Kinetics of the hydrolysis of mixed micelles of dipalmitoyllecithin with triton X-100 catalyzed by a phospholipase A2 from the venom of Agkistrodon halys blomhoffii

The pH dependence of kinetic parameters for the hydrolysis of mixed micelles of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (diC16PC) with Triton X-100, catalyzed by the intact and the N-terminal alpha-NH2-modified phospholipases A2 (PLA2s) of Agkistrodon halys blomhoffii, was studied at 25 degrees C and ionic strength 0.1 in the presence of saturating amounts of Ca2+. The pH dependence of the kinetic parameters for the hydrolysis of monodispersed diC6PC, catalyzed by the modified enzyme, was also studied under the same conditions, and the data were compared with the previous results for the intact enzyme [Teshima, K. et al. (1986) J. Biochem. 100, 1655-1662]. The pK values of the catalytic group, His 48, and Tyr 52 were found to shift from 5.55 to 7.00 and from 10.50 to 11.50, respectively, on binding of the micellar substrates to the enzyme. On the other hand, no participation of these ionizable groups was observed for the binding of the monodispersed substrate. On the basis of the present finding and the X-ray crystallographic studies on bovine pancreatic PLA2 [Dijkstra, B.W. et. al. (1981) J. Mol. Biol. 147, 97-123] and on a PLA2 of Crotalus atrox venom [Brunie, S. et al. (1985) J. Biol. Chem. 260, 9742-9749], the hydrogen-bonding of Tyr 73, which is involved in the lipid-water interface recognition site, to His 48 and Tyr 52 in the active center was strongly suggested to be important for the hydrolysis of micellar substrates.     

Ring Y chromosome: 45,X/46,X,r(Y) chromosome mosaicism in a phenotypically normal male with azoospermia

Summary Chromosome analysis of lymphocytes in a phenotypically normal male with azoospermia showed a mosaicism 45,X/46,X,r(Y). Seven other cases from the literature are discussed.     

Formation of aminopropylhomospermidine from homospermidine in yeasts and thermophilic bacilli

Abstract When the yeasts Saccharomyces cerevisiae, Candida albicans and Schizosaccharomyces pombe and the thermophilic bacteria Bacillus stearothermophilus and Bacillus acidocaldarius were cultured in the presence of homospermidine, a new compound accumulated in the cells within a few days. This compound was identified as aminopropylhomospermidine [NH₂(CH₂)₃NH (CH₂)₄NH(CH₂)₄NH₂] by gas chromatographymass spectrometry (GC-MS) and by the enzymatic cleavage method developed in our laboratories. This polyamine was not produced from homospermidine in Escherichia coli, Bacillus subtilis, Bacillus alkalophilus , or a eukaryotic protozoon, Tetrahymena pyriformis , none of which usually contains appreciable amounts of spermine. These findings suggest that the synthesis of aminopropylhomospermidine from homospermidine is mediated by spermine synthase.     

Photosynthetic and light-enhanced dark fixation of 14CO2 from the ambient atmosphere and 14C-bicarbonate infiltrated through vascular bundles in maize leaves

1. The capacity of light-enhanced dark fixation of ¹⁴CO₂ from theambient atmosphere decayed following time-course characteristicsof a first-order reaction (half-life, 1–2 min). The levelof phosphoenolpyruvate in maize leaves under CO₂-free air didnot decrease in the dark subsequent to preillumination. Theseresults indicate that phosphoenolpyruvate carboxylase is activatedin light and quickly inactivated in the following darkness.
2. Removal of oxygen from the atmosphere did not exert any effecton the products of light-enhanced dark fixation of ¹⁴CO₂ providedfrom the atmosphere, the major labeled compounds being malateand aspartate. This confirms that the transfer of carboxyl carbonof C₄-acids to form 3-phosphoglycerate is light-dependent.
3. WhenNaH¹⁴CO₃ solution was vacuum-infiltrated through vasculartissuesof maize leaves, the main initial photosynthetic ¹⁴CO₂fixationproducts were phosphate esters. This indicates thatby thistechnique, ¹⁴CO₂ could be directly provided to the bundlesheathcells, and was fixed via the reductive pentose phosphatecycle.On the other hand, the main initial ¹⁴CO₂-fixation productswere malate and aspartate even when ¹⁴CO₂ was provided throughvascular tissues in the dark immediately following preillumination.The possible regulatory mechanisms underlying the above findingsare discussed.

¹ This work was reported at the 4th International Congress onPhotosynthesis, Reading, September 1977. Request for reprintsshould be addressed to S. Miyachi, Institute of Applied Microbiology,University of Tokyo, Bunkyo-ku, Tokyo 113, Japan ² Present address: Okinawa Branch of Tropical Agriculture ResearchCenter, Ishigaki-shi, Okinawa 907, Japan. (Received October 28, 1977; )     

N1-aminopropylagmatine, a new polyamine produced as a key intermediate in polyamine biosynthesis of an extreme thermophile, Thermus thermophilus

In the extreme thermophile Thermus thermophilus, a disruption mutant of a gene homologous to speB (coding for agmatinase = agmatine ureohydrolase) accumulated N1-aminopropylagmatine (N8-amidino-1,8-diamino-4-azaoctane, N8-amidinospermidine), a new compound, whereas all other polyamines produced by the wild-type strain were absent from the cells. Double disruption of speB and speE (polyamine aminopropyltransferase) resulted in the disappearance of N1-aminopropylagmatine and the accumulation of agmatine. These results suggested the following. 1) N1-Aminopropylagmatine is produced from agmatine by the action of an enzyme coded by speE. 2) N1-Aminopropylagmatine is a metabolic intermediate in the biosynthesis of unique polyamines found in the thermophile. 3) N1-Aminopropylagmatine is a substrate of the SpeB homolog. They further suggest a new biosynthetic pathway in T. thermophilus, by which polyamines are formed from agmatine via N1-aminopropylagmatine. To confirm our speculation, we purified the expression product of the speB homolog and confirmed that the enzyme hydrolyzes N1-aminopropylagmatine to spermidine but does not act on agmatine.     

