Ecdysteroid regulation of the onset of cuticular melanization in allatectomized and Black mutant Manduca sexta larvae

The absence of juvenile hormone at the time of head cap slippage during the last-larval moult of the tabacco hornworm, Manduca sexta, causes deposition of premelanin granules into the outer regions of the newly forming endocuticle beginning 13 h later. These granules were found to contain an inactive phenoloxidase which becomes activated about 9 h later, 4 h before body melanization begins. The onset of melanization was not accelerated by melanization and reddish colouration hormone from Bombyx heads, extracts of pharate-adult corpora cardiaca or pharate-larval ventral nerve cords (sources of eclosion hormone), or extracts of pharate-larval suboesophageal ganglia or corpora cardiaca-corpora allata complexes. Instead the fall of the ecdysteroid titre to below 250 ng/ml 20-hydroxyecdysone equivalents appeared to be the cue that allowed melanization about 4.5 h later. Up to, but not after, this time both melanization and ecdysis could be delayed by exogenous 20-hydroxyecdysone in a dose-dependent fashion above 0.1 μg per larva. In vitro studies published elsewhere indicate that 20-hydroxyecdysone prevents the activation of the premelanin granules. Thus the granules can be deposited at the proper time in the newly forming endocuticle but their melanization is regulated by the declining ecdysteroid titre and it thus synchronized with other events occurring just before ecdysis.     

Thehalose mycolates from Nocardia asteroides,Nocardia farcinica,Gordona lentifragmenta and Gordona bronchialis

Isolation of glycolipids from Nocardia asteroides, N. farcinica, Gordona lentifragmenta and G. bronchialis, by column chromatography of lipid extracts on a 50% (w/w) mixture of silicic acid and silica gel H, is described. The isolated materials were partially characterized by infrared spectroscopy, optical rotation and refractive index measurements, and by identifying the products of alkaline hydrolysis. Analytical studies showed that the glycolipids released only trehalose in the aqueous phase while mycolic acids were the constituent fatty acids identified.The isolated lipids are trehalose esters in which the trehalose molecule is esterified with mycolic acids.     

Transport of [14C]Gly-Pro in a proline peptidase mutant of Salmonella typhimurium

The transport of [¹⁴C]Gly-Pro was examined using a mutant of Salmonella typhimurium (strain TN87) deficient in an X-Pro dipeptidase and an X-Pro-Y iminopeptidase. The dipeptide was taken up by one saturable transport system having a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of $\text{5.3 · 10}{}^{\mathrm{?7}}\text{M}$ and a $\text{V}$ of 1.4 nmol/mg dry wt cell per min. The uptake of Gly-Pro was not inhibited by amino acids or tripeptides and the transport system exhibited a rather broad side chain specificity for dipeptides. Dipeptides containing hydrophobic residues were the most potent inhibitors of this dipeptide transport system exhibiting $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ values between 10^?8 and 10^?7 M. In contrast, dipeptides containing glycine residues were particularly weak inhibitors. Finally, Gly-Pro was found to be in the intact form inside the cell and was concentrated more than 1000-fold.     

Semiquantitative paper chromatographic separation of chlorophylls a and b

[¹⁴C]Chlorophyll (chl) a has been utilized to demonstrate the contamination of chl b by (probably) oxidation products of chl a in thin-layer or paper chromatography. By circular chromatography of both chlorophylls as their pheophytins, the contamination of chl a (as pheophytin a) in chl b (as pheophytin b) may be reduced to 0.15–0.35.     

Characterisation of two ornithine-containing lipids from Erwina aroideae

An apparently pure ornithine-containing lipid (OCL) was isolated from Erwina aroideae by solvent extraction and thin-layer chromatography (TLC). However, selective hydrolysis of the lipid under acidic and basic conditions and analysis of hydrolysates by gas chromatography-mass spectrometry (GCMS) showed that two structurally similar OCL were in fact present. These lipids both contained a 3-hydroxyhexadecanoic acid moiety which was linked to ornithine by an amide group formed between the 2-amino group of ornithine and the carboxyl group of the acid. The two lipids, however, differ in the nature of the fatty acid bound through an ester linkage to the hydroxyl group of the 3-hydroxyhexadecanoic acid moiety. One lipid is the ester of hexadecanoic acid whereas the other lipid is the ester of octadecenoic acid. These lipids are present in approximately equal amounts.     

