Soluble Fraction Of Homogenates Research Articles

Digestion of invertebrate neuropeptides by preparations from the free-living nematodePanagrellus redivivus

Proteases in the soluble fraction of homogenates prepared from the free-living nematode Panagrellus redivivus hydrolysed the amidated invertebrate neuropeptides FMRFa and FLRFa, and nematode FMRFa-like peptides (FLPs) KPNFLRFa (FLP-1-H), APKPKFIRFa (FLP-5-A), KNEFIRFa (FLP-8), KPSFVRFa (FLP-9), RNKFEFIRFa (FLP-12) and KHEYLRFa (FLP-14) in vitro. Results were assessed by analysing reaction components with RP-HPLC, UV detection at 210 nm and peak integration. Based upon substrate peak size, more than 90% of most of the peptide substrates was consumed after 1 h at 27 degrees C, but digestion was not complete even with a crude protease mixture. Two peptides, FLP-12 and FLP-14, were significantly less susceptible to digestion than the others. FLP-12 was the least susceptible of all sequences (71% loss; P < 0.0001), while FLP-14 was digested less (84% loss; P < 0.0004) than all but FLP-12. Product peak digestion patterns of FLP-12, a second nonapeptide (FLP-5-A), and FMRFa, incubated with aminopeptidase (amastatin) and serine endoprotease (AEBSF) inhibitors, demonstrated highly specific behaviours of each sequence to protease cleavage. Amastatin significantly (P < 0.03) reduced digestion of FLP-12 (54% loss) and FMRFa (61% loss; P < 0.0005), but had no effect on FLP-5-A. AEBSF had no protective effect on FMRFa but significantly decreased hydrolysis of FLP-5-A (77% loss; P < 0.0001) and FLP-12 (59% loss; P < 0.03). The combination of both inhibitors had additive effects only for FMRFa (34% loss; P < 0.0005). Further analysis of FMRFa digestion using peptides with D-amino acid substitutions demonstrated nearly complete protection of FdMRFa (2% loss; P < 0.0001) from all proteolytic digestion, whereas digestion of FMRdFa was complete. Results suggest that in addition to aminopeptidase and serine proteases, both deamidase and aminopeptidase P participate in neuropeptide metabolism in P. redivivus.

Decrease of PKC precedes other cellular signs of calpain activation in area CA1 of the hippocampus after transient cerebral ischemia

One of the specific features of severe brain injury is an activation of calcium-dependent proteolysis by calpains. We have observed a significant increase of activity as early as 3h after the insult in a well defined model of delayed ischemic neuronal death in gerbil hippocampus. At 24h, the enzymatic activity transiently normalized, then increased again, following the place and time of selective cellular death in the CA1 region of hippocampus. The enhanced postischemic proteolysis resulted in concomitant cleavage of calpain-specific endogenous substrates like protein kinase C (PKC), fodrin and microtubule-associated protein-2 (MAP2). These effects were also time-dependent and restricted to the vulnerable, CA1 pyramidal neurons-containing the dorsal part (DP) of the hippocampus.We have also characterized the postischemic changes of six different isoforms of PKC. The vulnerable dorsal part of the hippocampus, but not its relative resistant abdominal part (AbP), exhibited a loss of PKCα, β, γ, and δ isoforms as early as 3h after ischemic insult. However, at this time, solely in the soluble fraction of homogenate. Later (72h), a further loss of the enzyme proteins, comprised the particulate fraction as well and resulted in an about 50% decrease of total PKCs in the vulnerable DP region. In the case of PKCα, the immunostaining pattern showed, in addition to the disappearance of the enzyme from the injured area, an extensive translocation into nuclei of the survived, ischemia-resistant neurones.The early decreases of PKC isoforms in the cytosol paralleled the transient calpain activation at 3h postischemia but substantially preceded the proteolysis of any other classical calpain substrates, such as fodrin and MAP2, being evidenced not earlier than 48–72h after the insult and restricted also to the vulnerable dorsal part.In conclusion, our results of the time-dependent effects of transient global cerebral ischemia on the calpain activity, levels and localization of its several substrates suggest, that calpain-mediated proteolysis is specifically involved in the early (induction) as well as in the late (execution) phases of delayed ischemic neuronal death in the CA1 hippocampus.

