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PubMed1975

Functional characterization of membrane components of cytotoxic peritoneal exudate T lymphocytes. II. Trypsin sensitivity of the killer cell receptor.

Studies were carried out to investigate the nature of the lymphocyte membrane receptor involved in the specific lysis of [51Cr]EL4 target cells by BALB/c cytotoxic peritoneal exudate lymphocytes (PEL) in vitro. In confirmation of earlier related studies, the cytolytic reaction is markedly inhibited by trypsin treatment (1 mg of trypsin/10(6) PEL/ml; 37 C for 30 min) of the PEL. Trypsinized PEL recover their cytolytic activity after 6 hr of incubation in trypsin-free medium at 37 C; recovery is suppressed at 4 C. Significantly, trypsinization of PEL inhibits their ability to adsorb to EL4 target monolayers, indicating that lymphocyte-target cell binding is a trypsinsensitive step of the cytolytic interaction. Specificity of binding for EL4 is restored upon recovery from trypsin treatment. In addition, trypsin treatment, under conditions sufficient to inhibit cytolysis, causes a drastic nonspecific deletion of iodinatable membrane species from the surface of PEL. Thus, a direct correlation between altered cytolytic function and membrane structure cannot be made. Interestingly, trypsinized PEL retain the capacity to kill EL4 to which they are agglutinated by concanavalin-A, suggesting that the lytic mechanism, as distinguished from the binding receptor, is still intact. Papain and pronase also have an inhibitory effect on cytolysis, while neuraminidase causes a significant augmentation of lymphocyte-mediated cytolysis.

Animals↗
PubMed1989

Kunitz trypsin inhibitor genes are differentially expressed during the soybean life cycle and in transformed tobacco plants.

We investigated the structure, organization, and developmental regulation of soybean Kunitz trypsin inhibitor genes. The Kunitz trypsin inhibitor gene family contains at least 10 members, many of which are closely linked in tandem pairs. Three Kunitz trypsin inhibitor genes, designated as KTi1, KTi2, and KTi3, do not contain intervening sequences, and are expressed during embryogenesis and in the mature plant. The KTi1 and KTi2 genes have nearly identical nucleotide sequences, are expressed at different levels during embryogenesis, are represented in leaf, root, and stem mRNAs, and probably do not encode proteins with trypsin inhibitor activity. By contrast, the KTi3 gene has diverged 20% from the KTi1 and KTi2 genes, and encodes the prominent Kunitz trypsin inhibitor found in soybean seeds. The KTi3 gene has the highest expression level during embryogenesis, and is also represented in leaf mRNA. All three Kunitz trypsin inhibitor genes are regulated correctly in transformed tobacco plants. Our results suggest that Kunitz trypsin inhibitor genes contain different combinations of cis-control elements that program distinct qualitative and quantitative expression patterns during the soybean life cycle.

Amino Acid Sequence↗
PubMed1981

Effects of trypsin on the cyclic AMP system of the pig skin.

Trypsin increased cyclic AMP levels of the pig skin. This effect was markedly potentiated by the cyclic nucleotide phosphodiesterase inhibitor theophylline. Without theophylline this trypsin-induced cyclic AMP accumulation was transient and the maximal accumulation was noted by 5 min. Soybean trypsin inhibitor inhibited this trypsin-induced cyclic AMP accumulation. After the trypsin treatment, marked acantholysis was noted histologically and the decreased responsiveness to other adenylate cyclase stimulators was seen. The decrease of the epinephrine response was most marked and that of histamine response was much less. Both low and high Km cyclic nucleotide phosphodiesterase activities were decreased by the trypsin treatment. However, at 5 min incubation time, when the increase in cyclic AMp level was most marked, the decrease in the phosphodiesterase activities was minimal. Trypsin seems to reveal its action through the proteolytic activation of adenylate cyclase system of the skin.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

Trypsin does not reconstitute responsiveness to lipopolysaccharide in the strain C3H/HeJ, but is a B-cell mitogen-like lipopolysaccharide, stimulating a different subpopulation.

