfish (Procambarus acutus acutus, Girard, 1852), under culture conditions

Length–weight (TL vs WWT) and chelae length–width (ChL vs ChW) relationships were described for juveniles, males and females, and for form I and form II males of Procambarus acutus acutus. The length–weight relationships for juveniles, form I, form II males, and females could be described as: WWT = 5 × 10⁻³ TL^3.09, WWT = 6 × 10⁻³ TL^3.61, WWT = 6 × 10⁻⁹ TL^3.26, and WWT = 6 × 10⁻⁴ TL^3.5, respectively. In all forms, growth was allometric (P < 0.05). The ancova test indicated that slopes and intercepts of the length–weight regressions were significantly different between sex and sexual stages. The regressions for chelae length–width relationships for form I and form II males, and females were: ChW = −0.81 + 0.27CL, ChW = −0.33 + 0.25CL, and ChW = −0.82 + 0.32CL, respectively. Although the slope and intercepts of regressions for ChL and ChW were similar for those of form I and form II males, the slopes and intercepts of regressions of females were significantly different from form I and form II males. No statistical difference was observed in mean ChL between form II males and females (P > 0.05), but a significant difference was detected in mean ChL between form I and form II males (P < 0.05) and form I and females (P < 0.05). Form I males had longer ChL than form II males and females. The same trend was observed in mean ChW for form I and form II males, but a significant difference was detected between form II males and females (P < 0.05). In addition, results indicated that chelae lengths and widths increased allometrically with total length (TL) for both sex and sexual stages.     

Rapid purification and thermostability of the cytoplasmic aspartate aminotransferase from carrot suspension cultures

Several isoenzymic forms of aspartate aminotransferase (AAT) have been identified in protein extracts from carrot (Daucus carota) cell suspension cultures. The cellular location of the major form (form I) of AAT in carrot suspension cultures was determined by heat inactivation, subcellular fractionation, and amino acid sequence analysis. In mammalian systems, there are two forms of AAT, a heat-stable cytoplasmic form and a heat-labile form in the mitochondria. The thermostability of three isoenzymes of carrot AAT was examined, and the results showed that form I was more thermostable than forms II or III. Organelles were separated in sucrose gradients by isopynic centrifugation. Activity for form I was identified in the soluble fractions and not in fractions containing peroxisomes, proplastids, or mitochondria. Form I was purified to homogeneity and endoproteolytically cleaved, and the peptide fragments were separated by reverse phase chromatography. Analysis of the sequence data from two of the polypeptides showed that the amino acid identity of form I is more conserved to the animal cytoplasmic AAT than to animal mitochondrial AAT sequences. These data strongly suggest that form I of AAT from carrot is the cytoplasmic isoenzyme. Additionally, a rapid purification scheme for form I of AAT from carrot is presented using selective heat denaturation and anion-exchange chromatography.     

Formation and crystallization of amylose–monoglyceride complex in a starch matrix

The formation of amylose–lipid complexes in a gelatinized potato starch matrix was investigated using potato starch and glycerol monopalmitin. These complexes exist in two forms, with the amounts of each of the forms being dependent on the temperatures and durations of the pre-treatments.

Differential scanning calorimetry (DSC) was used to analyze transition temperatures and melting enthalpies, and thereby determine the amount of the complexes in the samples. X-ray diffraction analysis was used to investigate their crystallinity.

In measurements with DSC, form I started to melt at 88.5°C, and form II at 112.9°C. When complex form II was preheated at 100 or 110°C, its melting point rose to 116.3 and 119.7°C, respectively, because of an annealing effect. The same phenomenon occurred with complex form I: when preheated at 90°C, its melting point rose to 96.8°C. The crystal formation of form II appeared to be slower when treated at 110°C than at 100°C. Their maximum melting enthalpies were reached after about 24 h and 4 h of preheating, respectively. In X-ray diffraction analyses, form II showed a V-pattern, but form I did not. This indicates that form II is more crystalline than form I. It was possible to transform form I into form II when it was heat treated, because form I was then partially or totally melted.

As a comparison, the charged substance cetyltrimethylammonium bromide created complex form I with amylose in the starch matrix, but not form II.     

