Subunit structure and immunological properties of wheat carboxypeptidase

Wheat carboxypeptidases I, II, III and IV from wheat seeds with isoelectric points of 4.8, 5.6, 6.0 and 6.5, respectively, were found to be homogeneous by the Ouchterlony double immunodiffusion technique using an antiserum of the enzyme III. In a previous paper [1], the native enzyme III (MW = 118 k) was separated into two 58 k subunits (MW = 58 k) and further divided into the 35 k and 25 k fragments (MW = 35 k and 25 k, respectively). The native enzyme III and the 58 k subunit produced a single precipitin line against the antiserum. The 35 k and 25 k fragments did not cross-react with the antiserum. The amino acid compositions of the 35 k and 25 k fragments were similar to each other. Amino-terminal amino acids of the 35 k and 25 k fragments were both glutamic acid. Carboxy-terminal groups of the 35k and 25k fragments were determined to be -(Gly, Ser)-Glu-OH and -Thr-Pro-Glu-OH, respectively. The Ouchterlony double immunodiffusion technique revealed the presence of a common antigen in a carboxypeptidase from wheat seeds and one from germinated wheat. Comparison of both enzymes is discussed.     

Acetylglutamate synthase from Neurospora crassa: structure and regulation of expression

A novel gene shuffle approach has been developed for investigating the functions of genes on the cytoplasmic linear DNA killer plasmids of Kluyveromyces lactis . By transplacing k2ORF5 from the larger plasmid pGKL2(k2) onto pGKL1(k1) we have shown this gene to be essential and functionally interchangeable between plasmids. Once transferred onto k1, k2ORF5 is fully able to complement a k2ORF5⁰ deletion on k2 in trans , giving rise to yeast strains containing only the two recombinant plasmid forms. Additionally, the in vivo product of k2ORF5 has been identified as a 19.5 kDa protein by transplacing an epitope-tagged k2ORF5 allele from k2 to k1. The ease of detection of the tagged ORF5 product in comparison to TRF1, the gene product of k2ORF10, indicates that Orf5p is one of the most abundant k2 products, implying structural rather than regulatory function.     

A new protein subunit k for RNA polymerase from Xanthomonas campestris pv. oryzae

During the purification of RNA polymerase from Xanthomonas campestris pv. oryzae, a new subunit named k was found to be associated with this enzyme. The removal of subunit k from holoenzyme by DEAE-cellulose column chromatography results in a decrease in specific activity of the enzyme. The readdition of subunit k to subunit k-depleted holoenzyme results in restoration of enzymatic activity. Subunit k increase the activity of RNA polymerase; the activation was in proportion to the concentration of subunit k added. Antiserum against holoenzyme devoid of subunit k was prepared. This antiserum did not react with purified subunit k; therefore, subunit k may not be the proteolytic fragment of the beta, beta', sigma, or alpha subunit. When this antiserum was used to precipitate RNA polymerase obtained from a crude extract of bacterial cells, subunit k was coprecipitated as determined by sodium dodecyl sulfate gel electrophoretic analysis. The molecular mass of subunit k is approximately 29 kDa, and the molar ratio of beta:beta':sigma:alpha:k was estimated to be 1:1:1:2:4. When native Xp10 DNA was used as template, subunit k stimulated subunit k-depleted holoenzyme, but not core enzyme. When the synthetic polynucleotide poly[d(A-T)] was used, subunit k activated both subunit k-depleted holoenzyme and core enzyme. Subunit k also activated the binding of RNA polymerase to template DNA.     

Alice grows up: canopy science in transition from Wonderland to Reality

This paper gives, for the first time, a complete history of rain forest canopy access, from the early years to the present day. This review is primarily from the European perspective, and explores the development of canopy access techniques, from low-tech methods such as single rope technique, to hi-tech approaches such as canopy cranes. In recent years, canopy science has moved away from pure exploration (the Wonderland phase) to tackling the practicalities of rigorous canopy research (Reality), and the underlying emphasis is now shifting from access to the upper canopy per se to conducting replicative and manipulative science. The paper concludes by advocating the integration of many access techniques (both hi-tech and low-tech) at selected research sites, and certain neglected key areas of research are highlighted, including the comparison of adjacent primary and logged forests.     