The effect of trypsin on mouse spleen cells and enriched B cells, added alone or together with lipopolysaccharide (LPS), was investigated. With trypsin, proliferation in serum free spleen cell cultures was 2-6 times greater than the background using cells from LPS responder strains, and 2-4 times the background with cells from the C3H/HeJ strain. Trypsin also induced the formation of a low number of IgM plaque forming cells (PFC). When added together with LPS, trypsin increased the proliferation caused by LPS alone by 10-50% with cells from LPS responder strains and by 50-100% with cells from the LPS non-responder strain C3H/HeJ. Trypsin enhanced proliferation in cultures maximally stimulated by LPS. The increased proliferation obtained when trypsin was added to LPS-stimulation of cells from the C3H/HeJ strain, was therefore not interpreted as a reconstitution of the LPS response. We conclude that trypsin has a moderate mitogenic effect on mouse B cells, stimulating the cells to proliferate and secrete IgM. The mechanism of action is unknown, but is different and independent from the action of LPS.

Animals↗
PubMed1976

Isolation of a trypsin-like enzyme from Streptomyces paromomycinus (paromotrypsin) by affinity adsorption through Kunitz inhibitor-sepharose.

A trypsin-like enzyme has been isolated from the filtrate of a Streptomyces rimosus forma paromomycinus culture. Purification involves acetone fractionated precipitation, ultrafiltration on a Diaflo UM 10 membrane and affinity adsorption on to Kunitz pancreatic trypsin inhibitor linked to Sepharose. The trypsin-like enzyme (paromotrypsin) appears homogeneous by zone electrophoresis on gelatinized cellulose acetate. Specific activity toward Tos-Arg-OMe, calculated from amino acid analysis, is about 220 mu mg-1. The overall yield in activity is about 30%. The molecular weight of the trypsin-like enzyme, determined by gel filtration, is around 22,000-25,000 daltons. Electrophoretic migration on cellulose acetate strips indicates an isoelectric point around 8. Amino acid composition has been determined; the protein comprises about 210 residues on the basis of a single histidine residue per molecule. Paromotrypsin is unstable in acidic medium and is not stabilized by calcium ions. Enzymic activity towards Bz-Argo-OEt is not increased by the addition of calcium ion in contrast to the activating effect observed on bovine trypsin. Paromotrypsin is inhbited by TLCK and NPGB; it interacts with naturally occurring bovine trypsin inhibitors such as soya bean and Kunitz pancreatic inhibitors, but not with chicken ovomucoid. Proteolytic specificity, examined by hydrolysis of oxidized Kunitz pancreatic inhibitor and characterization of resulting peptides, seems similar to that of bovine trypsin.

Adsorption↗
PubMed1978

Evidence for the inhibition of trypsin by thiols. The mechanism of enzyme-inhibitor complex formation.

Ehrlich ascites tumour cells contain a neutral protease, capable of solubilising fluorescein-labelled telopeptides from fluorescein-labelled polymeric collagen fibrils. The cells also contain an inhibitor for this enzyme and for trypsin. The enzymically inactive enzyme-inhibitor complex can be dissociated with the mercurial thiol agent, mersalyl, with the consequent regain of enzymic activity. The reactivated neutral protease and also trypsin can be inhibited by addition of thiols such as cysteine, mercaptoethanol and dithiothreitol. Trypsin can be protected from inactivation by the tumor inhibitor by addition of cystine or L-1-tosylamido-2-phenylethyl chloromethyl ketone(TosPheCH2Cl)-inactivated chymotrypsin. The evidence suggests that the inhibitor contains a reactive thiol group which exchanges with one or more significant disulphide bridges in trypsin and the neutral protease, resulting in enzyme-inhibitor complex formation and loss of activity. Similarly, thiols interact with these enzymes resulting in a corresponding loss of enzymic activity. The evidence obtained with Tos-PheCH2Cl-inactivated chymotrypsin, which reactivated previously inhibited trypsin and neutral protease, demonstrates that the active site of the enzyme is not involved in the interaction with the thiol of the inhibitor but that the significant disulphide bond in the enzyme is required for the maintenance of the active site conformation. This disulphide exchange mechanism is therefore a form of reversible allosteric control of proteolytic activity and has been shown to be distinct from the mechanism by which soya bean trypsin inhibitor interacts with trypsin.

Chymotrypsin↗
PubMed1985

Differential degradation of different benzodiazepine binding proteins by incubation of membranes from cerebellum or hippocampus with trypsin.