In Vivo and in Vitro binding of platinum to metallothionein

The in vivo binding of platinum to metallothionein (MT) has been observed in rat tissues following injections of the cis and trans isomers of DDP (dichlorodiammine-platinum(II)). Platinum in either cis-DDP or trans-DDP does not directly induce MT; platinum-MT is produced by the replacement of previously bound zinc in the protein. The binding of Pt(II) to MT depends on the availability of SH groups in MT. Preinjection with CdCl₂ significantly enhances the association of Pt(II) with MT fractions compared to the degree of association resulting from injections with either cis-DDP or trans-DDP without CdCl₂ pretreatment. In vitro experiments in which tissue extracts including a known (Cd,Zn)-MT were incubated with either cis-DDP or trans-DDP show that these isomers differ with respect to the transfer of Pt to MT; the equilibrium in both cases was reached when approximately 40% of the available Pt is bound to MT but with this equilibrium value attained in 2 h in the case of trans-DDP and only after 72 h in the case of cis-DDP. Pt-MTs were also formed by a series of incubation steps in which a native MT was used to prepare the apoprotein which was subsequently incubated with either cis-DDP or trans-DDP. Spectrophotometry established that a shoulder occurs at 285 nm for the Pt-MTs resulting from the incubation with either isomer. A competitive double-antibody radioimmunoassay for MT demonstrated that these Pt-MTs had complete cross-reactivity with a native (Cd,Zn)-MT. Gel filtration of tissue extracts after either in vivo or in vitro treatment with DDP showed that Pt was bound to a molecular species with properties characteristic of MT. These results were verified by atomic absorption spectrophotometry and polyacrylamide gel electrophoresis assays.     

Molecular characterization of choline acetyltransferase from Drosophila melanogaster

Purified acetyl-CoA: choline O-acetyltransferase (EC 2.3.1.6) from Drosophila melanogaster has been shown to contain two major polypeptides of 67 and 54 K Daltons. However, all enzyme activity is found in a single molecular weight form of approx 67 K Daltons as determined by sucrose gradient sedimentation and molecular exclusion chromatography. The latter showed both the 67 and 54 K Dalton polypeptides on polyacrylamide gel electrophoresis in sodium lauryl sulfate (10% acrylamide). Analysis of purified choline acetyltransferase on polyacrylamide gel electrophoresis in sodium lauryl sulfate (15% acrylamide) revealed the presence of an additional polypeptide at 13 K Daltons. Tryptic-peptide maps of the 67, 54 and 13 K Dalton components showed all three to be structurally related. In addition to several common tryptic peptides, the 13 K Dalton polypeptide contained three tryptic-peptides that were also found in the 67 K Dalton polypeptide, but were absent from the 54 K Dalton polypeptide. This evidence suggests that native Drosophila choline acetyltransferase may exist in two forms, one a single polypeptide chain with a molecular weight of 67 K Daltons and the other consisting of two noncovalently bound polypeptide chains with molecular weights of 54 and 13 K Daltons. The latter form is the major one isolated and may be generated by limited proteolysis of the single chain 67 K Dalton form.     

Purification and characterization of glucose-6-phosphate dehydrogenase from Aspergillus parasiticus

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was purified from mycelium of Aspergillus parasiticus (1-11-105 Whl). The enzyme had a molecular weight of 1.8 × 10⁵ and was composed of four subunits of apparently equal size. The substrate specificity was very strict, only glucose 6-phosphate and glucose being oxidized by NADP or thio-NADP. Zinc ion was a powerful inhibitor of the enzyme, inhibition being competitive with respect to glucose 6-phosphate, with K_i about 2.5 μm. Other divalent metal ions which also serve as inhibitors are nickel, cadmium, and cobalt. It is proposed that the stimulation of polyketide synthesis by zinc ion may be mediated in part by inhibition of glucose-6-phosphate dehydrogenase.     