Bovine colostric transforming growth factor-beta-like peptide that induces growth inhibition and changes in morphology of human osteogenic sarcoma cells (MG-63)

TGF-beta like peptide, termed TGF(BC-1), was partially purified from defatted and decaseinated bovine colostrum by a sequence of DEAE-Sephacel chromatography and Sephadex G-50 gel filtration in 1M acetic acid. TGF(BC-1) was distinct from well-known 25K TGF-beta in chemical properties: TGF(BC-1) was sensitive to acid ethanol extraction (Roberts et al., 1980). Its apparent molecular weight ranged from 21k to 11k by gel filtration and it was composed of low MW peptides (15k, 13k, 10k and 7.3k but not 25k) as examined by SDS-PAGE under non-reducing conditions. However, TGF(BC-1) shares some biological properties with the prototype TGF-b. TGF(BC-1) remarkably suppressed growth of osteogenic sarcoma cells (MG-63), and this was intriguingly accompanied by a striking change in morphology.     

Rational design of a drug for Alzheimer's disease with cholinesterase inhibitory and neuroprotective activity

The rate and duration of inhibition of recombinant human acetylcholinesterase (AChE) and human butyrylcholinesterase (BuChE) by nine N-methyl,N-alkyl derivatives of (R)-3-prop-2-ynylamino-indan, designed as potential treatment of Alzheimer's disease, was obtained from measurement of the carbamylation k(i) and decarbamylation k(3) rate constants. This also provided information about the rate of formation of the leaving group, 6-OH-(R)-3-prop-2-ynylamino-indan, designed as an MAO-B inhibitor with neuroprotective activity. The N-dimethyl derivative had the highest k(i) of the alkyl derivatives. Substitution of one N-methyl by N-ethyl resulted in a 14-fold decrease in k(i) and 28-fold decrease in k(3). A progressive increase in k(i) occurred as the length of the alkyl chain progressed from propyl to n-hexyl and cyclo-hexyl, with relatively little or no increase in k(3). Higher k(i) values than that of the dimethyl analogue were obtained with the N-aryl substitutes, N-phenyl and N-methoxy-phenyl. Six of the compounds had much higher k(i) values for BuChE than AChE, but the N-cyclo-hexyl and N-methoxy-phenyl compounds were inactive. However, an inverse relation was found between k(i) and the degree of brain AChE inhibition ex vivo after parenteral administration of the compounds in rats. This could have resulted from more rapid hydrolysis of the compounds with high k(i) values by esterases in blood and liver. Only the N-ethyl and N-propyl derivatives showed AChE and BuChE inhibitory activity in vivo of a suitably slow onset and long duration, together with MAO-B inhibition.     

Genetic manipulation of Kluyveromyces lactis linear DNA plasmids: gene targeting and plasmid shuffles

Genetic manipulation of yeast linear DNA plasmids, particularly of k1 and k2 from the non-conventional dairy yeast Kluyveromyces lactis, has been advanced by the recent establishment of DNA transformation-mediated one-step gene disruption and allele replacement techniques. These methods provide the basis for a strategy for the functional analysis of plasmid genes and DNA elements. By use of double selection regimens, these single-gene procedures have been extended to effect disruption of individual genes on plasmid k2 and transplacement of a functional copy onto plasmid k1, resulting in the production of yeast strains with an altered plasmid composition. This cytoplasmic gene shuffle system facilitates the introduction of specifically modified alleles into k1 or k2 in order to study the function, expression (from UCS promoters) and regulation of cytoplasmic linear plasmid genes. Additionally, identification, characterization and localization of plasmid gene products of interest are made possible by shuffling GFP-, epitope- or affinity purification-tagged alleles between k2 and k1. The gene shuffle approach can also be used for vector development and heterologous protein expression in order to exploit the biotechnical potential of the K. lactis k1/k2 system in yeast cell factory research.     