Dual response of calpain to rat brain postdecapitative ischemia

Calpains, Ca(2+)-dependent neutral proteinases (microM and mM Ca(2+)-sensitive), and their endogenous inhibitor calpastatin were examined in rat brain. Specific activity of m-calpain exceeded almost 10 times that of mu-calpain, and the both isoforms of calpain together with calpastatin were mainly located in the soluble fraction of homogenate. Acute postdecapitative ischemia of 15 min duration resulted in a gradual, time-dependent decrease of total mu-calpain activity (to 60% of control values) and in the moderate elevation of calpastatin activity (by 28%). The decrease of total mu-calpain activity coincided with its remarkable increase (above 300% of control values) in particulate fraction. In the case of m-calpain, the only observed effect of ischemia was its redistribution and, as a consequence, the elevation of activity in particulate fraction. The accumulation of breakdown products, resulting from calpain-catalyzed proteolysis of fodrin (as revealed by Western blotting) indicated activation of calpain under ischemia. The findings suggest that this rapid activation involves partial enzyme translocation toward membranes, and is followed (at least in acute phase) by mu-calpain downregulation and increased calpastatin activity.

Gibberellin-photoaffinity labeling of wild oat (Avena fatua L.) aleurone protoplasts

Aleurone protoplasts of wild oat (Avena fatua L.), and subcellular fractions isolated from them, were photoaffinity labeled using the synthetic gibberellin (GA) derivative GA4-17-yl-1′-(1′-thia)propan-3′-ol-4-azido-5-[125I]iodosalicylate. Labeled polypeptides were identified by electrophoresis under denaturing conditions followed by autoradiography. GA-photoaffinity labeling of both intact protoplasts and isolated subcellular fractions led to the covalent attachment of the reagent to many polypeptides. A 50 kD polypeptide in the soluble fraction of homogenates of aleurone protoplasts GA-photoaffinity labeled in vivo showed specific binding. The biologically active GA1, GA4 and GA4-17-yl-1′(1′-thia)propan-3′-ol-4-azidosalicylate completed for binding whereas the biologically inactive GA8 and GA34 did not. The GA-photoaffinity labeling characteristics of this polypeptide suggested that it might interact specifically with biologically active GAs in vivo. Attempts to detect specific GA-binding in in vitro GA-photoaffinity labeling experiments met with only limited success perhaps indicating the labile nature of specific binding observed in vivo. The potential of GA-photoaffinity labeling for identifying GA-binding proteins in aleurone and other GA-responsive tissues is discussed.

M-phase-specific protein kinase from mitotic sea urchin eggs: Cyclic activation depends on protein synthesis and phosphorylation but does not require DNA or RNA synthesis

Histone H 1 kinase (H 1K) undergoes a transient activation at each early M phase of both meiotic and mitotic cell cycles. The mechanisms underlying the transient activation of this protein kinase were investigated in mitotic sea urchin eggs. Translocation of active H 1K from particulate to soluble fraction does not seem to be responsible for this activation. H 1K activation cannot be accounted for by the transient disappearance of a putative H 1K inhibitor present in soluble fractions of homogenates. Aphidicolin, an inhibitor of DNA synthesis, and actinomycin D, an inhibitor of RNA synthesis, do not impede the transient appearance of H 1K activity. H 1K activation therefore does not require DNA or RNA synthesis. Fertilization triggers a rise in intracellular pH responsible for the increase of protein synthesis. H 1K activation is highly dependent on the intracellular pH. Ammonia triggers an increase of intracellular pH and stimulates protein synthesis and H 1K activation. Acetate lowers the intracellular pH, decreases protein synthesis, and blocks H 1K activation. Protein synthesis is an absolute requirement for H 1K activation as demonstrated by their identical sensitivities to emetine concentration and to time of emetine addition. About 60 min after fertilization, H 1K activation and cleavage become independent of protein synthesis. The concentration of p34, a homolog of the yeast cdc2 gene product which has been recently shown to be a subunit of H 1K, does not vary during the cell cycle and remains constant in emetine-treated cells. H 1K activation thus requires the synthesis of either a p34 postranslational modifying enzyme or another subunit. Finally, phosphatase inhibitors and ATP slow down in the in vitro inactivation rate of H 1K. These results suggest that a subunit or an activator of H 1K is stored as an mRNA in the egg before mitosis and that full activation of H 1K requires a phosphorylation.

