Isolation of brush-border membranes from rat and rabbit colonocytes: Is alkaline phosphatase a marker enzyme?

Summary A method for the isolation of brush-border membranes of large intestinal epithelial cells was developed, which is based on the purification of intact brush-border caps by Percoll^® density-gradient centrifugation followed by separation of the vesiculated brush-border membranes on sucrose gradients. The procedure has two major advantages in comparison to known methods: 1) its first step does not depend on the determination of marker enzymes and 2) the method is applicable to rats as well as rabbits without major modifications. Due to the lack of an accepted marker for the colonic brush-border membrane the validity of the isolation procedure was tested by its application to the small intestine. Rat small intestinal brush-border membranes were enriched 21-fold when compared to the homogenate. The method was used to evaluate alkaline phosphatase as a marker enzyme for the colonic brush-border membrane. The results suggest that alkaline phosphatase is not exclusively localized in the brush-border membrane since this enzyme was also associated with membranes having different physical properties.     

Toxigenic Potential of Some Fusarium Isolates from Southeast Asia

The isolation and characterization of 10 isolates of six Fusarium spp. from plant and soil samples collected in Southeast Asia is reported. The ability of these isolates to produce trichothecenes both in liquid cultures (CZ, GYEP, and MYRO) and on rice was assessed, and their toxigenic potential was examined by skin assay and gavage studies with culture filtrates. Although culture filtrates of all the isolates caused minor damage to test animals, only that of F. equiseti DAOM 189762 produced trichothecenes.     

A spin label ESR and saturation transfer-ESR study of archaebacteria bipolar lipids

A spin label study has been carried out on bipolar lipids extracted from Sulfolobus solfataricus, an extreme thermophilic archaebacterium growing at about 85°C and pH 3. These lipids are cyclic diisopranyl tetraether molecules, quite different from the usual fatty acid lipids. Two hydrolytic fractions of the membrane complex lipids have been studied: the symmetric lipid glycerol-dialkyl-glycerol-tetraether (GDGT) and the asymmetric lipid glyceroldialkyl-nonitol-tetraether (GDNT). The ESR spectra confirm the results previously obtained from calorimetric and X-ray diffraction experiments showing a polymorphic behaviour of these lipids and indicating the critical temperature ranges at which structural transitions occur. Moreover, the present study adds information on the dynamics of the different portions of the hydrophobic chain. ST-ESR measurements show correlation times ranging from 10^-8 s up to 10^-5 s, depending upon the lipid sample, the label position and the degree of hydration. At very high temperatures, i.e. the physiological temperatures of Sulfolobus solfataricus, the nonitol head groups of the asymmetric lipids form a strongly immobilized structure. Indeed, the molecular correlation times of the outermost hydrophobic portion of GDNT are higher, by a factor up to 10³, than those of usual monopolar lipids. Anisotropic motional behaviour is observed even at such very high temperatures. Possible biological implications are discussed.Abbreviations used are ESR electron spin resonance - St-ESR saturation transfer electron spin resonance - GDGT glyceroldialkyl-glycerol-tetracther - GDNT glycerol-dialkyl-nonitoltetraether - 5 SASL 12SASL and 16SASL, stearic acid spin labels, N-oxyl-4,4-dimethyloxazolidine derivatives of 5-ketostearic acid, 12-ketostearic acid and 16-ketostearic acid, respectively - DSC differential scanning calorimetry     

Polyamine-sensitive protein kinase from chick intestinal mucosa

Summary A cyclic nucleotide-independent protein kinase which phoshorylates preferentially acidic proteins such as casein or phosvitin was isolated from cytosol of chick duodenal mucosa. The enzyme was purified more than 633 fold to apparent homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite and by sucrose density gradient centrifugation. The native enzyme has a molecular weight of 131000 as measured by gel filtration. The enzyme is a complex protein containing three polypeptides of molecular weight of 39 000, 36 000 and 27 000. It behaves as a complex throughout its purification and gel filtration but its components are readily separated by electrophoresis in denaturing buffer. The 27 000 molecular weight band was selectively autophosphorylated when the enzyme was incubated in the presence of [-³²P]ATP.When casein was used as substrate, physiological concentrations of naturally occurring polyamines such as spermine and spermidine markedly stimulated enzyme activity. However with phosvitin as substrate polyamines were strong inhibitors of the enzyme activity. This contrasting effect on intestinal kinase activity was also apparent using cytoplasmic proteins as endogenous phosphate acceptors. A characterization of this differential effect is presented and some possible physiological implications are discussed.     

Synthesis and phosphorylation of cytoskeleton components in foetal,regenerating and adult normal rat hepatocytes during culture

Summary Detergent insoluble material (DIM) was prepared by gentle treatment with detergent from foetal, regenerating and adult normal rat hepatocytes cultured for various times. It retained to some degree the morphology of the cells. After incubation of intact cells with ³⁵S-methionine, most of the labelled DIM proteins were found to be components of the cytoskeleton. They included several cytokeratins, vimentin and actin. The synthesis rate varied with the age of animals and culture conditions. The high synthetic rate of vimentin in foetal and regenerating hepatocytes could be associated with cell proliferation. No correlation was found between cytokeratin synthesis and hepatocyte growth. Most of the cytoskeleton proteins could be phosphorylated in intact cells and in DIM from cultured hepatocytes. However the degree of phosphorylation of these proteins was not related to their synthetic rate. The decreased phosphorylation level in cultured adult rat hepatocytes could be related to the rapid loss of specific functions.     