When rat brain membranes were incubated with [3H]flunitrazepam in the presence of UV light, predominantly one protein (P51) was irreversibly labeled in cerebellum and at least two proteins (P51 and P55) were labeled in hippocampus. On digestion of membranes with increasing concentrations of trypsin up to 40% of radioactivity irreversibly bound to proteins was removed from the membranes. In addition, P51 was nearly completely degraded to a peptide with apparent molecular weight 39,000 and this peptide was further degraded to a peptide with apparent molecular weight 25,000. In contrast, protein P55 was only partially degraded by trypsin and yielded two proteolytic peptides with apparent molecular weights 42,000 and 45,000 which seemed to be rather stable against further attack by trypsin. Membranes treated with trypsin still had the capacity to bind [3H]-flunitrazepam reversibly with an affinity similar to that of membranes not previously treated with trypsin. When these membranes were irradiated with UV light, the same proteolytic peptides were detected as in membranes first photolabeled and then digested with trypsin. These results suggest a close association between reversible and irreversible benzodiazepine binding sites and indicate that membrane-associated proteins P51 and P55 are differentially protected against degradation by trypsin.

Affinity Labels↗
PubMed1982

Inhibition of trypsin and papain by sodium aurothiomalate mediated by exchange reactions.

Sodium aurothiomalate has been shown to participate in exchange reactions leading to the inhibition of trypsin; for this exchange to take place it was necessary to include in the test system a suitable thiol, such as N-acetyl-cysteine. Neither N-acetyl-cysteine nor aurothiomalate on their own had any inhibitory action on trypsin. The results indicate that aurothiomalate dissociates in the presence of a carrier to form thiosuccinate and gold. The gold is responsible for trypsin inhibition since independent experiments demonstrated that the total concentration of thiosuccinate was insufficient to cause the observed inhibition of trypsin. Bovine serum albumin was shown to act as a carrier in place of N-acetyl-cysteine. It is known that histidine in the active centre of trypsin binds heavy metal ions with consequent inhibition of the enzyme. In this study, imidazole was shown to act as a carrier for gold from aurothiomalate to trypsin resulting in inhibition. This inhibition by gold was reversed when higher concentrations of imidazole were added to the test system due to competition for the trypsin-bound gold by imidazole. Conversely, the thiol enzyme papain was re-activated in the presence of low concentrations of sodium aurothiomalate and inhibited by higher concentrations of this reagent in a biphasic manner. This observation will be discussed in relation to the dissociation of sodium aurothiomalate. These observations can also be explained in terms of exchange reactions involving thiols and free metal ions.

Acetylcysteine↗
PubMed2001

A comparison of serum trypsinogen-2 and trypsin-2-alpha1-antitrypsin complex with lipase and amylase in the diagnosis and assessment of severity in the early phase of acute pancreatitis.

OBJECTIVE: The aim of the study was to compare the recently introduced laboratory markers trypsinogen-2 and trypsin-2-alpha1 antitrypsin complex (trypsin-2-AAT) in serum with lipase and amylase in the diagnostic and prognostic evaluation of patients with acute pancreatitis (AP). METHODS: The analytes were measured on admission in 64 consecutive patients with AP and in 30 controls with acute abdominal disease of extrapancreatic origin. Twenty-one patients had severe and 43 mild AP. As reference methods we used serum amylase and C-reactive protein. RESULTS: In subjects with AP, elevated trypsinogen-2 values (> or = 90 microg/L) were observed in 63 patients (98%), trypsin-2-AAT values (> or = 12 microg/L) in 64 patients (100%), lipase values (> or = 200 U/L) in 64 patients (100%), and amylase values (> or = 300 IU/L) in 62 patients (97%). The diagnostic accuracy of the markers was evaluated by receiver operating characteristic (ROC) analysis. On admission, trypsinogen-2, trypsin-2-AAT, lipase, and amylase differentiated patients with AP from controls with high accuracy and ROC analyses showed similar areas under the ROC curves (AUC) for trypsinogen-2 (AUC 0.960), trypsin-2-AAT (0.948), lipase (AUC 0.947), and amylase (AUC 0.930). For differentiation between severe and mild AP, trypsin-2-AAT (AUC 0.805) was slightly better than trypsinogen-2 (AUC 0.792), and they were both clearly better than lipase (AUC 0.583), C-reactive protein (AUC 0.519), or amylase (AUC 0.632) (p < 0.05). CONCLUSIONS: All the markers studied showed high accuracy for differentiating between AP and extrapancreatic diseases. However, trypsinogen-2 and trypsin-2-AAT displayed the best accuracy for predicting a severe AP already at admission, which makes these markers superior for clinical purposes.

