Nonsedimentable Fraction Research Articles

Influence of heating treatment and membrane concentration on the formation of soluble aggregates

This study investigated the effect of thermal denaturation on milk concentrated by ultrafiltration (UF) with or without diafiltration (DF) at various time–temperature combinations. The non-sedimentable protein fractions present in milk after heating were analyzed using size exclusion chromatography and their composition was determined. The extent of whey protein denaturation and the composition of the soluble complexes depended not only on heating temperature and time, but also on the protein concentration. There was a greater extent of protein aggregation in concentrated milk compared to 1× milk, and there was a significant difference in the size and composition of the aggregates present in the non-sedimentable fraction depending on whether the milk was concentrated only by UF or if DF was also used. DF milk contained a higher concentration of non-sedimentable protein compared to UF milk. In all samples, κ-casein and β-lactoglobulin were the main proteins present in the soluble aggregates, but higher temperatures and longer heating times resulted in greater extents of denaturation of α-lactalbumin. This work clearly identified differences in the extent of aggregation between milk concentrated by UF or UF and DF, and unconcentrated milk, with important consequences on the processing functionality of caseins.

Total and soluble contents of calcium, magnesium, phosphorus and zinc in yoghurts

This paper examines the content of major minerals (calcium, magnesium, phosphorus and zinc) and its distribution between soluble (non-sedimentable) and micellar fractions of 16 commercial yoghurts from five different branches. These elements were determined by atomic absorption spectrometry after mineralisation by dry ashing. Total mineral contents showed wide intervals of variation (calcium 1090–2050 mg l −1, magnesium 101–177 mg l −1, phosphorus 878–1560 mg l −1 and zinc 4.0–7.3 mg l −1) due to the addition during manufacturing of different dairy products or fractions. Most of the minerals were found in the non-sedimentable fraction. Practically all calcium was measured in the supernatants of ultracentrifugation. Lower proportions of magnesium and zinc were determined in the non-sedimentable fraction (between 87 and 96%) whereas the lowest ones (63–77%) were found for phosphorus.

The Isolation from Human Seminal Plasma of a New Form of Soluble 5′-Nucleotidase

Soluble broad spectrum 5′-nucleotidase from human seminal plasma was purified to homogeneity by a combination of (NH4)2SO4precipitation, affinity chromatography, and gel filtration. The pure enzyme had a specific activity of 4800 nmol min−1mg−1. SDS–PAGE of purified enzyme preparation revealed a single polypeptide band of 53 kDa and a tetrameric structure of 203 kDa was proposed for the native enzyme. This form had modest preference for AMP as substrate; Mg2+and Mn2+were activators of the enzyme although its activity was not absolutely dependent on the presence of these exogenous bivalent cations. The enzyme, recovered in the nonsedimentable fraction of human seminal plasma, had a pH optimum of 7.5; ATP and ADP were inhibitors of mixed type, Piwas a potent inhibitor at nonphysiological concentrations, and Con A and adenosine 5-[α,β-methylene]diphosphate had no effect on the enzyme activity. The enzyme described here therefore has some unique properties between truly cytoplasmic and membrane-bound derived forms.

Isolation and characterization of endoglucanases 1 and 2 from Bacteroides succinogenes S85.

Two endoglucanases designated EG1 and EG2 were purified by column chromatography from the nonsedimentable extracellular culture fluid of Bacteroides succinogenes S85. They accounted for approximately 32 and 11%, respectively, of the total endoglucanase present in the nonsedimentable fraction. The most active enzyme (EG1) had a molecular weight of 65,000, pI of 4.8, and temperature and pH optima of 39 degrees C and 6.4, respectively. The Km for carboxymethyl cellulose was 3.6 mg/ml, and the Vmax was 84 U/mg. The major products of cellulose hydrolysis catalyzed by EG1 were cellotriose and cellobiose. EG2 was present as two components with molecular weights of 118,000 and 94,000. The two components had nearly identical cyanogen bromide peptide maps, thereby indicating that the 94,000-dalton component was a proteolytic degradation product of the 118,000-dalton enzyme. The larger component, which was more abundant in the culture fluid than the smaller form was, had a Km of 12.2 mg/ml and a Vmax of 10.4 U/mg. It was a basic protein with a pI of 9.4, a temperature optimum of 39 degrees C, and a pH optimum of 5.8. The major product of cellulose hydrolysis was cellotetraose. EG2 exhibited specific binding to acid-swollen cellulose, whereas EG1 did not, and neither of them had affinity for crystalline cellulose. Based on the substrate specificities and the affinities of the two enzymes for cellulose, we postulated that EG2 is involved in the early stages of cellulose hydrolysis and that EG1 is active primarily on the products arising from EG2.