Modulation of F1 cytotoxic potentials by graft-vs-host reaction. Cooperative non-H-2- and H-2D region-gene control of F1 natural resistance to graft-vs-host reaction-associated immunosuppression

Our previous study revealed that in F1 mice raised by crossing C3H/He or AKR/J mice with various H-2-congenic B10-series strains, parental H-2k spleen cells (SC) could not induce the graft-vs-host reaction (GvHR)-associated immunosuppression (GAIS). We also elucidated that a limited number of non-H-2 genes of parental C3H/He or AKR/J mice that had been incorporated into the F1 hybrids determined the F1 resistance to the GAIS, and the present study was done to explore the mechanism implicated in this type of F1 resistance to GAIS. SC from B10.AL mice carrying an rH-2 (K:k I:k S:k D:d) haplotype but not SC from H-2K B10.BR (k k k k) mice induced GAIS of in vitro CTL responses to third-party alloantigens in H-2k/d (C3H/He x B10.D2)F1 recipients mice. Further, SC from H-2k/a (C3H/He x B10.A)F1 mice carrying heterozygous C3H/B10 non-H-2 background but not SC from the same H-2k/a (B10.BR x B10.A)F1 mice but carrying homozygous B10/B10 background induced GAIS in H-2k/d (C3H/He x B10.D2)F1 recipients. Although C3H/He-, B10.BR-, and C3H.OH (d d d k)-SC were incapable of inducing GAIS in (C3H/He x B10.D2)F1 (k/d k/d k/d k/d) recipients, they were all good inducers of GAIS in (C3H.OH x B10.BR)F1 (d/k d/k d/k k/k) recipients. Exactly the same pattern of co-operative non-H-2 AKR and H-2D region-gene control of GAIS was observed on GvHR induced in H-2k/d (AKR/J x B10.D2)F1 recipients. These results suggest that the non-H-2 genes of C3H/He or AKR/J strain inhibit the functional expression of certain antigenic determinant(s) when it is encoded by heterozygous but not homozygous gene(s) linked tightly to H-2D region of k haplotype. Thus, the F1 resistance to GAIS is mediated by immune response of F1 recipients who miss the antigenic determinant(s) against that expressed on cell surface of GvHR-inducing T lymphocytes.     

Patterns of proteins synthesized in the R15 neuron of Aplysia. Temporal studies and evidence for processing

The time-course of changes in the pattern of newly synthesized proteins in the R15 neuron of the parietovisceral ganglion of Aplysia californica has been studied at 14 degrees C. 5% polyacrylamide gels containing sodium dodecyl sulfate (SDS) have been used to separate newly synthesized (leucine-labeled) proteins from the neuron. We have demonstrated that the pattern of newly synthesized proteins from the R15 neuron does not change significantly if 5-h pulses of labeled leucine are given during the first 72 h of in vitro incubation of the excised ganglion. However, the level of leucine incorporation begins to decline somewhere between 17 and 43 h after the ganglion is isolated; at 43 and 69 h the levels of incorporation fell to 29 and 10% of the initial level, respectively. A number of conclusions have been drawn from the use of a sequential, double-label type of experiment in the same cell. There is processing of SDS-soluble, 12,000-dalton (12k) material to 6,000-9,000-dalton (6-9k) material. These materials are the two major peaks on gels after long labeling periods and together account for about 35% of all newly synthesized proteins. After synthesis of 12k material, there is a gradual disappearance of 12k (half-life about 8 h) and simultaneous appearance of 6-9k material on the gels, as the postsynthesis "chase" period of ganglia incubation is increased. The processing of 12k to 6-9k material occurs even in the presence of anisomycin, a protein syntehsis inhibitor, during the chase period. While the rate of 12k to 6-9k conversion can vary from cell to cell, it appears to remain consistent within, and is characteristic of, any individual R15. We detect no circadian rhythm in either the rate of 12k synthesis or the rate of 12k to 6-9k processing with 5-h label periods. These results are discussed in relation to the roles of 12k and 6-9k material in the R15 neuron.     