Acute Disease↗
PubMed1987

Flow cytofluorometry of lysosomal acridine orange uptake by living cultured cells. Effect of trypsinization and starvation.

The uptake of the fluorescent, lysosomotropic weak base acridine orange (AO) by living cells in culture was studied by flow cytofluorometry. A mouse myeloma cell line (SP 2/0), growing in suspension, and an anchorage-dependent human malignant glioma cell line (U-251 MG), brought into suspension by trypsinization, were used. The consequences of trypsinization were also studied using static cytofluorometry. The lysosomal accumulation of AO by myeloma cells growing in suspension was found to be only moderately affected by starvation (i.e. incubation without medium change) for a period of up to five days. Trypsinization of the glioma cells after staining with AO caused pronounced release of the fluorescent dye while trypsinization before staining with AO did not significantly change the average lysosomal concentration of AO. We did, however, notice certain side effects of trypsinization in the form of both increased cellular green fluorescence and greater intercellular variability that reduce the validity of data obtained from cells detached by routine trypsinization. In conclusion, the condition of the lysosomal vacuome of living cultured cells growing in suspension may be studied by flow cytofluorometry after vital staining with the lysosomotropic weak base AO. Anchorage-dependent trypsinized cells, however, yield unsatisfactory results when examined in a flow cytofluorometer system and are better studied while still attached to their substratum, using static cytofluorometry.

Acridine Orange↗
PubMed1980

Stimulation of the sodium-potassium pump by trypsin in low potassium type erythrocytes of goats.

1. Treatment of low K goat red cells with trypsin stimulated the Na-K pump more than twofold. Dose dependence and time course experiments indicated a half-maximal stimulation at 1.6 mg trypsin/ml. (37 degrees C, 3 hr), and a maximum effect after 5 hr (10 mg/ml). 2. Trypsin had only a small and variable effect on the ouabain-insensitive component of K influx. 3. The Na-K pump activity of high K goat red cells was not affected by trypsinization. 4. When intracellular K was varied by the PCMBS technique, it was found that the trypsin stimulation was greatest (2-5-fold) in cells with the highest K (40 m-mole/1. cells) and lowest (1.1-fold) in cells with low K (<1 m-mole/1. cells). 5. The trypsin effect was reversed by nystatin treatment or hypotonic lysis. 6. Trypsin did not increase the number of ouabain-binding sites. 7. It is concluded that trypsinization modifies the L antigen in low K goat red cells to decrease the apparent internal affinity for K of the Na-K pump in these cells.

Animals↗
PubMed1971

Standardization of human diploid fibroblast cultivation: trypsinization procedure.

Human embryonic diploid lung fibroblasts were used to examine the influence of the trypsinizing procedure on the growth and adsorption of these cells. The best buffer for trypsinizing these cells was Hanks balanced salt solution containing 0.5% lactalbumin hydrolysate. Trypsinizing cells at 4 C gave better growth results than trypsinization at higher temperatures. The presence of antibiotics in the trypsin buffer increased the longevity of the cells. Cells initially trypsinized from tissue in phosphate-buffered saline without Ca(2+) or Mg(2+) and 0.33 m sucrose plus 10(-3)m Mg(2+) gave rise to better subsequent growth and adsorption than cells from tissue trypsinized in other buffers.

Adsorption↗
PubMed1996

Antibody-mediated inhibition of Aedes aegypti midgut trypsins blocks sporogonic development of Plasmodium gallinaceum.

The peritrophic matrix (PM) that forms around a blood meal is a potential barrier of Plasmodium development in mosquitoes. Previously, we have shown that to traverse the PM, Plasmodium ookinetes secrete a prochitinase and that an inhibitor of chitinase blocks further parasite development. Here we report that it is the mosquito trypsin that activates the Plasmodium prochitinase. Trypsin was identified as the chitinase-activating enzyme by two criteria: (i) trypsin activity and activating activity comigrated on one-dimensional gels, and (ii) activating activity and penetration of the PM by Plasmodium parasites were both hindered by trypsin-specific inhibitors. Subsequently, we examined the effect of antitrypsin antibodies on the parasite life cycle. Antibodies prepared against a recombinant blackfly trypsin effectively and specifically inhibited mosquito trypsin activity. Moreover, when incorporated into an infective blood meal, the antitrypsin antibodies blocked infectivity of Aedes aegypti mosquitoes by Plasmodium gallinaceum. This block of infectivity could be reversed by exogenously provided chitinase, strongly suggesting that the antibodies act by inhibiting prochitinase activation and not on the parasite itself. This work led to the identification of a mosquito antigen, i.e., midgut trypsin, as a novel target for blocking malaria transmission.

