Restriction fragment length polymorphisms detected in N-ras-related sequences of rats and their linkage analyses

Novel restriction fragment length polymorphisms (RFLPs) in inbred rats were revealed with the human N-ras gene as probe. Three fragments hybridizing to the probe were detected by Southern blot hybridization under highly stringent conditions, and one of the fragments showed variation in inbred rat strains. Furthermore, on hybridization under low-stringency conditions, an additional fragment hybridizing to the probe was observed, and this fragment also showed interstrain variation. These two variant fragments showed different distributions in 27 inbred rat strains and segregated in backcross progeny as codominant alleles of independent single autosomal loci. Therefore, the loci for these RFLPs were named Nras-1 and Nras-2, respectively. Analyses of linkages between the RFLPs and 11 other loci revealed that the Nras-2 locus was closely linked to the c locus (3.7 +/- 2.6%), which belongs to rat linkage group I.     

Chemical modification of coenzyme B12-dependent diol dehydrase with pyridoxal 5′-phosphate: Lysyl residue essential for interaction between two components of the enzyme

The apoenzyme of diol dehydrase was inactivated by modification with pyridoxal 5′-phosphate (pyridoxal-P). The inactivation was accompanied by appearance of a new peak at 425 nm which was shifted to 325 nm by reduction with NaBH₄. ?-N-Pyridoxyl lysine was detected by paper chromatography and paper electrophoresis from the hydrolysate of the NaBH₄-reduced enzyme-pyridoxal-P complex. The relationship of inactivation vs pyridoxal-P incorporation as well as kinetic experiments suggests that one lysyl residue per enzyme molecule was essential for catalytic activity, although two to three pyridoxal-P molecules were introduced into the almost completely inactivated enzyme molecule. Both 1,2-propanediol (substrate) and adenosylcobalamin (coenzyme) completely protected the enzyme from inactivation. The result of disc gel electrophoresis showed that the inactivation of diol dehydrase by pyridoxal-P results from irreversible dissociation of the enzyme into subunits upon pyridoxal-P modification. Therefore, it is suggested that this modifiable lysyl residue is essential for subunit interaction to form an active oligomeric enzyme. The inactivated enzyme restored activity by addition of excess component F, but not by S, suggesting that the essential lysyl residue is located in component F of the enzyme. Pyridoxal-P-modified enzyme was no longer able to bind cyanocobalamin (a competitive inhibitor of adenosylcobalamin).     

Reactive sulfhydryl groups of coenzyme B12-dependent diol dehydrase: Differential modification of essential and nonessential ones

The apoenzyme of diol dehydrase was inactivated by four sulfhydryl-modifying reagents, p-chloromercuribenzoate, 5,5′-dithiobis(2-nitrobenzoate) (DTNB), iodoacetamide, and N-ethylmaleimide. In each case pseudo-first-order kinetics was observed. p-Chloromercuribenzoate modified two sulfhydryl groups per enzyme molecule and modification of the first one resulted in complete inactivation of the enzyme. DTNB also modified two sulfhydryl groups, but modification of the second one essentially corresponded to the inactivation. In both cases, the inactivation was reversed by incubation with dithiothreitol. Cyanocobalamin, a potent competitive inhibitor of adenosylcobalamin, protected the essential residue, but not the nonessential one, against the modification by these reagents. By resolving the sulfhydryl-modified cyanocobalamin-enzyme complex, the enzyme activity was recovered, irrespective of treatment with dithiothreitol. From these results, we can conclude that diol dehydrase has two reactive sulfhydryl groups, one of which is essential for catalytic activity and located at or in close proximity to the coenzyme binding site. The other is nonessential for activity. Neitherp-chloromercuribenzoate- nor DTNB-modified apoenzyme was able to bind cyanocobalamin, whereas the iodoacetamide- and N-ethylmaleimide-modified apoenzyme only partially lost the ability to bind cyanocobalamin. The inactivation of diol dehydrase by p-chloromercuribenzoate and DTNB did not bring about dissociation of the enzyme into subunits. Total number of the sulfhydryl groups of this enzyme was 14 when determined in the presence of 6 m guanidine hydrochloride. No disulfide bond was detected.     