汉坦病毒结构蛋白的鉴定及其应用

对差速离心纯化的汉坦病毒R84-1毒株进行了SDS-PAGE和免疫印迹试验。发现有67k和43k两条蛋白区带能与汉坦病毒抗体起反应。经单克隆抗体鉴定,67k多肽可能属病毒囊膜蛋白,43k多肽未定。用免疫印迹法对出血热患者进行检测,初步证明野鼠型感染者具有上述两种蛋白抗原的抗体,大鼠型患者仅具有67k蛋白的抗体,这对出血热患者血清学分型有重要意义。     

Species-specific antigens ofMycoplasma hyopneumoniae and cross-reactions with other porcine mycoplasmas

Cell proteins from the porcine mycoplasmasMycoplasma hyorhinis, M. hyopneumoniae, andM. flocculare have been analyzed by SDS-gel electrophoresis and immunoblotting. The protein profiles ofM. hyopneumoniae andM. flocculare were similar, but the protein profile ofM. hyorhinis was quite different from the others. Antisera prepared against whole cells of the above three mycoplasmas were used in immunoblotting of electrophoretically separated antigens and in enzyme-linked immunosorbent assay. One major antigen, which had a molecular weight of 73 k, was found to be common to all three mycoplasmas. Another major antigen, with a molecular weight of 41 k, was common toM. hyopneumoniae andM. flocculare and may also be present inM. hyorhinis. Several antigens of comparatively high molecular weights (108 k, 102 k, 93 k, 89 k, and 87 k) seemed to be specific forM. hyopneumoniae. Three antisera prepared by immunization of rabbits with immunoprecipitates obtained by crossed immunoelectrophoresis ofM. hyopneumoniae were also used in blotting experiments. One of these antisera was found to be directed against the 73 k antigen common to the three porcine mycoplasmas investigated. The other two antisera were directed againstM. hyopneumoniae-specific antigens with molecular weights of 74 k, 58 k, 45 k, 44 k, and 38 k.     

Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional S6 kinase.

Recent studies have shown that the p70(s6k)/p85(s6k) signaling pathway plays a critical role in cell growth by modulating the translation of a family of mRNAs termed 5'TOPs, which encode components of the protein synthetic apparatus. Here we demonstrate that homozygous disruption of the p70(s6k)/p85(s6k) gene does not affect viability or fertility of mice, but that it has a significant effect on animal growth, especially during embryogenesis. Surprisingly, S6 phosphorylation in liver or in fibroblasts from p70(s6k)/p85(s6k)-deficient mice proceeds normally in response to mitogen stimulation. Furthermore, serum-induced S6 phosphorylation and translational up-regulation of 5'TOP mRNAs were equally sensitive to the inhibitory effects of rapamycin in mouse embryo fibroblasts derived from p70(s6k)/p85(s6k)-deficient and wild-type mice. A search of public databases identified a novel p70(s6k)/p85(s6k) homolog which contains the same regulatory motifs and phosphorylation sites known to control kinase activity. This newly identified gene product, termed S6K2, is ubiquitously expressed and displays both mitogen-dependent and rapamycin-sensitive S6 kinase activity. More striking, in p70(s6k)/p85(s6k)-deficient mice, the S6K2 gene is up-regulated in all tissues examined, especially in thymus, a main target of rapamycin action. The finding of a new S6 kinase gene, which can partly compensate for p70(s6k)/p85(s6k) function, underscores the importance of S6K function in cell growth.     

The insulin-induced signalling pathway leading to S6 and initiation factor 4E binding protein 1 phosphorylation bifurcates at a rapamycin-sensitive point immediately upstream of p70s6k.

Employing specific inhibitors and docking-site mutants of growth factor receptors, recent studies have indicated that the insulin-induced increase in 40S ribosomal protein S6 and initiation factor 4E binding protein 1 (4E-BP1) phosphorylation is mediated by the mTOR/FRAP-p70s6k signal transduction pathway. However, it has not been resolved whether the phosphorylation of both proteins is mediated by p70s6k or whether they reside on parallel pathways which bifurcate upstream of p70s6k. Here we have used either rapamycin-resistant, kinase-dead, or wild-type p70s6k variants to distinguish between these possibilities. The rapamycin-resistant p70s6k, which has high constitutive activity, was able to signal to S6 in the absence of insulin and to prevent the rapamycin-induced block of S6 phosphorylation. This same construct did not increase the basal state of 4E-BP1 phosphorylation or protect it from the rapamycin-induced block in phosphorylation. Unexpectedly, the rapamycin-resistant p70s6k inhibited insulin-induced 4E-BP1 phosphorylation in a dose-dependent manner. This effect was mimicked by the kinase-dead and wild-type p70s6k constructs, which also blocked insulin-induced dissociation of 4E-BP1 from initiation factor 4E. Both the kinase-dead and wild-type constructs also blocked reporter p70s6k activation, although only the kinase-dead p70s6k had a dominant-interfering effect on S6 phosphorylation. Analysis of phosphopeptides from reporter 4E-BP1 and p70s6k revealed that the kinase-dead p70s6k affected the same subset of sites as rapamycin in both proteins. The results demonstrate, for the first time, that activated p70s6k mediates increased S6 phosphorylation in vivo. Furthermore, they show that increased 4E-BP1 phosphorylation is controlled by a parallel signalling pathway that bifurcates immediately upstream of p70s6k, with the two pathways sharing a common rapamycin-sensitive activator.     