An exo-beta-1,3-galactanase having a novel beta-1,3-galactan-binding module from Phanerochaete chrysosporium

An exo-beta-1,3-galactanase gene from Phanerochaete chrysosporium has been cloned, sequenced, and expressed in Pichia pastoris. The complete amino acid sequence of the exo-beta-1,3-galactanase indicated that the enzyme consists of an N-terminal catalytic module with similarity to glycoside hydrolase family 43 and an additional unknown functional domain similar to carbohydrate-binding module family 6 (CBM6) in the C-terminal region. The molecular mass of the recombinant enzyme was estimated as 55 kDa based on SDS-PAGE. The enzyme showed reactivity only toward beta-1,3-linked galactosyl oligosaccharides and polysaccharide as substrates but did not hydrolyze beta-1,4-linked galacto-oligosaccharides, beta-1,6-linked galacto-oligosaccharides, pectic galactan, larch arabinogalactan, arabinan, gum arabic, debranched arabinan, laminarin, soluble birchwood xylan, or soluble oat spelled xylan. The enzyme also did not hydrolyze beta-1,3-galactosyl galactosaminide, beta-1,3-galactosyl glucosaminide, or beta-1,3-galactosyl arabinofuranoside, suggesting that it specifically cleaves the internal beta-1,3-linkage of two galactosyl residues. High performance liquid chromatographic analysis of the hydrolysis products showed that the enzyme produced galactose from beta-1,3-galactan in an exo-acting manner. However, no activity toward p-nitrophenyl beta-galactopyranoside was detected. When incubated with arabinogalactan proteins, the enzyme produced oligosaccharides together with galactose, suggesting that it is able to bypass beta-1,6-linked galactosyl side chains. The C-terminal CBM6 did not show any affinity for known substrates of CBM6 such as xylan, cellulose, and beta-1,3-glucan, although it bound beta-1,3-galactan when analyzed by affinity electrophoresis. Frontal affinity chromatography for the CBM6 moiety using several kinds of terminal galactose-containing oligosaccharides as the analytes clearly indicated that the CBM6 specifically interacted with oligosaccharides containing a beta-1,3-galactobiose moiety. When the degree of polymerization of galactose oligomers was increased, the binding affinity of the CBM6 showed no marked change.     

Preparation of gemini-type amphiphiles bearing cyclitol head groups and their application as high-performance modifiers for lipases

Five gemini-type amphiphiles bearing cyclitol head groups, which have abundance of axial hydroxy groups, are newly synthesized. The syntheses are based on a common mixed anhydride method utilizing N,N'-[iminobis(trimethylene)]bisquinamide, prepared from iminobispropylamine and quino-1,5-lactone, and dialkyl N-(3-carboxypropanoyl)-L-glutamates as polar and hydrophobic components, respectively. Candida rugosa lipase (CRL) and Pseudomonas cepacia lipase (PCL) are co-lyophilized with these synthesized gemini-type amphiphiles, and their transesterification activities in organic solvents are evaluated. The modified PCL and CRL prepared by using each amphiphile showed highly enhanced and moderately enhanced enzyme activity, respectively. These results are discussed in terms of the increased preferential exclusion of the hydrophilic heads of the amphiphile and of the topological view of the amphiphile.     

Identification of cut8+ and cek1+, a novel protein kinase gene, which complement a fission yeast mutation that blocks anaphase.

The fission yeast Schizosaccharomyces pombe [corrected] temperature sensitivity cut8-563 mutation causes chromosome overcondensation and short spindle formation in the absence of sister chromatid separation. The cut8-563 mutation allows cytokinesis before the completion of anaphase, thus producing cells with a cut phenotype. The cut8+ gene product may be required for normal progression of anaphase. Diploidization occurs at the restrictive temperature, and 60 to 70% of the cells surviving after two generations are diploid. These phenotypes are reminiscent of those of budding yeast (Saccharomyces cerevisiae) ctf13 and ctf14 (ndc10) mutations. The cut8+ gene, isolated by complementation of the mutant, predicts a 262-amino-acid protein; the amino and carboxy domains are hydrophilic, while the central domain contains several hydrophobic stretches. It has a weak overall similarity to the budding yeast DBF8 gene product. DBF8 is an essential gene whose mutations result in delay in mitotic progression and chromosome instability. Anti-cut8 antibodies detect a 33-kDa polypeptide. Two multicopy suppressor genes for cut8-563 are identified. They are the cut1+ gene essential for nuclear division, and a new gene (designated cek1+) which encodes a novel protein kinase. The cek1+ gene product is unusually large (1,309 amino acids) and has a 112-amino-acid additional sequence in the kinase domain. The cek1+ gene is not an essential gene. Protein phosphorylation by cek1 may facilitate the progression of anaphase through direct or indirect interaction with the cut8 protein.