Purification of nuclei from a flagellate protozoan, Trypanosoma brucei

A simple and rapid technique is described for the isolation of nuclei from the flagellate protozoan Trypanosoma brucei. Cells were disrupted by nitrogen cavitation in the presence of hexylene glycol which enhances nuclear stability. Isolated nuclei were separated from nuclei trapped with cytoskeletal fragments and flagellae by Percoll density gradient centrifugation. Centrifugation in a medium with increased sucrose concentration greatly improved the separation of free nuclei from those trapped in cell debris. The isolation procedure resulted in the recovery of 34% of the nuclei present in the whole cell suspension. Electron microscopic and chemical analysis indicated that the recovered nuclei were in good condition and were highly purified.     

The carotenoid band shift in reaction centers from Rhodopseudomonas sphaeroides

A specific carotenoid associated with reaction centers purified from Rhodopseudomonas sphaeroides shows an optical absorbance change in response to photochemical activity, at temperatures down to 35 K. The change corresponds to a bathochromic shift of 1 nm of each absorption band. The same change is induced by either chemical oxidation or photo-oxidation of reaction center bacteriochlorophyll (P-870). Reduction of the electron acceptor of the reaction center, either chemically or photochemically, does not cause a carotenoid absorbance change or modify a change already induced by oxidation of P-870. The change of the carotenoid spectrum can therefore be correlated with the appearance of positive charge in the reaction center. In these studies we observed that at 35 K the absorption band of reaction center bacteriochlorophyll near 600 nm exhibits a shoulder at 605 nm. The resolution into two components is more pronounced in the light-dark difference spectrum. This observation is consistent with our earlier finding, that the “special pair” of bacteriochlorophyll molecules that acts as photochemical electron donor has a dimer-like absorption spectrum in the near infrared.     

Chemical and catalytic properties of the peroxisomal acyl-coenzyme A oxidase from Candida tropicalis

The peroxisomal acyl-CoA oxidase has been purified from extracts of the yeast Candida tropicalis grown with alkanes as the principal energy source. The enzyme has a molecular weight of 552,000 and a subunit molecular weight of 72,100. Using an experimentally determined molar extinction coefficient for the enzyme-bound flavin, a minimum molecular weight of 146,700 was determined. Based on these data, the oxidase contains eight perhaps identical subunits and four equivalents of FAD. No other β-oxidation enzyme activities are detected in purified preparations of the oxidase. The oxidase flavin does not react with sulfite to form an N(5) flavin-sulfite complex. Photochemical reduction of the oxidase flavin yields a red semiquinone; however, the yield of semiquinone is strongly pH dependent. The yield of semiquinone is significantly reduced below pH 7.5. The flavin semiquinone can be further reduced to the hydroquinone. The behavior of the oxidase flavin during photoreduction and its reactivity toward sulfite are interpreted to reflect the interaction in the N(1)-C(2)O region of the flavin with a group on the protein which acts as a hydrogen-bond acceptor. Like the acyl-CoA dehydrogenases which catalyze the same transformation of acyl-CoA substrates, the oxidase is inactivated by the acetylenic substrate analog, 3-octynoyl-CoA, which acts as an active site-directed inhibitor.     

Purification and physicochemical properties of an extra-cellular cycloamylose (cyclodextrin) glucanotransferase from Bacillus macerans

An extracellular cycloamylose (cyclodextrin) glucanotransferase (EC 2.4.1.19) from Bacillus macerans was purified to homogeneity by adsorption on starch, ammonium sulfate fractionation, column chromatography on DEAE-cellulose, and gel filtration on Sephadex G-100. The enzyme had a molecular weight of 67,000 and consisted of one polypeptide chain. The isoelectric point was pH 5.4. Temperature and pH optima were 60° and 5.45.8, respectively. The purified enzyme was quite stable at 50° (pH 6.0), but lost ≈80% of its activity at 60° for 30 min (pH 6.0). Prolonged digestion by trypsin did not affect the catalytic properties of the enzyme. The K_m for starch was 5.7 mg/ml.     