Saponin-like substances inhibit α-galactosidase production in the endosperm of fenugreek seeds

When fenugreek (Trigonella foenum-graecum L.) endosperms plus testa (endosperms), which had been isolated from 5-h-imbibed seeds, were incubated for at least 2 h under germination conditions, they leaked substances which, like exogenous abscisic acid (ABA), inhibited the production of fenugreek endosperm -galactosidase. However, unlike ABA, 8 h treatment with these inhibitors had no effect on fenugreek endosperms which had been isolated from 15-h-imbibed seeds and leached for 2 h. This indicated that either their inhibitory action was on processes which were related to the production of -galactosidase and had been completed by this time, or that there might be factors present which inactivate these inhibitors. It was also concluded that the action of the endosperm leachate could not be attributed to the presence of ABA. The activity of the leachate decreased when it originated from endosperms imbibed for periods longer than 25 h and thin-layer chromatography (TLC) of extracts from these endosperms showed decreased contents of the leachable inhibitors as imbibition proceeded. From the seed leachate, which had a TLC pattern and inhibitory action similar to that of the endosperm, were isolated three substances which, when applied to endosperms, inhibited the production of -galactosidase activity. According to their chromatographic behaviour and their reaction with specific reagents, there are strong indications that these substances are saponins. These diffusible saponin-like substances were located in both endosperm and perisperm and their physiological role is discussed.Abbreviations ABA abscisic acid - PEG polyethylenglycol - TLC thin-layer chromatography We wish to thank the Alexander S. Onasis Public Benefit Foundation for a grant to K.Z. and Dr. J.S.G. Reid (University of Stirling, Scotland) for a kind gift of fenugreek seeds.     

Proton inventories constitute reliable tools in investigating enzymatic mechanisms: application on a novel thermo-stable extracellular protease from a halo-alkalophilic Bacillus sp

A novel protease designated protease-A-17N-1, was purified from the halo-alkalophilic Bacillus sp. 17N-1, and found active in media containing dithiothreitol and EDTAK(2). This enzyme maintained significant activity from pH 6.00 to 9.00, showed optimum k(cat)/K(m) value at pH 7.50 and 33 degrees C. It was observed that only specific inhibitors of cysteine proteinases inhibited its activity. The pH-(k(cat)/K(m)) profile of protease-A-17N-1 was described by three pK(a)s in the acid limb, and one in the alkaline limb. Both are more likely due t3o the protonic dissociation of an acidic residue, and the development and subsequent deprotonation of an ion-pair, respectively, in its catalytic site, characteristic for cysteine proteinases. Moreover, both the obtained estimates of rate constant k(1) and the ratio k(2)/k(-1) at 25 degrees C, from the temperature-(k(cat)/K(m)) profile of protease-A-17N-1, were found similar to those estimated from the proton inventories of the same parameter, verifying the reliability of the latter methodology. Besides, the bowed-downward proton inventories of k(cat)/K(m), as well as the large inverse SIE observed for this parameter, in combination with its dependence versus temperature, were showed unambiguously that k(cat)/K(m) = k(1). Such results suggest that the novel enzyme is more likely to be a cysteine proteinase functioning via a general acid-base mechanism.     