Accumulation pattern of arachin and its subunits in maturation of groundnut seeds

The accumulation pattern of arachin and its subunits in growinggroundnuts was investigated. Soluble proteins were extractedfrom the kernels at twelve different stages of maturation (4–16weeks after pegging). Fractionation showed arachin, conarachinII, 5S and 2S protein components with sucrose gradient centrifugation.Ten weeks after pegging, only 35% of the maximum amount of arachinhad accumulated, whereas conarachin II was 85%, the 5S component89%, and the 2S component 76%. Arachin, however, increased rapidlyin the later stage of maturation. No change in the subunit ratioin arachin during seed growth was observed on the patterns ofsodium dodecyl sulfate-gel electrophoresis and gel isoelectricfocusing in the presence of urea. The ratio of the arachin subunitscontained in urea-extractable fraction of the kernels was constantthroughout seed development and was consistent with the subunitratio in arachin. On the other hand, the arachin subunits inthe free forms, if any, accounted for less than 1% of the associatedarachin subunits. Probably, the arachin subunits synthesizedin equimoles are associated into arachin without individualdeposition and are accumulated as arachin associates in growingseeds. (Received July 17, 1980; )     

Synthesis of biologically active cyclic peptides

1. The extent of racemization and the coupling yield in peptide synthesis were studied under high dilution conditions. The azide method yielded the best results. 2. Five linear penta-peptide precursors related to gramicidin S were subjected to cyclization in order to study how the difference in the sequence influences the yield and the ratio of cyclic dimer to monomer. The azide with the sequence of -L -Pro-L -Val-L -Orn(Z)-L -Leu-D -Phe- afforded diZ-gramicidin S in a high yield of 63%. 3. Alternaria mali toxin III, a cyclotetradepsipeptide phytotoxin, was synthesized. The activated linear tetradepsipeptide containing a D -Dap(Z) (N³-Z-D -2,3-diaminopropionic acid) residue at the N-terminus afforded the cyclic precursor (53%). The Dap residue in the precursor was converted into a ΔAla residue by Hofmann degradation to give the desired product.     

Purification and properties of endo-alpha-1,3-glucanase from a Streptomyces chartreusis strain.

An enzyme hydrolyzing the water-insoluble glucans produced from sucrose by Streptococcus mutans was purified from the culture concentrate of Streptomyces chartreusis strain F2 by ion-exchange chromatography on diethylaminoethyl cellulose and carboxymethyl cellulose columns and gel filtration on Bio-Gel A-1.5m. The purification achieved was 6.4-fold, with an overall yield of 27.3%. Electrophoresis of the purified enzyme protein gave a single band on a sodium dodecyl sulfate-polyacrylamide gel slab. Its molecular weight was estimated to be approximately 68,000, but there is a possibility that the native enzyme exists in an aggregated form or is an oligomer of the peptide subunits, have a molecular weight larger than 300,000. The pH optimum of the enzyme was 5.5 to 6.0, and its temperature optimum was 55 degrees C. The enzyme lost activity on heating at 65 degrees C for 10 min. The enzyme activity was completely inhibited by the presence of 1 mM Mn2+, Hg2+, Cu2+, Ag2+, or Merthiolate. The Km value for the water-insoluble glucan of S. mutans OMZ176 was an amount of glucan equivalent to 1.54 mM glucose, i.e., 0.89 mM in terms of the alpha-1,3-linked glucose residue. The purified enzyme was specific for glucans containing an alpha-1,3-glucosidic linkage as the major bond. The enzyme hydrolyzed the S. mutans water-insoluble glucans endolytically, and the products were oligosaccharides. These results indicate that the enzyme elaborated by S. chartreusis strain F2 is an endo-alpha-1,3-glucanase (EC 3.2.1.59).     

Studies in the Cyperaceae of Thailand I. New and otherwise noteworthy species of Fimbristylis

Six Thai species ofFimbristylis, including two new ones, are taxonomically discussed, and some range extensions into Burma are reported. Described as new areFimbristylis kernii from the neighborhood ofF. hookeriana, andF. smitinandii, a clearcut species of the sectionAbildgaardia.