Multiplicity of extracellular β-(1,4)-endoglucanases of Bacteroides succinogenes S85

During growth on 0.2% (w/v) microcrystalline cellulose, Bacteroides succinogenes S85 produces endoglucanase activity which can be separated by centrifugation into sedimentable and nonsedimentable fractions. The sedimentable activity, after solubilization with Triton X-100, was resolved into four components by ion-exchange chromatography and these were further fractionated by nondenaturing polyacrylamide gel electrophoresis (PAGE). The nonsedimentable activity contained three enzymic components as determined by gel filtration. Like the preparations derived from the sedimentable fraction, these components yielded a multiplicity of endoglucanases when electrophoresed under nondenaturing conditions. The fractions obtained by ion-exchange chromatography and by gel filtration were assayed for endoglucanase activity by both viscometric assays and reducing sugar production using carboxymethylcellulose as the substrate. Plots of the fluidity change in the enzyme–substrate preparation in relation to reducing sugar production revealed the presence of two distinct groups of endoglucanases differing in catalytic activity. Two of the components from the nonsedimentable fraction had more exoglucanase-like activity than either the third nonsedimentable fraction or any of the four fractions derived from the sedimentable material. These two enzymes could be further differentiated on the basis of glucose production from microcrystalline cellulose and by their relative activity toward p-nitrophenyl cellobioside, a chromogenic substrate.

Salvage of pyrimidine nucleosides by Trichomonas vaginalis

Trichomonas vaginalis is incapable of de novo pyrimidine biosynthesis because it cannot incorporate bicarbonate, aspartate or orotate into its pyrimidine nucleotides or nucleic acids. The organism can salvage exogenous cytidine > uridine > uracil and thymidine, and incorporate them into the nucleotide pool. A portion of cytidine is converted to CMP, CDP and CTP by cytidine phosphotransferase and nucleotide kinases. Some cytidine and most of uracil are, however, converted first to uridine by cytidine deaminase and uridine phosphorylase respectively; uridine is then incorporated into UMP, UDP and UTP by uridine phosphotransferase and nucleotide kinases. The two phosphotransferases, found mainly in the non-sedimentable fraction of T. vaginalis, provide the main avenue of pyrimidine salvage. No significant levels of pyrimidine phosphoribosyl transferase or nucleoside kinases can be detected in the extract. T. vaginalis has no appreciable dihydrofolate reductase or thymidylate synthetase; it grows normally in millimolar concentrations of methotrexate, pyrimethamine, or trimethoprim, and cannot incorporate labels from exogenous uracil or uridine into DNA. It has an enzyme thymidine phosphotransferase in the sedimentable fraction which converts thymidine to TMP. Thymidine salvage in T. vaginalis is thus totally isolated from the rest of the pyrimidine salvage.

Metabolism of xenobiotic compounds by enzymes in cell extracts of the fungus Cunninghamella elegans.

Cell extracts of the filamentous fungus Cunninghamella elegans contain epoxide hydrolase (EC 3.3.2.3), glutathione S-transferase (EC 2.5.1.18) and UDP-glucuronosyltransferase (EC 2.4.1.17) activities. Epoxide hydrolase activity was determined with p-nitrostyrene oxide as substrate and was shown to be associated with the 100 000 g pellet obtained from disrupted mycelia. Glutathione S-transferase activity was demonstrated with 1-chloro-2,4-dinitrobenzene and p-nitrobenzyl chloride as substrates. The presence of two or more glutathione S-transferase activities was indicated by different activity ratios for the two substrates in different extracts, and by distinct thermal denaturation curves. UDP-glucuronosyltransferase activity with 3-hydroxybenzo[a]pyrene as substrate was found only with the non-sedimentable fraction prepared from ruptured mycelia.