Relationship between the two immunologically distinguishable forms of glucocerebrosidase in tissue extracts

Extracts of human spleen contain two immunologically distinguishable forms of glucocerebrosidase: form I is precipitable by polyclonal or monoclonal anti-(placental glucocerebrosidase) antibodies, whereas form II is not [Aerts, J. M. F. G., Donker-Koopman, W. E., Van der Vliet, M. F. K., Jonsson, L. M. V., Ginns, E. I., Murray, G. J., Barranger, J. A., Tager, J. M. & Schram, A. W. (1985) Eur. J. Biochem. 150, 565-574]. The proportion of form II glucocerebrosidase was high in extracts of spleen, liver and kidney and low in extracts of brain, placenta and fibroblasts. Furthermore, the proportion of form II enzyme was higher in a detergent-free aqueous extract of spleen than in a Triton X-100 extract of total spleen or splenic membranes. When form II glucocerebrosidase in a splenic extract was separated from form I enzyme by immunoaffinity chromatography and stored at 4 degrees C, a gradual conversion to form I enzyme occurred. The conversion was almost immediate if 30% (v/v) ethylene glycol was present. In the denatured state both forms of glucocerebrosidase reacted with anti-(placental glucocerebrosidase) antibodies. Form I glucocerebrosidase was stimulated by sodium taurocholate or sphingolipid-activator protein 2 (SAP-2), whereas form II enzyme exhibited maximal activity in the absence of the effectors. The pH activity profile of form II glucocerebrosidase was almost identical to that of form I enzyme in the presence of SAP-2. In the native state, form I glucocerebrosidase had a molecular mass of 60 kDa whereas that of form II glucocerebrosidase was about 200 kDa. After gel-permeation high-performance liquid chromatography of splenic extracts, the fractions with form II glucocerebrosidase contained material cross-reacting with both anti-(placental glucocerebrosidase) and anti-(SAP-2) antibodies. Preincubation of form I glucocerebrosidase with SAP-2 at pH 4.5 led to masking of the epitope on glucocerebrosidase reacting with monoclonal anti-(placental glucocerebrosidase) antibody 2C7. Furthermore, preincubation of form I glucocerebrosidase with monoclonal antibody 2C7 prevented activation of the enzyme by SAP-2. We propose that form I glucocerebrosidase is a monomeric form of the enzyme, whereas form II glucocerebrosidase is a high-Mr complex of the enzyme in association with sphingolipid-activator protein 2.     

alpha-D-mannosidase forms in chicken liver

Two forms (I and II) of alpha-D-mannosidase have been separated by ion-exchange chromatography on DEAE-cellulose from embryonic chicken liver. A third form (III), which is absent in embryos, was also separated from 4-day-old chickens. The optimum pH of form I is at pH 5.0. Form II is named "neutral" because it shows maximal activity at pH 6.5. The optimum pH of form III is 4.5. Forms I and III are heat-stable at 50 degrees C for 1 hr, whereas form II is very unstable under these conditions. Zn2+ and Mg2+ have been found to increase the alpha-D-mannosidase activity of forms I and II. In contrast, Co2+ increases mannosidase I activity and inhibits form II from 18-day-old embryos. alpha-Methyl-D-mannoside, N-acetyl-D-mannosamine and D-mannosamine were found to be inhibitors of both forms I and II. "Neutral" mannosidase was also inhibited by chloride. Competitive inhibition by D-mannose was also studied and Ki values are given.     

Changement de structure de la poly-L-proline à l'état solide par hydratation: Etude par spectrométrie infra-rouge

Films of poly-L -proline I and II have been hy drated by water vapor and their infrared spectra analysed between 1250 and 3500 cm^?1. Forms I and II behave differently. Water binds to the carbonyl of form II through hydrogen bridges and, when released by drying, leaves the polypeptide conformation unchanged. In form I, complex formation with water seems to proceed through a different mechanism involving more than the carbonyls. Hydration of form I yields a partial irreversible transition to form II. Successive hydration and drying of the film brings the transition to completion. This I → II transition in the solid state has a cooperative character; its mechanism is discussed. Poly-L -prolines I and II also absorb vapors of aliphatic alcohols but not change in conformation is observed.     

Reconstitution of bovine procarboxypeptidase A-S6 from the free subunits

The three subunits I, II, and III of bovine procarboxypeptidase A separated by reversible dimethylmaleylation can reassociate to form the reconstituted complexes I + II, I + III, and I + II + III. Since the association II + III is not possible, subunit I appears to play a central role in the formation of the complex. It is suggested that subunit I possesses two independent and specific sites for the recognition of subunits II and III. The liberation of subunit I from any of the complexes was observed to increase its activability, although to a lesser extent than predicted by assays carried out with the succinylated protein. By contrast, the bound form of subunit II was activated faster than the free form. The potential activity of the bound form and the activity of the preformed endopentidase were also higher, suggesting a conformational change induced by association. This suggestion was fully supported by the observed modifications of the heat stability and intrinsic fluorescence spectrum of the subunit resulting form association.     

The iron-sulfur clusters in the two related forms of mitochondrial NADH: ubiquinone oxidoreductase made by Neurospora crassa.