Purification and chemical characterization of polysaccharides obtained from Lampteromyces japonicus by concanavalin A-sepharose affinity chromatography

Two classes of neutral polysaccharide which could not be separated from each other by conventional methods were isolated from the fungus, Lampteromyces japonicus, by affinity chromatography using concanavalin A-Sepharose. The polysaccharide retained on the concanavalin A-Sepharose column was eluted with 0.05 M methyl α-d-mannopyranoside and appeared to be α-mannan, while that which passed through the column was virtually all β-glucan.Both polysaccharides were subjected to Smith-type degradation, methylation, acetolysis and glucosidase treatment. The results indicated that the α-mannan contained predominantly α-(1 → 2)-linked side chains branching from an α-(1 → 6)-linked backbone at the (1 → 2,6)-linked mannopyranosyl residues. Galactose was attached to approximately one-quarter of the non-reducing mannose terminals. The β-glucan seemed to contain mainly (1 → 6)-linked side chains branching from a (1 → 3)-linked backbone at the (1 → 3,6)-linked glucopyranosyl residues.     

Chemical investigations on the gum exudate from sajna (Moringa oleifera)

The purified, whole-gum exudate from the drum-stick plant (Moringa oleifera) was found to contain l-arabinose, d-galactose, d-glucuronic acid, l-rhamnose, d-mannose, and d-xylose in the molar ratios of ～ 14.5:11.3:3:2:1:1. A homogeneous, degraded-gum polysaccharide consisting of d-galactose, d-glucuronic acid, and d-mannose in the molar ratios of ～ 11.7:3.9:1, was obtained on mild hydrolysis of the whole gum with acid. Permethylation studies were conducted on the whole gum, the degraded gum, and their carboxyl-reduced products, and the results were in good agreement with those obtained from periodate oxidation followed by Smith degradation. Also, isolation and characterization of the oligosaccharides obtained from the mother liquor during preparation of the degraded gum, and by graded hydrolysis of the degraded gum, were achieved. On the basis of the results obtained from these studies, a tentative structure was assigned to the average repeating-unit of the gum.     

A complex polysaccharide from the seeds of anthocephalus indicus a. rich

The seeds of Anthocephalus indicus contain a water-soluble polysaccharide composed of D-xylose, D-mannose, and D-glucose in the molar ratios 1:3:5. Methylation analysis afforded 2,3,4-tri-O-methyl-D-xylose, 2,3,6,-tri-O-methyl-D-mannose, 2,3,6-tri-O-methyl-D-glucose, 2,3-di-O-methyl-D-glucose, and 2,3,4,6-tetra-O-methyl-D-glucose in the molar ratios 7:21:12:15:8. Periodate oxidation and methylation data indicated 22.5% and 21.9% of end groups, respectively. The above findings, together with the results of partial hydrolysis with acid, indicate the polysaccharide to consist of a linear chain of (1→4)-linked β-D-mannosyl and β-D-glucosyl residues to which α-D-xylosyl and β-D-glucosyl groups are attached by (1→6)-linkages.     

Structural investigations on the lipopolysaccharide isolated from Vibrio cholera,Inaba 569 B

On hydrolysis, the purified lipopolysaccharide (LPS) isolated from Vibrio cholera, Inaba 569 B, yielded glucose, mannose, a heptose behaving like d-glycero-l-manno-heptose and one behaving like d-glycero-l-gluco-heptose, 2-amino-2-deoxy-glucose, and glucuronic acid in the molar ratios of ～9:4:5:1:2:5. Studies on the LPS, the polysaccharide (PS), and carboxyl-reduced LPS showed that the PS has a branched structure, with (1→2)-linked mannopyranosyl and a heptopyranosyl, and (1→4)-linked glucopyranosyluronic and 2-amino-2-deoxyglucopyranosyl residues in the interior part of the molecule, and glucopyranosyl and heptopyranosyl residues as nonreducing end-groups.