Lysosomal hydrolases in the heterotopically isotransplanted heart undergoing atrophy

The potential role of lysosomal enzymes in the regression of cardiac mass was investigated in heterotopic “empty beating” isotransplanted hearts undergoing atrophy. The transplants were located in the abdomen of an inbred recipient rat while their coronaries were retrogradely perfused from the abdominal aorta and drained into the inferior vena cava. The animals were sacrificed 4, 13, 90 and 360 days after the operation and the decreasing weights of the beating transplants indicated progressive atrophy. The activities of cathepsin D, acetyl-β- d-glucosaminidse and p-nitrophenylphosphatase were determined in subcellular fractions of left ventricular homogenates of the transplanted and recipient hearts. The activities of all three enzymes were elevated in the transplants, most significantly during the first few days after the operation as compared to the recipients. This difference gradually diminished with time and disappeared by the third post-operative month, except in the case of cathepsin D where it persisted. The subcellular distribution of the lysosomal enzymes of the transplant indicated a shift into the non-sedimentable fraction during the first two post-operative weeks. The above changes in the activities and in the distribution of the enzymes paralleled the progressive loss of myocardial tissue, which was mainly the consequence of the hypodynamic function of the transplant. No changes were found in the sub-cellular distribution of recovered proteins between the transplanted and the recipient hearts. No differences were found in the activities of the enzymes between hearts of normal and sham-operated rats which served as controls.

Lectin Activity in Entamoeba histolytica trophozoites

A lectin (carbohydrate-binding protein) has been found in extracts of a number of axenically grown trophozoites of Entamoeba histolytica strains. The strains grown in TYI-S-33 medium (Diamond et al., Trans. R. Soc. Trop. Med. Hyg. 72: 431-432, 1978) were HK-9, 200:NIH, and HM-1:IMSS. Strain HU-1:MUSC (HSC) was grown monoxenically in the same medium. The amoebic lectin agglutinated glutaraldehyde-fixed erythrocytes. This activity was pH dependent and heat and oxidation sensitive, and was destroyed by proteolysis upon autoincubation. The relative agglutinating potency of the different strains of amoebae was investigated. Strain HSC had the highest specific activity (210 U/mg of protein), and strain HM-1 had the lowest (14 U/mg). One unit of hemagglutinating activity is defined as the amount of lectin present in 1 ml of extract which will agglutinate 1 ml of 4% erythrocytes. Upon subcellular fractionation of the lectin present in extracts of strain HK-9, two-thirds of the activity was detected in the soluble, nonsedimentable (100,000 x g, 60 min) fraction. Partial hydrolysate of chitin was found to inhibit the hemagglutinating activity. Among the oligosaccharides of N-acetylglucosamine, the trimer and tetramer were the most potent inhibitors. The lectin was purified approximately 300-fold by a one-step affinity chromatography on a chitin column. The loading and elution from the column were based on the pH dependence of the lectin activity.

Lysosomal alterations in autolyzing rabbit heart

Sequential biochemical and immunohistochemical lysosomal alterations were assessed in autolyzing ventricular myocardium incubated at 37°C in a humidified atmosphere for 15 min to 2 h. A profound redistribution of cathepsin D activity into the nonsedimentable fraction of homogenates could be demonstrated after only 15 min of autolysis, with further increases over the next 105 min. Nonsedimentable activity of n-acetyl-β, d-glucosaminidase was also significantly increased early after the onset of autolysis, but the distribution of acid phosphatase activity was not significantly altered. Immunohistochemical localization of cathepsin D revealed dramatic loss of granular staining in myocytes during the first hour of autolysis. Between 15 and 30 min, lysosomes became enlarged and halos of fluorescent material appeared, representing diffusion of cathepsin D from lysosomes into the myoplasm. By 60 min most cathepsin-D-positive organelles had vanished and the enzyme was instead distributed diffusely throughout the cell. The early redistribution of cathepsin D observed by biochemical and immunohistochemical techniques occurred before electron microscopic indications of cell death became apparent. Thus, autolyzing heart may provide a useful model for studying the possible role of lysosomal enzymes in provoking myocardial cell injury and necrosis.

