Some Factors Which Affect the Enzymatic Digestion of Ribonucleic Acid

Kalnitsky, George; Hummel, John P.; Dierks, Christa

doi:10.1016/s0021-9258(18)70040-9

Cited by 290 publications

(20 citation statements)

References 13 publications

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“…Measurement of RNase A Activity. RNase A activity was measured following an assay described by Kalnitsky et al 26 and Voziyan et al 27 after some modifications. One hundred microliters of RNase A (treated with the test solutions as described above; final concentration of 30 μg/mL) was mixed with 100 μL of 1% RNA.…”

Section: Continuous Incubation Ofmentioning

confidence: 99%

Structure- and Concentration-Specific Assessment of the Physiological Reactivity of α-Dicarbonyl Glucose Degradation Products in Peritoneal Dialysis Fluids

Distler

Georgieva

Kenkel

et al. 2014

Chem. Res. Toxicol.

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In peritoneal dialysis (PD), glucose degradation products (GDPs), which are formed during heat sterilization of dialysis fluids, lead to structural and functional changes in the peritoneal membrane, which eventually result in the loss of its ultrafiltration capacity. To determine the molecular mechanisms behind these processes, the present study tested the influence of the six major α-dicarbonyl GDPs in PD fluids, namely, glyoxal, methylglyoxal, 3-deoxyglucosone (3-DG), 3-deoxygalactosone (3-DGal), 3,4-dideoxyglucosone-3-ene (3,4-DGE), and glucosone with respect to their potential to impair the enzymatic activity of RNase A as well as their effects on cell viability. For comprehensive risk assessment, the α-dicarbonyl GDPs were applied separately and in concentrations as present in conventional PD fluids. Thus, it was shown that after 5 days, glucosone impaired RNase A activity most distinctly (58% remaining activity, p < 0.001 compared to that of the control), followed by 3,4-DGE (62%, p < 0.001), 3-DGal (66%, p < 0.001), and 3-DG (76%, p < 0.01). Methylglyoxal and glyoxal caused weaker inactivation with significant effects only after 10 days of incubation (79%, 81%, p < 0.001). Profiling of the advanced glycation end products formed during the incubation of RNase A with methylglyoxal revealed predominant formation of the arginine modifications imidazolinone, CEA/dihydroxyimidazoline, and tetrahydropyrimidine at Arg10, Arg33, Arg39, and Arg85. Particularly, modification at Arg39 may severely affect the active site of the enzyme. Additionally, structure- and concentration-specific assessment of the cytotoxicity of the α-dicarbonyl GDPs was performed. Although present at very low concentration, the cytotoxic effect of PD fluids after 2 days of incubation was exclusively caused by 3,4-DGE (14% cell viability, p < 0.001). After 4 days of incubation, 3-DGal (13% cell viability, p < 0.001), 3-DG (24%, p < 0.001), and, to a lower extent, glyoxal and methylglyoxal (both 57%, p < 0.01) also reduced cell viability significantly. In conclusion, 3,4-DGE, 3-DGal, and glucosone appear to be the most relevant parameters for the biocompatibility of PD fluids.

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Section: Continuous Incubation Ofmentioning

confidence: 99%

Structure- and Concentration-Specific Assessment of the Physiological Reactivity of α-Dicarbonyl Glucose Degradation Products in Peritoneal Dialysis Fluids

Distler

Georgieva

Kenkel

et al. 2014

Chem. Res. Toxicol.

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“…RNase A activity was determined by its ability to hydrolyze yeast RNA (Sigma Chemical Co .) according to the method of Kalnitsky, Hummel, and Dierks (30) .…”

Section: Assays For Rnase a And Lysozyme Activitymentioning

confidence: 99%

Degradation of proteins microinjected into IMR-90 human diploid fibroblasts.

Neff¹,

Bourret²,

Miao³

et al. 1981

The Journal of Cell Biology

120

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Erythrocyte ghosts loaded with ' 25 1-labeled proteins were fused with confluent monolayers of IMR-90 fibroblasts using polyethylene glycol . Erythrocyte-mediated microinjection of ' 25 1-proteins did not seriously perturb the metabolism of the recipient fibroblasts as assessed by measurements of rates of protein synthesis, rates of protein degradation, or rates of cellular growth after addition of fresh serum.A mixture of cytosolic proteins was degraded after microinjection according to expected characteristics established for catabolism of endogenous cytosolic proteins . Furthermore, withdrawal of serum, insulin, fibroblast growth factor, and dexamethasone from the culture medium increased the degradative rates of microinjected cytosolic proteins, and catabolism of long-lived proteins was preferentially enhanced with little or no effect on degradation of shortlived proteins . Six specific polypeptides were degraded after microinjection with markedly different half-lives ranging from 20 to 320 h . Degradative rates of certain purified proteins (but not others) were also increased in the absence of serum, insulin, fibroblast growth factor, and dexamethasone .The results suggest that erythrocyte-mediated microinjection is a valid approach for analysis of intracellular protein degradation. However, one potential limitation is that some microinjected proteins are structurally altered by the procedures required for labeling proteins to high specific radioactivities. Of the four purified proteins examined in this regard, only ribonuclease A consistently showed unaltered enzymatic activity and unaltered susceptibility to proteolytic attack in vitro after iodination .Intracellular protein degradation is a fundamentally important process occurring in all organisms from bacteria to humans (5,20,23,24,53) . The continued breakdown and replacement of proteins allows the cell to regulate concentrations of specific enzymes as well as to alter overall protein content in response to changing physiological demands. The major areas of current study in protein degradation can be divided into three broad topics : (a) the influence of polypeptide structure on protein half-lives (20, 23), (b) the physiological regulation of protein degradative rates (5,20,24), and (c) the mechanisms by which proteins are degraded within cells (5,24,53) . Despite considerable recent progress in each ofthese areas, many ofthe major questions in this field of research remain unanswered.Microinjection offers several advantages for analysis of protein degradation over more conventional approaches in which endogenous cellular proteins are radiolabeled and their deg- radative rates determined (I1, 34, 51). Microinjection of mammalian cells in culture has been achieved using microneedles (6,55) or by inducing fusion of the recipient cell with erythrocyte ghosts containing the protein to be microinjected (6,34,51) . Most studies of protein degradation have used erythrocytemediated microinjection because sufficient numbers of cells can be microinject...