A new assay system to classify non-mating mutants and to distinguish between vegetative cell and gamete in Chlamydomonas reinhardtii

A new system is described for the classification of various types of non-mating mutants of Chlamydomonas reinhardtii. This system makes use of two simple assays: one for cell body-agglutinin (Saito et al. 1985), and another for the cell wall lytic enzyme (Matsuda et al. 1987). Both assays use the soluble fraction of homogenates from nitrogen-starved cells. Many non-mating mutants previously isolated were analysed and divided into four main phenotypic classes. This assay method is also a valuable tool for distinguishing between vegetative cells and gametes. Without this assay, verifying gametogenesis is especially difficult in agglutinin-and flagella-deficient cells. We found that wild-type cells, which had been grown for 7 days on TAP plates and lacked flagella, contain neither cell body-agglutinin activity nor lytic enzyme activity in the soluble fraction of their cell homogenates, indicating that these cells can be classified as vegetative. When suspended in nitrogen-free liquid medium, the two activities developed rapidly in parallel to the acquisition of mating ability: a maximum level was reached within two hours.

Preparation and analysis of mass amounts of germin: demonstration that the protein which signals the onset of growth in germinating wheat is a glycoprotein

(1) A rapid method (2–3 weeks) is described for preparing milligram amounts of germin, in high yield, from kilogram quantities of germinated wheat embryos. An ammonium-sulphate fraction of proteins in the low-speed supernatant from an embryo homogenate is suspended in buffer for pepsin digestion, and the resulting digest is filtered through Ultrogel AcA34 to obtain germin, virtually free of contamination (&lt;1%) by other proteins.(2) An acid hydrolysate of germin contains a full complement of the 20 different amino acids. The proportions of half-cystine, tyrosine, and tryptophan are conspicuously low (&lt;1.5%), the proportions of aspartic acid and glycine (each ca. 11%) as well as proline (ca. 8%) are relatively high, and the cumulative proportion of hydrophobic amino acids (valine, leucine, isoleucine, phenylalanine) is also high (ca. 25%). The N-terminal sequence of germin, as determined by Edman degradation, is enriched with respect to aspartic acid, which occupies 5 of the first 15 positions.(3) There are two forms of germin, one (G) being the dominant species in the soluble fraction of homogenates of germinated embryos and the other (G′) being the dominant species in a fraction obtained by rinsing growing embryos. Consistent differences between the amino-acid compositions of G and G′ were not observed. Similarly, wherever identification was possible, the N-terminal amino-acid sequences (residues 1–22) of G and G′ were not different.(4) The polymeric and monomeric forms of germin (G or G′) give a positive Schiff reaction, characteristic of glycoproteins, when electrophoretically separated in sodium dodecyl sulphate (SDS) – poly aerylamide. Treatment with trifluoromethanesulfonic acid affirmed the glycoprotein character of germin, as did labeling, in vivo, from exogeneously supplied tritiated sugars.(5) The G and G′ forms of germin were among the most heavily labeled proteins seen in SDS–polyacrylamide separations if wheat-embryo proteins were labeled, in vivo, from exogenous supplies of tritiated mannose, glucosamine, or fucose. Differences between the patterns of glucosamine labeling and fucose labeling of G and G′ suggest the possibility that the two forms differ with respect to their carbohydrate components.