Effects of disulphides and α-oxoglutarate on nuclear thiol formation and thiol content of chromatin in lysed rat spleen nuclei

1. 1. During incubation of lysed rat spleen nuclei, there was a noted increase in total SH, accompanied by a slight decrease of chromatin SH and a more pronounced increase in SH of the non-sedimentable fraction. 2. 2. During incubation, chromatin nucleolytic cleavage was completely inhibited by 1 mM cystamine and 1 mM oxidized glutathione (GSSG). 3. 3. In nucleus lysates, glutathione reductase activity dependent on NADPH, and disulphide reductase NADH-dependent reducing disulphides of the non-sedimentable fraction, but not affecting GSSG, were demonstrated. 4. 4. In the presence of 1 mM GSSG there was a transfer of SH from the non-sedimentable fraction to the rat spleen chromatin, while 1 mM 6,8-thioctic acid exerted an opposite effect. Cystamine (1 mM) was found to decrease SH content of either chromatin and the non-sedimentable fraction. 5. 5. Reduced glutathione (GSH) and cysteamine (MEA) and CoA did not change chromatin SH. 6. 6. In the presence of α-oxoglutarate, as in the presence of GSSG, an increase in chromatin SH was found, indicating the role of dihydrolipoate in the control of chromatin SH level.

Phosphoric monoester hydrolase-like activity in the odontoblast-predentine region of intact and carious human teeth

Coronal odontoblast-predentine tissue was taken from intact teeth and from teeth with various types of occlusal caries, and the specific phosphoric monoester hydrolase activity in the non-sedimentable (20,200 g ) fraction was measured at alkaline and acid pH, using p-nitrophenyl phosphate as a substrate. A significant increase of activity similar to alkaline phosphatase (AlPase) was observed under enamel and initial dentine caries lesions. The enzyme activity was about equal to that of the intact tissue when the overlying lesions were arrested, subsiding significantly where large advanced lesions were concerned. As AlPase-like activity was maximal in active enamel and initial dentine lesions, it was concluded that the enzyme has an important role in protein matrix formation. The activity of acid phosphatase (AcPase) was approximately 10–15 times weaker than that of AlPase in the intact odontoblast-predentine area and was increased under advanced dentine lesions. The appearance of AcPase-like activity probably indicated metabolic changes related to reparative dentine formation and partly reflected the level of degradation of severely injured odontoblasts by the autolytic release of lysosomal enzymes. The phosphoric monoester hydrolases both in intact and in carious teeth probably originate from the host tissue.

Sodium dodecyl sulphate-polyacrylamide-gel electrophoretic detection of two high molecular weight proteins associated with tobacco mosaic virus infection in tobacco

Proteins extracted from tobacco mosaic virus (TMV)-infected tobacco plants were analyzed by electrophoresis in sodium dodecyl sulphate (SDS)-polyacrylamide gels. A protein of ca. 150,000 daltons, not detectable in healthy plants, appeared in leaves infected for 2 days or more. A second, somewhat smaller protein (ca. 130,000 daltons) was found to accumulate in greater amounts; in the latter case, extracts of healthy plants also contained a protein migrating to the same position on the gels. The larger protein was detected in the 2500 and 20,000 g pellets of tissue homogenates fractionated by differential centrifugation, and the smaller one in the nonsedimentable (soluble) fraction.

Biosynthesis of starch: Formation of a glucoproteic acceptor by a potato non-sedimentable preparation

1. 1. A non-sedimentable fraction of potato tuber has been found to catalyze [ 14C] glucose transfer from [ 14C] glucose 1-phosphate to an endogenous proteic acceptor in the absence of added primer. This transfer is activated by Mn 2+. 2. 2. The labeled glucosylated product formed is trichloroacetic acid insoluble and sensitive to proteolytic and amylolytic digestions. It appears to be a glucoprotein with glucosyl chains bound to the peptide portion of the molecule through an unknown linkage. 3. 3. The carbohydrate portion of the glucoprotein can be released by prolonged incubations with the enzymatic preparation, and becomes in turn, trichloroacetic acid soluble and alcohol precipitable. 4. 4. Both products, the glucoprotein as well as the α-1,4-glucan that seems to arise from the enzymatic cleavage of the former, can be used as primers by the transglucosylating system with ADP[ 14C] glucose, UDP[ 14C] glucose or GDP[ 14C] glucose as glucosyl donors. The results presented in this paper are the first demonstration of soluble glucosyl transferases with the same glucose donor specificity to that of the particulate starch synthetase. 5. 5. This report presents further evidence in favor of the assumption of a glucoproteic intermediate in α-1,4-glucan synthesis initiation.