Aedes↗
PubMed1996

Trypsin activation pathway of rotavirus infectivity.

The infectivity of rotaviruses is increased by and most probably is dependent on trypsin treatment of the virus. This proteolytic treatment specifically cleaves VP4, the protein that forms the spikes on the surface of the virions, to polypeptides VP5 and VP8. This cleavage has been reported to occur in rotavirus SA114fM at two conserved, closely spaced arginine residues located at VP4 amino acids 241 and 247. In this work, we have characterized the VP4 cleavage products of rotavirus SA114S generated by in vitro treatment of the virus with increasing concentrations of trypsin and with proteases AspN and alpha-chymotrypsin. The VP8 and VP5 polypeptides were analyzed by gel electrophoresis and by Western blotting (immunoblotting) with antibodies raised to synthetic peptides that mimic the terminal regions of VP4 generated by the trypsin cleavage. It was shown that in addition to arginine residues 241 and 247, VP4 is cleaved at arginine residue 231. These three sites were found to have different susceptibilities to trypsin, Arg-241 > Arg-231 > Arg-247, with the enhancement of infectivity correlating with cleavage at Arg-247 rather than at Arg-231 or Arg-241. Proteases AspN and alpha-chymotrypsin cleaved VP4 at Asp-242 and Tyr-246, respectively, with no significant enhancement of infectivity, although this enhancement could be achieved by further treatment of the virus with trypsin. The VP4 end products of trypsin treatment were a homogeneous VP8 polypeptide comprising VP4 amino acids 1 to 231 and a heterogeneous VP5, which is formed by two polypeptide species (present at a ratio of approximately 1:5) as a result of cleavage at either Arg-241 or Arg-247. A pathway for the trypsin activation of rotavirus infectivity is proposed.

Amino Acid Sequence↗
PubMed1987

Pretreatment of human platelet membranes with trypsin abolishes GTP but not Na+ effects on alpha 2-adrenoreceptor-agonist interactions.

The affinity of many types of membrane-bound receptors coupled negatively to adenylate cyclase is regulated by divalent and monovalent cations and by guanine nucleotides (GTP). We used alpha 2-adrenoreceptors of human platelets as a model system to find out the effect of limited proteolysis with trypsin on the regulation of the alpha 2-adrenoreceptor-agonist interactions by GTP and Na+. We found that partial proteolysis of the membranes with trypsin for 3 min at 35 degrees C reduced specific [3H]yohimbine binding to platelet membranes to 40-50% of control. The following characteristics of the receptors remaining after proteolysis were similar to those of untreated membranes: affinity for the agonist and antagonists, stereospecificity, and kinetic properties. Trypsin also did not modify the ability of the receptor's change from a high to low affinity state in the presence of Na+. These findings suggested that the capability of the receptors to recognize the ligand and their ability to undergo a conformational change in the presence of the agonist were retained despite a reduction in the total number of receptors by trypsin. However, the modulation of the receptor--agonist interactions by GTP or Mg2+ was lost in the trypsin-pretreated membranes, while the modulation by Na+ remained intact. It is suggested that the loss of GTP or Mg2+ effects on receptor--ligand interactions produced by trypsin may be due to trypsin-induced disruption of subunits (alpha i, beta gamma) interactions of Gi protein.

Adrenergic alpha-Agonists↗
PubMed2004

Membrane-bound ICAM-1 is upregulated by trypsin and contributes to leukocyte migration in acute pancreatitis.