Multiple proteins and subgenomic mRNAs may be derived from a single open reading frame on tobacco mosaic virus RNA

It has previously been shown that messenger activity for a protein of Mr = ca. 30k exists in RNA fractions extracted from particles of either native or alkali stripped U1 TMV, or from cowpea strain TMV, that are smaller than full genomic length. Analysis of sucrose gradient fractions containing this activity reveals a number of slightly smaller template activities directing synthesis of proteins between 18.5k and 29k in size. All of these messenger activities, including that for the 30k protein, respond to cap analogues in anomalous ways. Discrete RNA species that include active mRNAs for these proteins can be demonstrated in the same fractions by labelling with preparations of vaccinia capping enzyme and [alpha-32P] GTP without prior beta-elimination. Detailed analysis of three of these proteins (of Mr's ca. 30k, 29k and 23k) by peptide mapping and translation of purified vaccinia-labelled RNA demonstrates that all three are unrelated to the large early TMV proteins, but are related to each other in such a way as to form a nested set with staggered N termini and identical C termini. mRNAs of chain lengths ca. 1900 and 1500 bases direct synthesis of the 30k and 23k proteins respectively, an mRNA of about 1850 bases directs both 29k and (perhaps because of cross-contamination) 30k synthesis. Initiation codons for the 29k and 23k proteins have been mapped at positions 4960-4962 and 5191-5193 respectively on TMV RNA. Since all three encapsidated templates have similar properties we conclude that either there is a family of 30k-related proteins with unusual mRNAs, or that none of these in vitro translation products are directed by physiological templates.     

Design of a low‐density SNP chip for the main Australian sheep breeds and its effect on imputation and genomic prediction accuracy

Genotyping sheep for genome‐wide SNPs at lower density and imputing to a higher density would enable cost‐effective implementation of genomic selection, provided imputation was accurate enough. Here, we describe the design of a low‐density (12k) SNP chip and evaluate the accuracy of imputation from the 12k SNP genotypes to 50k SNP genotypes in the major Australian sheep breeds. In addition, the impact of imperfect imputation on genomic predictions was evaluated by comparing the accuracy of genomic predictions for 15 novel meat traits including carcass and meat quality and omega fatty acid traits in sheep, from 12k SNP genotypes, imputed 50k SNP genotypes and real 50k SNP genotypes. The 12k chip design included 12 223 SNPs with a high minor allele frequency that were selected with intermarker spacing of 50–475 kb. SNPs for parentage and horned or polled tests also were represented. Chromosome ends were enriched with SNPs to reduce edge effects on imputation. The imputation performance of the 12k SNP chip was evaluated using 50k SNP genotypes of 4642 animals from six breeds in three different scenarios: (1) within breed, (2) single breed from multibreed reference and (3) multibreed from a single‐breed reference. The highest imputation accuracies were found with scenario 2, whereas scenario 3 was the worst, as expected. Using scenario 2, the average imputation accuracy in Border Leicester, Polled Dorset, Merino, White Suffolk and crosses was 0.95, 0.95, 0.92, 0.91 and 0.93 respectively. Imputation scenario 2 was used to impute 50k genotypes for 10 396 animals with novel meat trait phenotypes to compare genomic prediction accuracy using genomic best linear unbiased prediction (GBLUP) with real and imputed 50k genotypes. The weighted mean imputation accuracy achieved was 0.92. The average accuracy of genomic estimated breeding values (GEBVs) based on only 12k data was 0.08 across traits and breeds, but accuracies varied widely. The mean GBLUP accuracies with imputed 50k data more than doubled to 0.21. Accuracies of genomic prediction were very similar for imputed and real 50k genotypes. There was no apparent impact on accuracy of GEBVs as a result of using imputed rather than real 50k genotypes, provided imputation accuracy was >90%.     