Germin. Compartmentation of the protein, its translatable mRNA, and its biosynthesis among roots, stems, and leaves of wheat seedlings

(1) Onset of growth during germination of the isolated wheat embryo is allied with the emergence of a protein we have called germin. This study was undertaken to learn if germin is present and synthesized in the root, stem, and leaf during postgerminative growth of the wheat seedling.(2) Seedlings were grown from mature wheat grains on water-soaked filter paper and organs excised at various times were pulse labeled with [35S]methionine. Germin is synthesized in all organs at all times between 1.5 and 7 days.(3) During early seedling development (1.5 days), the fraction of [35S]methionine incorporated into germin, relative to other proteins, is much greater in the case of stem than other organs, either in vivo or when cell-free protein synthesis is directed by bulk RNA from different organs.(4) During late seedling development (4–7 days), when root growth is greater than stem growth, total isotope incorporation into germin is much greater in roots than stem.(5) Quasi-quantitative estimates of germin in pepsin-treated soluble fractions of homogenates were made by visual comparison of dye binding when gels were stained by Coomassie blue. The quantity of germin (ca. 1 mg/100 g) keeps pace with growth in spite of a 100-fold increase in fresh weight during transformation of the germinating embryo into a 7-day seedling.(6) During early seedling growth (1.5 days), germin is concentrated in the stem, but later (7 days), after extensive growth of the root but not the stem, the amount of germin in the root is about 1/2 and in the leaf is about 1/10 as great as in stem, on a fresh-weight basis, but more nearly equal on a per-organ basis owing to high mass proportions of the leaf and root relative to stem.(7) The dye-staining technique following pepsin treatment of soluble proteins has been used to detect germin in the stems of other cereal (rye, barley, oat) seedlings (3–8 days).

Separation of β-d-galactosidases in rabbit tissues: Genetics of neutral β-d-galactosidase

Three different types of beta-D-galactosidase (EC 3.2.1.23) could be distinguished in rabbit tissues using electrophoretic procedures. (1) Acid beta-D-galactosidase with a low mobility and maximal activity at pH 3-5 was found in the particulate fraction of various tissue homogenates. This enzyme hydrolyzed 4-methylumbelliferyl-D-galactoside, but no activity against other glycoside substrates could be demonstrated. The enzyme was inhibited by galactono-(1 leads to 4)-lactone. (2) Lactose-hydrolyzing beta-D-galactosidase with an intermediate mobility was found only in juvenile small intestine. Most of the activity was found in the particulate fraction of the cell. The enzyme hydrolyzed several other synthetic glycoside substrates besides lactose. It was most active at pH 5-6 and strongly inhibited by glucono-(1 leads to 5)-lactone but not much affected by galactono-(1 leads to 4)-lactone. (3) Neutral beta-D-galactosidase with a fast mobility and maximal activity at pH 6-8 was found in the soluble fraction of homogenates from liver, kidney, and small intestine. This enzyme also showed a broad substrate specificity; it possessed activity against aryl-beta-D-glucoside, -fucoside, and -galactoside substrates but not against lactose. The enzyme was strongly inhibited by glucono-(1 leads to 5)-lactone and (less) by galactone-(1 leads to 4)-lactone. Neutral beta-D-galactosidase and neutral beta-D-glucosidase (EC 3.2.1.21) are probably identical enzymes in the rabbit. Individual variation, in both electrophoretic mobility and activity, was found for neutral beta-D-galactosidase. Genetic analysis of the electrophoretic variants revealed that two alleles at an autosomal locus are responsible for this variation.

A beta-D-galactoside binding protein from electric organ tissue of Electrophorus electricus.