Modulation of cyclic nucleotide-independent protein kinase from chick intestine by naturally occurring polyamines and mucopolysaccharides

Summary Chick duodenal mucosa contains an endogenous factor which is capable to inhibit selectively a homologous polyamine-sensitive protein kinase. The inhibitor was partially purified and characterized, and it was found to contain typical mucopolysaccharidic components.Glycosidases digestion studies, selective degradation analysis and spectrophotometric titrations with metachromatic dyes indicated that the inhibitor preparation contained two major moieties identified as heparin-like and heparan sulfate-like structures. In chick intestine the inhibitor was specific for polyamine-sensitive protein kinase since selectively interacted with it and was inert towards other cAMP-independent and cAMP-dependent protein kinases. The inhibitory effect of the endogenous factor was counteracted by naturally occurring polyamines such as spermine. The order of potency of various polyamines was: spermine > thermine spermidine diamines. The release of inhibition by addition of physiological concentrations of spermine was also apparent when using cytosolic proteins as endogenous phosphate acceptors. These results suggest that a possible role of polyamine in the regulation of polyamine-sensitive protein kinase in the intestine is to protect the enzyme from the inhibitory action of endogenous heparinoids.     

Dilution and acidification effects during the spring flood of four Swedish mountain brooks

The seasonal variation in water chemistry was followed during 1980 and 1981 in four mountain brooks in the southern part of Swedish Lapland. In the area investigated the soil is calcareous and the brook water is very well buffered during the major part of the year, with alkalinity varying between 0.4 and 1.0 milliequivalents per liter and with pH values about 7.5. These years the snow had a pH of approximately 5.2, which was considerably higher than has been reported from adjacent areas in the lower, coniferous region. During snowmelt the water discharge increased drastically, and although the net transport of bicarbonate increased, alkalinity showed low values due to dilution with meltwater. pH decreased, but not further than to 6.3–6.5, far from values reported in 1979 (pH less than 5), apparently due to the comparatively clean snow. A slight deficit in alkalinity, as compared to the nonmarine calcium and magnesium content, points to an acidification impact on the area. During maximum runoff some chemical variables, like aluminium, iron, nitrogen and phosphorus, behaved reversely to what might be expected during dilution and reached maxima in concentrations. It is concluded that the extreme runoff characteristics of high mountain areas make brook water more sensitive to acid precipitation than might be expected when regarding only the calcareous properties of soil and bedrock.     

Structural Genes for a Newly Recognized Acetolactate Synthase in Escherichia coli K-12

Evidence is reported that shows the presence in Escherichia coli K-12 of a newly found acetolactate synthase. This enzyme is the product of two genes, ilvH and ilvI, both located very close to leu. Amber mutations have been found in both genes and therefore their products are polypeptides. Mutations in the ilvH gene cause the appearance of an acetolactate synthase activity which is relatively resistant to valine inhibition and can be separated by adsorption on hydroxylapatite from another activity present in the extract and more sensitive to valine inhibition than the former. A mutant altered in the ilvI gene was isolated among the revertants sensitive to valine inhibition of an ilvH mutant. Such a mutant lacks the resistant acetolactate synthase. A temperature-sensitive revertant of the ilvI mutant contained a temperature-sensitive acetolactate synthase. Thus ilvI is the structural gene for a specific acetolactate synthase. The activity of the ilvH gene product has been measured by adding an extract containing it to a purified ilvI acetolactate synthase, which, upon incubation, became more sensitive to valine inhibition. Conversely, a valine-sensitive acetolactate synthase (the product of the ilvH and the ilvI genes) became more resistant to valine inhibition upon incubation with an extract of a strain containing a missense ilvH gene product.     

The effect of post-heparin lipolytic activity on plasma lipoproteins

Summary Electron microscopy of negatively stained plasma shows that intravenous administration of heparin to healthy humans results in a fast degradation of very low density lipoproteins (VLDL), which are very rich in triglycerides (50% w/w). VLDL are also rapidly digested by post-heparin lipolytic activity when serum from non-heparinized subjects is incubated at 27°C in vitro with post-heparin serum of healthy controls.Abbreviations VLDL very low density lipoproteins - LDL low density lipoproteins - HDL high density lipoproteins - LP-X lipoprotein-X, abnormal lipoprotein characterizing obstructive jaundice     

Kinetics of denaturation of DNA

The kinetics of denaturation of DNA have been studied by relaxation techniques. Examination of the terminal relaxation times for a variety of DNA's under a variety of conditions has shown that DNA denaturation is principally a hydrodynamically limited process. Measurements within the helix–coil transition have demonstrated that the experimentally measured terminal relaxation times are a function of the following: (1) position in the helix–coil transition; (2) ionic strength of the solvent; (3) solvent viscosity; (4) DNA concentration; (5) molecular weight; (6) number and position of single-strand breaks. The dependence of the terminal relaxation time on the above mentioned factors can be attributed to hydrodynamic effects. Thus a hydrodynamic model for DNA unwinding is required. The model which best fits the data involves the assumption of a rotational frictional coefficient independent of molecular weight. This assumption is suggested by the fact that the relaxation time is proportional to the first power of the molecular weight.