Sequential elution of proteins in small volumes by preparative polyacrylamide gel electrophoresis

A simple flexible method for separation of proteins by polyacrylamide gel electrophoresis and sequential elution into dialysis bags has been devised. The system was applied to isolation of three glycoproteins from the peritoneal fluid of mice bearing Ehrlich ascites tumor.     

Identification of mitochondrial proteins on two-dimensional electrophoresis gels of extracts of adult Drosophila melanogaster

Several hundred proteins have been resolved on two-dimensional gels of extracts of [³⁵S]methionine-labeled adult Drosophila melanogaster. 27 of these polypeptides disappear from the gel pattern after feeding the K⁺ ionophore nonactin. These proteins have been identified as mitochondrial, since the two-dimensional gel pattern of extracts of isolated mitochondria correlates well with the pattern of the proteins missing from that of nonactin-treated flies. Nine new proteins also appear on the two-dimensional gels of the extracts from the nonactin-treated flies. Apparently, these nine proteins are precursors of the mature mitochondrial forms. These particular data support the concept that processing of many of the cytoplasmically synthesized mitochondrial proteins requires a specific membrane potential, and that some of these proteins are modified intramitochondrially. However, using [³⁵S]methionine incorporation techniques, not all labeled polypeptides disappear from mitochondria during such treatment. Feeding similarly radiolabeled flies with chloramphenicol, an inhibitor of mitochondrial protein synthesis, results in the disappearance of only one protein from the gel pattern with the concurrent appearance of a ‘new’ high-molecular-weight polypeptide. Collectively, these data show that a specific group of [³⁵S]methionine-labeled mitochondrial proteins can be identified by selective inhibition of mitochondrial function in whole cell protein maps of adult D. melanogaster.     

A near-infrared method for studying hydration changes in aqueous solution: Illustration with protease reactions and protein denaturation

Proteins undergoing protease reactions, heat denaturation, or interactions with sodium dodecyl sulfate (SDS) were used to demonstrate the effectiveness of a near-infrared method for the quantitative study of changes in hydration or water binding during such processes. The spectra of different proteins showed that the liberation of $\text{COO}{}^{\mathrm{?}}\text{and NH}{}_3{}^{\mathrm{+}}$ groups during a protease reaction is associated with a large increase in hydration and excluded volume. On the basis of experiments with model compounds, other spectral changes, including development of continuum absorbance between 1.55 and 1.85 μm and a band with a peak near 2.1 μm, were also attributed to the liberation of these groups. After heat denaturation or in the presence of SDS, the rate of proteolytic hydrolysis was markedly increased, consistent with the view that some preliminary denaturation is necessary for protease activity. The validity of the hydration changes calculated for protease reactions was supported by model studies with l-lysine, and with poly-l-lysine before and after hydrolysis. The near-infrared spectrum of the protein substrate with no added protease was largely unaffected by heat treatment alone, indicating that the hydration as such was not changed to a large extent by the structural modifications of denaturation. In contrast to the protease reaction, the interactions between SDS and the proteins resulted in a decrease in hydration. Results of this paper are compared with those obtained from other methods. Some unique advantages of the near-infrared method for the study of hydration changes during reactions in aqueous solution are described.     

Patterns of product inhibition for bacterial nitrite reductase

PO +Dehydrophenylalanine (delta Phe) having the E-configuration (delta EPhe ; phenyl and C = O cis) was incorporated into [Leu5]-enkephalin in order to restrict its conformation. Compared with the Z-isomer, in the radio-ligand receptor binding assays, [D-Ala2, delta EPhe4 , Leu5] enkephalin showed drastically decreased potency for the delta and mu opiate receptors, i.e., 260- and 150-fold loss of affinity, respectively. The results strongly indicate that the opiate receptors require the Z-configuration (phenyl and C = O, trans) of the delta Phe4 residue and may require a specific interrelationship between the aromatic rings of the Tyr1 and Phe4 residues in the molecule for binding. The conformation of [Leu5]-enkephalin specific for the delta receptors was analyzed and a comparison made with its crystal structure recently elucidated.     

