Distribution of 59- and 55-kDa Catalase in Dark- and Light-grown Pumpkin and Various Other Plant Tissues

The catalase molecule in germinating pumpkin cotyledons is synthesizedas a precursor (59-kDa) form, whose relative molecular massis larger than the mature enzyme (55-kDa). Although both typesof molecules are localized in the microbodies, the 59-kDa specieshas been shown to be present predominantly in the leaf peroxisomesisolated from green cotyledons, while the 55-kDa species ispredominantly in the glyoxysomes from etiolated cotyledons [Yamaguchiet al. (1984) Proc. Natl. Acad. Sci. USA, 81: 4809]. We examinedthe distribution of the 59- and 55-kDa catalase molecules indark- and light-grown tissues of pumpkin seedlings as well asin other plant species, using the immunoblotting technique.The ratios of the 59- and 55-kDa catalase species differed inthe pumpkin tissues examined. Light interferes with the conversionof the 59-kDa precursor to the 55-kDa form, especially in thecotyledons. The effect of light was less pronounced in the rootsand hypocotyls, indicating that the light regulation of theconversion is tissue-specific. Dark- and light-grown cotyledonsfrom cucumber and watermelon seedlings showed a similar lightregulation, suggesting that cucurbitaceous plants possess similarlight-regulatory mechanism. From the analysis of catalase proteinfrom various plant tissues, a limited correlation between molecularforms of catalase and different microbody populations was observed. (Received September 6, 1986; Accepted December 4, 1986)     

Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H

To cleave RNA molecules using RNase H in a site-specific manner, a short deoxyoligonucleotide (3-5mer) joining with 2'-O-methyl oligonucleotide(s) was designed as a DNA splint to be used. Model experiments were carried out using ribooligonucleotide substrates (9 and 18 mer). It was found that the use of this type of splints (9 mer) causes a unique cleavage by RNase H. For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU. This method will have a variety of applications for the study of RNA.     

Accumulation pattern of amino acid substitutions in protein evolution

Summary A simple method for the evolutionary analysis of amino acid sequence data is presented and used to examine whether the number of variable sites (NVS) of a protein is constant during its evolution. The NVSs for hemoglobin and for mitochondrial cytochrome c are each found to be almost constant, and the ratio between the NVSs is close to the ratio between the unit evolutionary periods. This indicates that the substitution rate per variable site is almost uniform for these proteins, as the neutral theory claims. An advantage of the present analysis is that it can be done without knowledge of paleontological divergence times and can be extended to bacterial proteins such as bacterial c-type cytochromes. It is suggested that the NVS of cytochrome c has been almost constant even over the long period (ca. 3.0 billion years) of bacterial evolution but that at least two different substitution rates are necessary to describe the accumulated changes in the sequence. This two clock interpretation is consistent with fossil evidence for the appearance times of photosynthetic bacteria and eukaryotes.     

Phosphorylation and Inactivation of Brain Glycogen Synthase by a Multifunctional Calmodulin-Dependent Protein Kinase

Glycogen synthase was partially purified from canine brain to about 70% purity. The purified enzyme showed differences from the properties of the skeletal muscle enzyme with respect to molecular weights of the holoenzyme and subunit and phosphopeptide mapping. The multifunctional calmodulin-dependent protein kinase from the brain phosphorylated brain glycogen synthase with concomitant inactivation of the enzyme. Although about 1.3 mol of phosphate/mol subunit was maximally incorporated into glycogen synthase, 0.4 mol of phosphate/mol subunit was sufficient for the maximal inactivation of the enzyme. The results indicate that brain glycogen synthase is regulated in a calmodulin-dependent manner similarly to the skeletal muscle enzyme, but that the brain enzyme is different from the skeletal muscle enzyme.     

Taxonomic studies ofAsarum sensu lato

The floral vascular anatomy of 12 species representing each ofAsarum s. str.,Asiasarum, Geotaenium, Heterotropa andHexastylis are compared to clarify intergeneric relationships. The five genera have basically similar structures in floral morphology and vasculature, and consistently have a six-carpelled compound ovary and the associated similar placental vasculature. They show, however, a significant difference in the position and the constituent of the “ventral” carpellary bundles in the placental axis betweenAsiasarum-Heterotropa-Hexastylis andAsarum-Geotaenium. InAsiasarum, Heterotropa andHexastylis the ventral bundles of each carpel are basically free and antilocular as expected in the least specialized compound ovary of angiosperms; in contrast, inAsarum andGeotaenium the ventral carpellary bundles are antiseptal and heterogenous (i.e., formed by the lateral fusion of ventral bundles of adjacent carpels). Shared probable apomorphic floral vasculature, as well as shared single style-column, suggests the closest mutual relationships betweenAsarum andGeotaenium. In terms of floral morphology and anatomy,Asiasarum, Heterotropa andHexastylis retain plesiomorphies. Possible chromosomal evolution in the related genera is also discussed.     

A low-temperature-sensitive intermediate state between S2 and S3 in photosynthetic water oxidation deduced by means of thermoluminescence measurements

The temperature dependence of S-state transitions in Photosystem II was measured by means of thermoluminescence using two different protocols for low-temperature flash excitation: protocol A, “last flash at low temperature”, and protocol B, “all flashes at low temperature”. Comparison of the temperature-dependence curves obtained by these two protocols revealed a marked difference particular for the three-flash experiments. The difference was attributed to the formation of a low-temperature sensitive precursor state between S₂ and S₃. The state is formed by two flash illumination given at −5 to −50°C, spontaneously transforms to normal S₃ on dark warming, and is not converted to S₀ by the 3rd flash. The precursor state was tentatively assigned to an S₃ in which H⁺ release is not completed.     

Hypersomatostatinemia in chronic renal failure

Plasma concentrations of somatostatin-like immunoreactivity (SLI) were determined in uremic patients on maintenance hemodialysis. Plasma SLI levels were significantly (p less than 0.001) elevated in 26 diabetic uremic patients (67.1 +/- 6.8 pg/ml, mean +/- SE) and in 24 non-diabetic uremic patients (43.5 +/- 7.2 pg/ml), when compared with 60 healthy subjects (5.0 +/- 0.7 pg/ml). Paired pooled plasma from uremic patients before and after hemodialysis was subjected to a reverse-phase octadecasilyl-silica (C-18) cartridge and then the extract was gel filtered on a Sephadex G-25 column (1.6 X 90 cm). Both elution profiles showed two peaks of SLI which coeluted with synthetic somatostatin (SS)-28 and SS-14 markers, respectively. The SS-28-like immunoreactivity (LI) peak, which was estimated by using SS-14 as a reference standard, was 3-fold larger than that for SS-14 LI. On the basis of immunoequivalency of the two components in the present assay, SS-28 LI constitutes approximately 75% of circulating somatostatin. In conclusion, plasma SLI is substantially high in uremic patients of both diabetic and non-diabetic etiology and the SS-28 is a predominant form of circulating SLI in these patients, probably, in part, for a lower clearance of this molecule.     

Structure and function relationship of staphylocoagulase

The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of -thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by -chymotrypsin. Limited -chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (K_d=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (K_d=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (K_d=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH₂-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH₂-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH₂-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, -chymotrypsin, and elastase.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.