Fetal mouse hearts: a model for studying ischemia.

A new experimental model for the study of two important aspects of ischemia, namely, oxygen and substrate deprivation, is proposed: the intact, beating fetal mouse heart in organ culture. This model offers long-term stability, ease and reproducibility of preparation, and the ability to manipulate experimental conditions. Hearts deprived of oxygen and glucose ceased beating immediately. After 3-4 hr of deprivation, biochemical and ultrastructural changes consistent with ischemic injury were evident. These include depletion of ATP and glycogen levels, loss of cytoplasmic enzymes, and extensive swelling and disruption of mitochondrial structure. Glucose and insulin partially protected against ATP depletion. Upon resupply of oxygen and glucose , beating resumed immediately, ATP levels rapidly increased to control levels and, consistent with this, mitochondrial structure returned toward normal. During the recovery phase autophagic vacuoles containing damaged mitochondria and myofibrils were seen, indicating that repair mechanisms were activated. Consistent with this, the proportion of lysosomal enzymes that were present in the nonsedimentable fraction of the tissue homogenate increased. We conclude that the cultured fetal mouse heart is a model useful for studying myocardial responses to anoxia and/or substrate deprivation and for assessing interventions designed to limit damage or to stimulate repair after ischemic injury.

Steroid β-d-Glucosidase: PURIFICATION OF THE ENZYME FROM RABBIT LIVER

Abstract The steroid β-d-glucosidase of rabbit liver has been purified to electrophoretic homogeneity and its molecular weight estimated by comparison of its behavior with that of standard proteins on electrophoresis and on gel filtration. The values obtained by the two methods were 51,300 and 58,000, respectively. Detailed studies of the hydrolysis of glucosides of phenolic and of alcoholic steroid hydroxyl groups, and of p-nitrophenylglucoside, indicate that one enzyme, contained in the nonsedimentable fraction of the cell, is responsible for the hydrolysis of all these glucosides.

Separation and Localization of Cell Wall Layers of a Gram-Negative Bacterium

The cell envelope of a marine pseudomonad as seen in thin section by electron microscopy has the double-membrane structure typical of other gram-negative bacteria. Cells washed with a solution containing Na(+), K(+), and Mg(++) at their concentrations in the growth medium, when suspended briefly in 0.5 m sucrose, lost 13% of their hexosamine in a form nonsedimentable by centrifugation at 73,000 x g. Since the resulting cells in thin section appeared unchanged, it was concluded that the material released was derived from a nonstaining, loosely bound outer layer. This same layer could be removed from the cells by washing with 0.5 m NaCl. A second nonsedimentable fraction was released after successive suspension of the cells in 0.5 m sucrose. Since this material was released only when the outer double-track structure had broken, it was concluded that it arose from a layer immediately underlying the latter layer. The three layers differed in their content of hexosamine and protein. None of the layers released contained muramic or diaminopimelic acid. The cell form remaining was rod shaped and appeared in thin section to be bounded only by its cytoplasmic membrane. This form contained all the muramic and diaminopimelic acid in the cell. Treatment with lysozyme released the muramic and diaminopimelic acid and converted the rod form to a protoplast, indicating that in the rod form (mureinoplast) a thin layer of peptidoglycan is located on the outside surface of the cytoplasmic membrane. Thus, five separate layers have been detected in the cell envelope of this marine pseudomonad.

Proteins of vesicular stomatitis virus: kinetics and cellular sites of synthesis.