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“…Enzymatic activities were measured as follows: amylase was determined according to Bernfeld (12); lipase was measured using a nephelometric procedure with olive oil emulsion (13); ribonuclease was determined according to Kalnitsky et al (14); tryptic activity was measured using TAME-HC1 as substrate (15); and chymotryptic activity was measured with N-benzoyl-L-tyrosine ethyl ester (BTEE) as substrate (16). Procarboxypeptidase A and B activities were assessed using hippuryl phenylalanine and hippuryl arginine, respectively (17).…”

Section: Measurement Of Actual and Potential Enzyme Activities Separated By Isoelectric Focusingmentioning

confidence: 99%

“…Amylase was present in two forms differing in isoelectric point on IEF gels and usually appeared as an apparent pair of doublets in SDS gels (spots [14][15][16][17]. Lipase activity was present in a minor acidic form (pI 4.3) and as a major acidic species (spot 11, pI 6.0).…”

Section: Characterization Of Discharged Secretory Proteins From Normal Rat Pancreasmentioning

confidence: 99%

Biochemical analysis of secretory proteins synthesized by normal rat pancreas and by pancreatic acinar tumor cells.

Iwanij

Jamieson

1982

The Journal of Cell Biology

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We have examined the secretogogue responsiveness and the pattern of secretory proteins produced by a transplantable rat pancreatic acinar cell tumor. Dispersed tumor cells were found to discharge secretory proteins in vitro when incubated with hormones that act on four different classes of receptors: carbamylcholine, caerulein, secretin-vasoactive intestinal peptide, and bombesin. With all hormones tested, maximal discharge from tumor cells was only about one-half that of control pancreatic Iobules, but occurred at the same dose optima except for secretin, whose dose optimum was 10-fold higher. Biochemical analysis of secretory proteins discharged by the tumor cells was carried out by crossed immunoelectrophoresis and by two-dimensional isoelectric focusing-SDS polyacrylamide gel electrophoresis. To establish a baseline for comparison, secretory proteins from normal rat pancreas were identified according to enzymatic activity and correlated with migration position on two-dimensional gels.Our results indicate that a group of basic polypeptides including proelastase, basic trypsinogen, basic chymotrypsinogen, and ribonuclease, two out of three forms of procarboxypeptidase B, and the major lipase species were greatly reduced or absent in tumor cell secretion. In contrast, the amount of acidic chymotrypsinogen was notably increased compared with normal acinar cells. Although the acinar tumor cells are highly differentiated cytologically and express functional receptors for several classes of pancreatic secretagogues, they show quantitative and qualitative differences when compared with normal pancreas with regard to their production of secretory proteins.The acinar cell of the mammalian pancreas is highly differentiated and adapted for the synthesis, packaging, and discharge of secretory proteins. The anatomic organization of organdies within the cell expedites the secretory process in that the protein synthetic apparatus is located in the basal pole of the cell while secretory granules accumulate in the apical pole. Under physiologic conditions, hormones interact with receptors located on the basal plasmalemma, which in turn leads to granule release at the apical membrane. Specialized regions of the plasma membrane may play a role in directing vectorial release of secretory proteins. Evidence for such specialization includes differences in intramembrane particle distribution between basolateral and apical membranes as shown by freezefracture electron microscopy (1) as well as by marked differences in distribution of lectin binding sites (2). As we have shown in previous papers (3, 4), rat pancreatic acinar tumor cells lack polarized organization of the plasmalemma as deter-734 mined by either freeze-fracture techniques or lectin binding. Though the acinar tumor ceils contain the cellular machinery for the production and packaging of secretory proteins, it is not known whether the lack of polar organization of the plasmalemma compromises their response to peptide secretagogues.In this investigation we sough...

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Some Factors Which Affect the Enzymatic Digestion of Ribonucleic Acid

Cited by 290 publications

References 13 publications

Structure- and Concentration-Specific Assessment of the Physiological Reactivity of α-Dicarbonyl Glucose Degradation Products in Peritoneal Dialysis Fluids

Structure- and Concentration-Specific Assessment of the Physiological Reactivity of α-Dicarbonyl Glucose Degradation Products in Peritoneal Dialysis Fluids

Degradation of proteins microinjected into IMR-90 human diploid fibroblasts.

Biochemical analysis of secretory proteins synthesized by normal rat pancreas and by pancreatic acinar tumor cells.

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