In acute pancreatitis, ICAM-1 is upregulated in various organs and contributes to the development of organ injury. To investigate the effects of pancreatic proteases on ICAM-1 expression and their role in the early process of leukocyte migration, human umbilical vein endothelial cells (HUVECs) were incubated with serum subjected to limited trypsin digestion and Wistar rats were injected with trypsin. Significant upregulation of membrane-bound ICAM-1 was seen on HUVECs incubated with trypsinated serum. Likewise, soluble ICAM-1 increased in the supernatant of HUVECs. Changes of membrane-bound ICAM-1 and soluble ICAM-1 were maximal with high concentrations of trypsin. HUVECs incubated with TNF-alpha (positive control) showed similar changes. In the pancreas and lungs of animals infused with trypsin, ICAM-1 and leukocyte sequestration were increased compared with controls. Reflecting the relevance of protease-induced ICAM-1 expression in leukocyte migration, leukocyte-endothelium interaction, as assessed by intravital microscopy, was markedly increased by trypsin. Inhibition of ICAM-1 ameliorated these changes significantly. In conclusion, trypsinated serum induces the upregulation of both membrane-bound ICAM-1 on endothelial cells and soluble ICAM-1. These changes contribute to the early steps of leukocyte migration in acute pancreatitis. The role of soluble ICAM-1 remains to be investigated.

Acute Disease↗
PubMed1995

Activities of trypsin and lipase in duodenal aspirates of healthy preterm infants: effects of gestational and postnatal age.

Lipase and trypsin activities were estimated in preprandially aspirated duodenal juice of preterm infants with gestational ages between 29 and 32 weeks (group I, n = 33) or between 33 and 36 weeks (group II, n = 22) during the first 6 weeks of postnatal life. The results were compared with the enzyme activities measured in 2- to 6-year-old children. There were no significant differences of the mean lipase or trypsin activities between the two groups lipase; group I 13.7 +/- 7.9, group II 15.9 +/- 9.8 U/ml; trypsin: group I 7.9 +/- 4.7, group II 8.5 +/- 5.1 U/ml). The activities of both enzymes increased significantly and similarly in both groups with postnatal age (lipase: group I r = 0.732, p < 0.01, group II r = 0.743, p < 0.01; trypsin: group I r = 0.705, p < 0.01, group II r = 0.669, p < 0.01). At the end of the study the lipase activities of both groups reached approximately 35% of the values found in the older children, whereas the trypsin activities reached the reference values within the 1st month of life. The results indicate an asynchronously age-related development of lipase and trypsin. The development of the lipase activity is delayed in comparison to the trypsin activity which should be considered in the nutritional management of preterm infants.

Aging↗
PubMed1978

Selective release of excreted DNA sequences from phytohemagglutinin-stimulated human peripheral blood lymphocytes. Effects of trypsin and divalent cations.

We studied the synthesis of excreted DNA sequences and their release from phytohemagglutinin-stimulated human peripheral blood lymphocytes under conditions permitting optimal cell growth. Cells were labeled by constant exposure to low specific activity [3H]thymidine. Excreted DNA sequences were synthesized during the period of logarithmic cell growth and moved slowly from the high molecular weight chromosomal DNA fraction into the low molecular weight cell DNA fraction (Hirt supernate) from which they could be specifically released by treating the cells briefly with small amounts of various proteases; 1 microgram/ml trypsin for 5 min was optimal. On day 5 of culture, 13.3 +/- 6.9% of the total cellular acid-precipitable [3H]thymidine was released by this treatment. Trypsin-induced release was partially and reversibly inhibited by incubating the cells for 16 h with 5 mM dibutyryl-cyclic AMP. Cells incubated in the absence of divalent cations spontaneously released this Hirt supernatant DNA; after maximal release had occurred under these circumstances, additional trypsin treatment caused no further release of DNA. Trypsin-induced DNA release could be completely and reversibly inhibited by incubating the cells in the presence of 10 mM calcium. Trypsin-released DNA was isolated and analyzed by reassociation kinetics. A major component, representing 54% of the DNA, reassociated with a C0t1/2 of 68 mol.s/liter (the value at which DNA association is 50% complete). The reassociation of this DNA was studied in the presence of an excess of DNA isolated from stimulated lymphocytes on day 3 in culture, and in the presence of an excess of resting lymphocyte DNA. The high molecular weight fraction of day-3 cell DNA contained three times more copies of the trypsin-released DNA major component as compared to resting lymphocyte DNA. Hirt supernatant DNA isolated from day-5 stimulated lymphocytes reassociated in an intermediate component representing 34% of the DNA with a Cot1/2 of mol.s/liter; after cells were treated with trypsin, this component could no longer be identified in the Hirt supernatant fraction, presumably because it had been released into the incubation medium. These data describe a quantitatively reproducible system with which synthesis and release of excreted DNA sequences can be studied.

Bucladesine↗