Differential substrate behaviour of phenol and aniline derivatives during oxidation by horseradish peroxidase: kinetic evidence for a two-step mechanism

The catalytic constant (k(cat)) and the second-order association constant of compound II with reducing substrate (k(5)) of horseradish peroxidase C (HRPC) acting on phenols and anilines have been determined from studies of the steady-state reaction velocities (V(0) vs. [S(0)]). Since k(cat)=k(2)k(6)/k(2)+k(6), and k(2) (the first-order rate constant for heterolytic cleavage of the oxygen-oxygen bond of hydrogen peroxide during compound I formation) is known, it has been possible to calculate the first-order rate constant for the transformation of each phenol or aniline by HRPC compound II (k(6)). The values of k(6) are quantitatively correlated to the sigma values (Hammett equation) and can be rationalized by an aromatic substrate oxidation mechanism in which the substrate donates an electron to the oxyferryl group in HRPC compound II, accompanied by two proton additions to the ferryl oxygen atom, one from the substrate and the other the protein or solvent. k(6) is also quantitatively correlated to the experimentally determined (13)C-NMR chemical shifts (delta(1)) and the calculated ionization potentials, E (HOMO), of the substrates. Similar dependencies were observed for k(cat) and k(5). From the kinetic analysis, the absolute values of the Michaelis constants for hydrogen peroxide and the reducing substrates (K(M)(H(2)O(2)) and K(M)(S)), respectively, were obtained.     

Isolation and characterization of recoverin-like Ca(2+)-binding protein from rat brain.

Rat brain was found, by immunoblot analysis, to have a protein of Mr 23,000 (P23k) that was clearly different from recoverin and was labeled with an antiserum raised against the NH2-terminus of recoverin. P23k could not be detected by an antiserum raised against the COOH-terminus of recoverin. Blots with 45Ca demonstrated that P23k bound Ca2+. This calciprotein was further purified by Ca(2+)-dependent hydrophobic interaction and ion-exchange chromatography. In SDS polyacrylamide gel electrophoresis, P23k had an apparent Mr of 21,000 in the presence of 10 microM Ca2+ and 23,000 in the absence of Ca2+ (0.1 mM EGTA). The isoelectric point of P23k was 5.6. Ca(2+)-binding analysis indicated that P23k bound 2 moles of Ca2+ per mole of protein and had two binding sites with dissociation constants of 13 microM and 0.2 microM. Purified P23k bound to the crude membrane fractions from the cerebellum, cerebrum and retina in a Ca(2+)-dependent manner. Partial amino acid sequence analysis of proteolytic fragments of P23k revealed the sequence homology between P23k and recoverin. These results suggested that P23k may act as a Ca(2+)-sensitive regulator by forming a complex with its target on the membrane.     

Chamber properties from transmitral flow: prediction of average and passive left ventricular diastolic stiffness.

A chamber stiffness (K(LV))-transmitral flow (E-wave) deceleration time relation has been invasively validated in dogs with the use of average stiffness [(DeltaP/DeltaV)(avg)]. K(LV) is equivalent to k(E), the (E-wave) stiffness of the parameterized diastolic filling model. Prediction and validation of 1) (DeltaP/DeltaV)(avg) in terms of k(E), 2) early rapid-filling stiffness [(DeltaP/DeltaV)(E)] in terms of k(E), and 3) passive (postdiastasis) chamber stiffness [(DeltaP/DeltaV)(PD)] from A waves in terms of the stiffness parameter for the Doppler A wave (k(A)) have not been achieved. Simultaneous micromanometric left ventricular (LV) pressure (LVP) and transmitral flow from 131 subjects were analyzed. (DeltaP)(avg) and (DeltaV)(avg) utilized the minimum LVP-LV end-diastolic pressure interval. (DeltaP/DeltaV)(E) utilized DeltaP and DeltaV from minimum LVP to E-wave termination. (DeltaP/DeltaV)(PD) utilized atrial systolic DeltaP and DeltaV. E- and A-wave analysis generated k(E) and k(A). For all subjects, noninvasive-invasive relations yielded the following equations: k(E) = 1,401. (DeltaP/DeltaV)(avg) + 59.2 (r = 0.84) and k(E) = 229.0. (DeltaP/DeltaV)(E) + 112 (r = 0.80). For subjects with diastasis (n = 113), k(A) = 1,640. (DeltaP/DeltaV)(PD) - 8.40 (r = 0.89). As predicted, k(A) showed excellent correlation with (DeltaP/DeltaV)(PD); k(E) correlated highly with (DeltaP/DeltaV)(avg). In vivo validation of average, early, and passive chamber stiffness facilitates quantitative, noninvasive diastolic function assessment from transmitral flow.