Divalent cation changes in cerebellar Purkinje cells after climbing fiber deafferentation.

A secondary ion mass spectrometry (SIMS) microscope was used to detect intracellular stores of calcium, magnesium, sodium and potassium. Measurements were made in semithin sections of fixed tissues of normal and climbing fiber deafferented cerebellar cortex. Quantitative data were collected from 150 microns diameter image fields in the molecular and granule layers. The results indicate smaller quantities of both calcium and magnesium in the deafferented cerebellar cortex compared to the normals, the molecular as well as the granule layer being affected. The results are discussed in terms of the usefulness and limitations of the SIMS microscope for histological preparations.     

Effects of denervation on the contents of cholesterol and membrane systems involved in muscle contraction in rabbit fast-twitch sarcotubular system.

Denervated fast-twitch rabbit muscles were progressively losing their fresh weight and the yield of sarcotubular protein was increasing. The activity of Ca(2+)-ATPase was affected but very slightly, the basal Mg(2+)-ATPase and the Mg(2+)-ATPase/Ca(2+)-ATPase ratio however increased together with a simultaneous depression of the membrane-bound acetylcholinesterase activity. We did not observe any differences in density properties of sarcotubular fractions between control and denervated muscle. However, a relative enrichment in SM and H fraction could be seen after denervation with small changes in the content of the Ca(2+)-pump protein, increased levels of calsequestrin and cholesterol, mostly in the heavy and the SM fraction. After denervation the binding sites for 3H-PN-200-110 did not show any changes in receptor affinity, but the number of putative Ca(2+)-channels increased twice along with a depression of 3H-ouabain binding sites. We suggest that the denervation of fast-twitch muscle leads to the hypertrophy of the junctional sarcoplasmic reticulum and the T-system. Changes in the cholesterol content, in the number of putative Ca(2+)-channels and in Na+, K(+)-ATPase can affect the muscle contraction.     

The sulphydryl groups of ox brain and liver glutamate dehydrogenase preparations and the effects of oxidation on their inhibitor sensitivities

Glutamate dehydrogenase preparations from several sources have been shown to have suffered limited proteolysis during purification. This proteolysis has been previously shown to involve removal of the N-terminal tetrapeptide and to result in changes in the regulatory properties of the enzyme. In the present work the previously unidentified N-terminal residue of the unproteolysed enzyme from ox brain and liver is shown to be cysteine. The thiol group of this residue is masked in the native enzyme but it becomes accessible after reduction. Exposure of solutions of the unproteolysed enzyme to air oxidation causes large changes in its sensitivity to inhibition by the antipsychotic drug perphenazine, GTP and by high concentrations of NADH. No such changes occurred in the behaviour of preparations of the enzyme that had suffered proteolysis during purification under these conditions.Special issue dedicated to Dr. Santiago Grisolia.     

The relation between the variability of polygenic morphological and monogenic biochemical traits as exemplified by Karakul sheep

A quantitative method for investigation of relationship between polygenic and monogenic traits has been proposed. It is based on examination of relationship between frequencies of distribution classes of an adaptive quantitative trait and frequencies of certain genetic character in the same classes. The method permits to locate a gene marker within a space of quantitative trait values. Using adaptively significant morpho-anatomic traits, it is possible to estimate indirectly adaptive values of gene markers under consideration, since, in accordance with the concept of adaptive norm, "average" phenotypes have maximal fitness, whereas deviative phenotypes transgress the bounds of the optimum. As a genetical character, genotype of certain biochemical locus, individual heterozygosity range or interlocus combinations of alleles could be used. The method has been applied to newborn Astrakhan lambs. Principal component analysis has been used to obtain complex characterization for six constitutional characters. Some regularities in location of homo- and heterozygous genotypes of the transferrin locus within a space of morphological characters' values have been revealed.     

Glycosylation at specific sites of erythropoietin is essential for biosynthesis, secretion, and biological function

The glycoprotein hormone erythropoietin (Ep), the primary regulator of erythropoiesis, is synthesized by the kidney and secreted as the mature protein with three N-linked and one O-linked oligosaccharide chains. To investigate the role(s) of each carbohydrate moiety in the biosynthesis and function of Ep, we have used oligonucleotide-directed mutagenesis of a cDNA for human Ep to alter the amino acids at each of the carbohydrate attachment sites. Each mutated cDNA construct was expressed in stably transfected sublines of a kidney cell line, baby hamster kidney. We show, by preventing attachment of N-linked carbohydrate at asparagines 38 or 83, or preventing O-linked glycosylation at serine 126, that glycosylation of each of these specific sites is critical for proper biosynthesis and secretion of Ep. Fractionation of cellular extracts demonstrated that the mutant proteins lacking glycosylation at each of these three sites, (38, 83, and 126) were associated mainly with membrane components or were degraded rapidly. Less than 10% of these three mutant proteins were processed properly and secreted from the cells. The Ep protein lacking N-linked glycosylation at asparagine 24 is synthesized and secreted as efficiently as native Ep. The carbohydrates at positions 24 and 38 may be involved in the biological activity of Ep, since the absence of either of the oligosaccharide side chains at these positions reduced the hormone's biological activity.     