Two related forms of the respiratory-chain complex, NADH: ubiquinone oxidoreductase (Complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large, piericidin-A-sensitive form, which consists of some 23 different nuclear- and 6-7 mitochondrially encoded subunits. Cells grown in the presence of chloramphenicol make a small, piericidin-A-insensitive form which consists of only approximately 13 nuclear-encoded subunits. The subunits of the small form are either identical or similar to nuclear-encoded subunits of the large form. The iron-sulfur clusters in these two forms of Complex I are characterized by redox potentiometry and EPR spectroscopy. The large form of Complex I contains four EPR-detectable iron-sulfur clusters, N1, N2, N3 and N4, with the spin concentration of the individual clusters equivalent to the flavin concentration, similar to the mammalian counterparts. The small Complex I contains clusters N1, N3 and N4, but it is devoid of cluster N2. A model of the electron-transfer route through the large form of Complex I has been derived from these findings and an evolutionary pathway which leads to the emergence of large Complex I is discussed.     

Electron microscopy of simian virus 40 DNA configuration under denaturation conditions.

After isolation, the DNA of simian virus 40 appeared as a negative supertwist (form I) or as an open circle with at least one single-strand scission (form II). Under the denaturation conditions usually applied, such as heating in the presence of formaldehyde or application of alkali, form I molecules could appear as "relaxed" circles without single-strand scissions (form I') containing denatured sites not visible under the electron microscope. Form II molecules, under these denaturation conditions, showed partial or complete strand separations allowing the construction of denaturation maps. By using a modified denaturation procedure, i.e., heating of isolated SV40 DNA in the presence of dimethyl sulfoxide and formaldehyde followed by keeping the DNA in this denaturation solution at room temperature for periods up to 3 weeks, partially denatured relaxed circles without single-strand scissions were produced (form I'D) in addition to completely denatured form II molecules. The absence of single-strand scissions in form I'D molecules was demonstrated by a second heat treatment, which did not change the configuration of this molecular form. Form I'D molecules, in contrast to form I', contained denatured sites clearly discerible under the electron microscope. This combined application of two subsequent denaturation steps (denaturation by heating followed by denaturation at room temperature and neutral pH) showed that the molecular configuration I'D originated in two steps. The heating procedure produced molecules not distinquishable by electron microscopy from form I. In contrast to form I, these molecules were assumed to possess "preformed" denaturation sites (form I). Further treatment of form I molecules with denaturation solution at room temperature finally transformed them into convalently closed, relaxed, partially denatured circles exhibiting strand separations easily measurable on electron micrographs (form I'D). Denaturation maps of form I'D molecules were constructed by computer and compared with denaturation maps derived from partially denatured form II molecules. From these denaturation maps it can be concluded that the melting of base pairs occurring during the transition of simian virus 40 DNA form I into form I'D also preferentially happened at sites rich in the bases adenosine and thymine.     

DNA synthesis in polyoma virus infection. IV. Mechanism of formation of closed-circular viral DNA deficient in superhelical turns.

A marked reduction in the rate of viral DNA synthesis is accompanied by an alteration to the superhelicity of progeny DNA in polyoma virus-infected cells in which protein synthesis has been inhibited by cycloheximide. Viral DNA molecules formed in the presence of cycloheximide consist predominantly of closed-circular monometric species (referred to as form Ic) characterized by a decreased superhelix density, corresponding to deltasigmao = 0.0195, as compared to form I DNA by propidium diiodide-cesium chloride isopycnic analysis. Form Ic is synthesized on pre-existing form I templates without the intervention of progeny form I as an intermediate. It is concluded that inhibition of protein synthesis results in the alteration of some process in the closure of daughter DNA that leads to a marked reduction of superhelical turns of progeny molecules. About two-thirds of form Ic molecules return to the form I conformation upon reversal of cycloheximide inhibition by a mechanism independent of DNA replication.     

Patterns of strongly protein-associated simian virus 40 DNA replication intermediates resulting from exposures to specific topoisomerase poisons

Exposure of infected CV-1 cells to specific type I and type II topoisomerase poisons caused strong protein association with distinct subsets of simian virus 40 (SV40) DNA replication intermediates. On the basis of the known specificity and mechanisms of action of these drugs, the proteins involved are assumed to be the respective topoisomerases. Camptothecin, a topoisomerase I poison, caused strong protein association with form II (relaxed circular) and form III (linear) viral genomes and replication intermediates having broken DNA replication forks but not with form I (superhelical) viral DNA or normal late replication intermediates which were present. In contrast, type II topoisomerase poisons caused completely replicated forms and late viral replication forms to be tightly bound to protein--some to a greater extent than others. Different type II topoisomerase inhibitors caused distinctive patterns of protein association with the replication intermediates present. Both intercalating and nonintercalating type II topoisomerase poisons caused a small amount of form I (superhelical) SV40 DNA to be protein-associated in vivo. The protein complex with form I viral DNA was entirely drug-dependent and strong, but apparently noncovalent. The protein associated with form I DNA may represent a drug-stabilized "topological complex" between type II topoisomerase and SV40 DNA.