FIVE VIRAL PEPTIDES SYNTHESIZED IN L CELLS INFECTED WITH VESICULAR STOMATITIS (VS) VIRUS WERE IDENTIFIED BY POLYACRYLAMIDE GEL ELECTROPHORESIS AND DESIGNATED AS FOLLOWS: nucleoprotein N, a membrane glycoprotein G, a membrane surface protein S, and two nonstructural proteins NS1 and NS2. A slowly migrating minor structural protein L also present in infected cells is probably an aggregate. Incorporation of (3)H-amino acids into each viral protein could be detected by the 2nd hr after infection and even earlier for protein N which is synthesized in the greatest amount. There was no evidence of regulation of viral protein synthesis at the transcriptive level; nonstructural and structural proteins were synthesized throughout the cycle of infection. Short pulses of (3)H-amino acids revealed no uncleaved precursor peptides that could be chased into structural peptides. Proteins N and S were chased into released virions but protein G was apparently incorporated into virions as it was being synthesized. VS viral proteins of infected cells were released by mechanically disrupting cytoplasmic membrane by nitrogen decompression and fractionated by high-speed centrifugation. Protein NS1 was present in the nonsedimentable cytoplasmic fraction throughout the cycle of infection. The nucleoprotein N was recovered primarily from the nonsedimentable fraction early in infection but aggregated into a sedimentable component, presumably the nucleocapsid, later in infection. Proteins G and S were always present in the sedimentable fraction of mechanically disrupted infected cells, presumably in association with plasma membrane. Exposure of infected cells to the membrane-dissolving agent, digitonin, resulted in solubilization of most of protein G and all of protein S but not of protein N. These experiments are compatible with the hypothesis that VS viral proteins G and S are synthesized at and inserted into plasma membrane which envelopes a nucleocapsid core to form the VS virion.

Nucleotidase activities in the soluble fraction of rat liver homogenate: Partial purification and properties of a 5′-nucleotidase with pH optimum 6.3

(NH 4) 2SO 4 fractionation of the nonsedimentable fraction of rat liver homogenates indicated that two 5′-nucleotidases (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) with pH optima around 6 were present. One of the enzymes had a broader specificity for 5′-nucleotides than the other, which acted mainly on dTMP and dUMP. A procedure is described for purifying the former enzyme to the stage where it is essentially free of nonspecific phosphatases (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1 and EC 3.1.3.2). The method involves (NH 4) 2SO 4 fractionation and precipitation by dialysis at pH 6.0. Chromatography on DEAE-cellulose and partial heat inactivation indicated that only one 5′-nucleotidase was present in the preparation. The enzyme had a pH optimum at 6.3, it was Mg 2+ dependent, and had a lower K m value for IMP ( K m = 0.2 mM) than for any of the other nucleotides tested. Evidence is presented indicating that the enzyme is identical with the 5′-nucleotidase isolated recently from rat liver acetone powder by Itoh, Mitsui and Tsushima ( J. Biochem. Tokyo, 63 (1968) 165). The enzyme is localized in the soluble cell cytoplasm and has properties different from the 5′-nucleotidases detected previously in particulate structures.

Persistent cyclic herpes simplex virus infection in vitro. II. Localization of virus, degree of cell destruction, and mechanisms of virus transmission.

Hampar, Berge (National Institute of Dental Research, Bethesda, Md.). Persistent cyclic herpes simplex virus infection in vitro. II. Localization of virus, degree of cell destruction, and mechanisms of virus transmission. J. Bacteriol. 91:1959-1964. 1966. The localization of virus, degree of cell destruction, and mechanisms of virus transmission in persistent herpes simplex virus-infected cultures were studied. The major fraction of infectious virus was associated with the medium and a minor fraction was associated with the attached cells. Virus in the medium was further separable into a sedimentable (cellular) fraction and a nonsedimentable (extracellular) fraction. The sedimentable fraction was comprised of cellular debris, most of which appeared to contain viral antigen, and intact cells of which less than 10% contained infectious virus. Cell destruction during the cycle involved more than 99.9% of the maximal number of cells present. Infection could be transmitted by extracellular virus, cell-to-cell transfer, and reattachment of infectious cellular material. The results indicated that transmission by reattachment was probably mediated through the cellular debris rather than the intact cells.