Comparison of the amino acid sequences of human protamines HP2 and HP3 and of intermediate basic nuclear proteins HPS1 and HPS2. Structural evidence that HPS1 and HPS2 are pro-protamines

Two intermediate nuclear basic proteins HPS1 and HPS2 were isolated from human sperm. They were characterized by their electrophoretic mobility in acid-urea gels, their amino acid composition, and their peptide maps after digestion by endoproteinase Lys-C and by endoproteinase Glu-C. Their amino-terminal amino acid sequences have also been determined. The structural data thus obtained suggest that HPS1 and HPS2 are precursors of human protamines HP2 and HP3.     

A Raman spectroscopic study on the interaction of an ion-channel protein with a phospholipid in a model membrane system (gramicidin A/L-alpha-lysophosphatidylcholine)

The interaction of the ion channel polypeptide gramicidin A with the L-alpha-lysophosphatidylcholine micelles in a membrane state association (approximative molar ratio 1:9) was investigated by Raman spectroscopy. Studies were carried out over the spectral ranges of 700-1700 cm-1 and 2800-3100 cm-1 at 10 degrees C. The Raman spectrum of L-alpha-lysophosphatidylcholine micelles indicated a disordered structure of the lipid acyl chains by the high intensities of the gauche conformation vibrations. Changing from the micellar phase to the membrane state of association with gramicidin A, the intensities of all-trans stretching modes increased whereas the intensities of gauche conformation vibrations decreased, reflecting the emergence of ordered lipid chains. Hydrophobic interactions between the acyl chains and the polypeptide side chain residues were demonstrated. The absence of modifications in intensities of the very strong tryptophan vibrations in the complex spectrum indicated that, if the tryptophan-stacking interactions suggested by some authors exist, they are very weak ones.     

Specific inhibition of human granulocyte elastase with peptide aldehydes

The kinetic features of human granulocyte elastase, chymotrypsin, porcine pancreatic elastase and elastomucoproteinase were compared. Amino acyl ester substrates were assayed and Km and kcat values were defined. Aldehyde analogues of the p-nitroanilide substrates designed for granulocyte elastase as optimal for Km appeared to be potent inhibitors. Suc-D-Phe-Pro-valinal (Ki = 40 microM) was found to inhibit granulocyte elastase competitively and specifically when measured with synthetic substrates, and the Ki was 3 microM with the natural protein substrate, elastin.     

Metabolic incorporation of 9-(2-anthryl)-nonanoic acid, a new fluorescent and photoactivable probe, into the membrane lipids of Chinese hamster ovary cells

9-(2-Anthryl)-nonanoic acid is a new fluorescent and photoactivable probe, which has been designed for studying the lateral diffusion rate and the lateral distribution of lipids in biological membranes by means of the anthracene photodimerization reaction. It is shown that this anthracene fatty acid is metabolically incorporated into the glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol) of the eukaryotic Chinese hamster ovary cells in culture. Under our culture conditions (Eagle's minimal essential medium plus delipidized fetal calf serum) this incorporation proceeded with a very good rate (up to 45 mol/100 mol, after two days culture) and could be easily modulated depending on the way the cells were fed with the anthracene fatty acid. It occurred to a similar extent at the sn-1 (55 +/- 5%) or at the sn-2 (45 +/- 5%) position on the phospholipid glycerol backbone, without any degradation or elongation. No double labelling at the sn-1 and sn-2 positions was detected. Although incorporation of the anthracene fatty acid affected the cell growth rate (generation time of 48 h compared to a generation time of 21 h for control cells) it did not bring about cell mortality. This incorporation took place not only into the phospholipids but also into the triglycerides with, as a consequence, the appearance of strongly fluorescent lipid vesicles inside the cells. It affected the whole cell fatty acid composition by slightly increasing the amount of palmitic acid and markedly decreasing the amount of stearic and oleic acids.