Proliferation and differentiation of chick skeletal muscle cells cultured in a chemically defined medium

By using the technique of nuclear transplantation in Paramecium [1], amicronucleate and renucleate clones were prepared in P. caudatum. The major differences between amicronucleate and micronucleate cells in the vegetative stage are elongation of cell cycle time, decrease in food vacuole formation, and shortening of the buccal cavity in the amicronucleate cells. These characteristics of amicronucleate cells are closely related with the absence of micronucleus, because all of these abnormalities were cured when the micronucleus was transplanted again into the amicronucleate. It is evident that the germinal micronucleus plays an important role not only during the sexual cycle but also in vegetative growth. Elongation of the cell cycle time in amicronucleates was also observed in P. bursaria and P. jenningsi.     

The carbon-13 nuclear magnetic resonance spectra of four eudesmane sesquiterpenols

The ¹³C NMR spectra of geosmin, selina-4(14),7(11)-diene-99-ol and two dihydroeudesmol isomers have been obtained and the individual resonances assigned. Several different empirical correlations developed by others have been combined in simple calculations to predict chemical shift values for sesquiterpenols of the eudesmane group.     

Chemical composition and ultrastructure of the epicuticular wax in four mutants of Pisum sativum (L)

The action of mutations affecting the epicuticular wax of Pisum sativum has been investigated at the chemical and ultrastructural level. Upper and lower surfaces of the leaves were found to differ markedly in both ultrastructure and chemistry. Mutations affected primarily either the lower (wa, wb and wsp) or the upper surface (wlo), but some effects of all 4 genes could be seen on both surfaces. Specific biochemical lesions could be implied for wsp and wa but the chemical effects of wb and wlo were more diffuse. Generally a close relation between chemical composition and crystallite form of the wax was evident throughout the work.     

5-Hydroxytryptamine-Stimulated Inositol Phospholipid Hydrolysis in the Mouse Cortex Has Pharmacological Characteristics Compatible with Mediation via 5-HT2 Receptors but This Response Does Not Reflect Altered 5-HT2 Function After 5,7-Dihydroxytryptamine Lesioning or Repeated Antidepressant Treatments

5-Hydroxytryptamine (5-HT; 3 x 10(-8)-1 x 10(-5)M) produced a dose-dependent increase in phosphatidylinositol/polyphosphoinositide (PI) turnover in mouse cortical slices, as measured by following production of 3H-labelled inositol phosphates (IPs) in the presence of 10 mM LiCl. Analysis of individual IPs, in slices stimulated for 45 min, indicated substantial increases in inositol monophosphate (IP1; 140%) and inositol bisphosphate (IP2; 95%) contents with smaller increases in inositol trisphosphate (IP3; 51%) and inositol tetrakisphosphate (IP4; 48%) contents. The increase in IP3 level was solely in the 1,3,4-isomer. This response was inhibited by the nonselective 5-HT antagonists methysergide, metergoline, and spiperone. It was also inhibited by the selective 5-HT2 antagonists ketanserin and ritanserin but not by the 5-HT1 antagonists isapirone, (-)-propranolol, or pindolol. 5-HT-stimulated IP formation was also unaltered by atropine, prazosin, and mepyramine. Lesioning brain 5-HT neurones using 5,7-dihydroxytryptamine (5,7-DHT; 50 micrograms i.c.v.) produced a 210% (p less than 0.01) increase in the number of 5-HT2-mediated head-twitches induced by 5-methoxy-N,N-dimethyltryptamine (2 mg/kg). However, 5,7-DHT lesioning had no effect on 5-HT-stimulated PI turnover in these mice. Similarly, an electroconvulsive shock (90 V, 1 s) given five times over a 10-day period caused an 85% (p less than 0.01) increase in head-twitch responses but no change in 5-HT-stimulated PI turnover. Decreasing 5-HT2 function by twice-a-day injection of 5 mg/kg of zimeldine or desipramine (DMI) produced 50% (p less than 0.01) and 56% (p less than 0.01), respectively, reductions in head-twitch behaviour.(ABSTRACT TRUNCATED AT 250 WORDS)     

Betulachrysoquinone hemiketal: A p-benzoquinone hemiketal macrocyclic compound produced by Phanerochaete chrysosporium

Betulachrysoquinone hemiketal was isolated from pre-extracted wood of Betula lutea Michx. inoculated with Phanerochaete chrysosporium Burds. Acid-catalysed hydrolysis of betulachrysoquinone hemiketal produced betulachrysoquinone which was shown to be 2-hydroxy-6-(13′-hydroxytetradecanyl)-p-benzoquinone.