Extracts of electric organ tissue of Electrophorus electricus contain a saccharide-binding protein, named electrolectin, which agglutinates trypsin-treated rabbit erythrocytes and is specifically inhibited by disaccharides containing nonreducing terminal beta-D-galactosyl residues. Electrolectin seems at least partially membrane-bound but is also found in soluble fractions of homoge-nates from which it can be purfied by affinity chromatography on cross-linked and desulfated agarose (ECD-Sepharose) as a protein of molecular weight 33,000. About 400 mg of electrolectin are present per kg of tissue. It has an affinity for lactose of 1.0 mM-1 and 5.5mM-1 as estimated, respectively, by hapten inhibition and fluorescence spectroscopy. Studies on the distribution of beta-D-galactoside-binding activity in animal tissues reveal particularly high levels in sheletal muscle tissue and in cultures of embryonic skeletal muscle and neuroblastoma cells.

Dephosphorylation of Pyrimidine Nucleotides in the Soluble Fraction of Homogenates from Normal and Regenerating Rat Liver

The dephosphorylation of the pyrimidine nucleotides UMP, CMP, dUMP, dCMP, and dTMP was determined in the soluble fraction of homogenates from normal rat liver and from regenerating liver during an 8‐day period after partial hepatectomy. The dephosphorylations were assayed at optimal pH in the presence of optimal concentrations of Mg++ ions and substrate. β‐Glycerophosphate was included among the substrates for measurement of non‐specific phosphatase activity. The intracellular origin of the enzymes was investigated by determining the rate of their appearance in the soluble fraction during homogenization and by estimating the rate at which the lysosomes were disrupted.The results showed that the dephosphorylation of the deoxyribonucleotides was mainly due to non‐specific acid phosphatase activity. This enzyme and the alkaline phosphatase activity were found to be localized in the soluble space of the liver cell. The dephosphorylation of the ribonucleotides was due largely to a specific 5′‐nucleotidase. The enzyme was adsorbed or loosely bound to some sedimentable cell constituent at the time of cell disruption, but it was quantitatively released into the soluble fraction during 2 minutes homogenization.During liver regeneration the acid and alkaline phosphatase activities showed slight variations which could not be related to the growth rate of the liver. In contrast, the 5′‐nucleotidase activity showed distinct, cyclic variations which were inversely related to the growth rate. The variations in the 5′‐nucleotidase activity were strikingly similar to the activity variations of uracil reductase and N‐carbamoyl‐β‐alanine amidohydrolase observed previously in regenerating liver.

The Effect of X-Irradiation on the Activities of Some Lysosomal Hydrolases of Heart Tissue

The location of the initial lesions produced by ionizing radiations in living systems has been investigated by many radiobiologists. One result of radiation injury is the increase in enzymic activities frequently observed in irradiated organisms. Bacq and Alexander (1) suggested that this effect could be explained on the basis that radiations break down internal barriers by altering the cellular and intracellular membranes with consequent release of enzymes. Recently it has been shown (2) that membrane-bound organelles containing hydrolytic enzymes are found in the cytoplasm of cells in many tissues: these particles have been named lysosomes. According to the enzyme release theory, if an ionizing radiation produces damage to the intracellular membranes, and therefore also to the membranes of these lysosomes, a release of the lysosomal enzymes should be expected after irradiation. This hypothesis seems to be supported by some experimental evidence. An increase of acid DNase (deoxyribonucleate 3'-nucleotidohydrolase, E.C. 3.1.4.6) (lysosomal enzyme) has been reported, for instance, a few hours after irradiation in the serum and in the urine of animals treated with X-rays (3, 4). Furthermore, this nuclease and the acid RNase (polyribonucleotide 2-oligonucleotide-transferase (cyclizing), E.C. 2.7.7.16), which is also a lysosomal enzyme, appear to be increased in the soluble fraction of homogenates of tissues derived from animals treated with X-rays (5, 6). Zeman et al. (7) have also demonstrated, by histochemical reactions, that, in mice brain irradiated with microbeams of accelerated deuterons, lysosomal enzymes such as acid phosphatase (o-phosphoric monoester phosphohydrolase, E.C. 3.1.3.2), B3-glucuronidase (3-D-glucuronide glucuronohydrolase, E.C. 3.2.1.31), and cathepsin c (E.C. 3.4.4.9) become evident. The positive histochemical reaction appears along the path of the ionizing particles.