33-kDa C-terminal heparin binding fragment of fibronectin promotes attachment and spreading of hepatocytes

The possibility of interaction of hepatocytes with the heparin binding domain of Fibronectin was examined. Rat hepatocytes adhered to coverslips coated with the 33-kDa heparin binding fragment of the C-terminal region of plasma fibronectin. When different concentrations of the heparin binding fragment were used to coat coverslips and used as substratum, cell attachment showed saturation kinetics. Half the maximum attachment was observed at 30–40 min after seeding of cells. The cells became flat after 2–3 h indicating that they spread on the heparin binding domain as they do on intact fibronectin. Among the different glycosaminoglycans tested, maximum inhibition of attachment was observed for heparin. However it was not possible to completely inhibit attachment even at high concentrations. These results indicate that hepatocytes interact with fibronectin not only through the Arg-Gly-Asp-containing cell binding fragment, but also through the heparin binding domain of fibronectin and, further, that there exist heparin-dependent and heparin-independent mechanisms of interaction of cells with the 33-kDa heparin binding fragment of fibronectin     

Differential effects of heparin, fibronectin, and laminin on the phosphorylation of basic fibroblast growth factor by protein kinase C and the catalytic subunit of protein kinase A

Basic fibroblast growth factor (FGF) is synthesized as a phosphoprotein by both bovine capillary endothelial and human hepatoma cells in culture. Because basic FGF is characterized by its high affinity for heparin and its association in vivo with the extracellular matrix, we examined the possibility that the phosphorylation of this growth factor by purified protein kinase C (PK-C) and the catalytic subunit of cAMP- dependent protein kinase-A (PK-A) can be modulated by components of the extracellular matrix. Heparin and other glycosaminoglycans (GAGs) inhibit the ability of PK-C to phosphorylate basic FGF. In contrast, heparin can directly increase the phosphorylation of basic FGF by PK-A. While fibronectin, laminin, and collagen IV have no effect on the ability of PK-C to phosphorylate basic FGF, they all can inhibit the effects of PK-A. Thus, there is a differential effect of extracellular matrix-derived proteins and GAGs on the phosphorylation of basic FGF. The enhanced phosphorylation of basic FGF that is mediated by heparin is associated with a change in the kinetics of the reaction and the identity of the amino acid targeted by this enzyme. The amino acids that are targeted by PK-C and PK-A have been identified by phosphopeptide analyses as Ser64 and Thr112, respectively. In the presence of heparin, basic FGF is no longer phosphorylated by PK-A at the usual PK-A consensus site (Thr112), but instead is phosphorylated at the canonical PK-C site (Ser64). Accordingly, heparin inhibits the phosphorylation of basic FGF by PK-C presumably by masking the PK-C dependent consensus sequence surrounding Ser64. Thus, when basic FGF is no longer phosphorylated by PK-A in the receptor binding domain (Thr112), it loses the increased receptor binding ability that characterizes PK-A phosphorylated basic FGF. The results presented here demonstrate three novel features of basic FGF. First, they identify a functional effect of the binding of heparin to basic FGF. Second, they establish that the binding of heparin to basic FGF can induce structural changes that alter the substrate specificity of protein kinases. Third, and perhaps most important, the results demonstrate the existence of a novel interaction between basic FGF, fibronectin, and laminin. Although the physiological significance of this phosphorylation is not known, these results clearly suggest that the biological activities of basic FGF are regulated by a complex array of biochemical interactions with the proteins, proteoglycans, and glycosaminoglycans present in the extracellular milieu and the cytoplasm.     

Mutation of the H-helix in antithrombin decreases heparin stimulation of protease inhibition

Blood clotting proceeds through the sequential proteolytic activation of a series of serine proteases, culminating in thrombin cleaving fibrinogen into fibrin. The serine protease inhibitors (serpins) antithrombin (AT) and protein C inhibitor (PCI) both inhibit thrombin in a heparin-accelerated reaction. Heparin binds to the positively charged D-helix of AT and H-helix of PCI. The H-helix of AT is negatively charged, and it was mutated to contain neutral or positively charged residues to see if they contributed to heparin stimulation or protease specificity in AT. To assess the impact of the H-helix mutations on heparin stimulation in the absence of the known heparin-binding site, negative charges were also introduced in the D-helix of AT. AT with both positively charged H- and D-helices showed decreases in heparin stimulation of thrombin and factor Xa inhibition by 10- and 5-fold respectively, a decrease in affinity for heparin sepharose, and a shift in the heparin template curve. In the absence of a positively charged D-helix, changing the H-helix from neutral to positively charged increased heparin stimulation of thrombin inhibition 21-fold, increased heparin affinity and restored a normal maximal heparin concentration for inhibition.     

Preparation of sodium deoxycholate (DOC) conjugated heparin derivatives for inhibition of angiogenesis and cancer cell growth

We describe new DOC (sodium deoxycholate)-heparin nanoparticles for in vivo tumor targeting and inhibition of angiogenesis based on chemical conjugation and the enhanced permeability and retention (EPR) effect. Heparin has been used as a potent anticoagulant agent for 70 years, and has recently been found to inhibit the activity of growth factors which stimulate the smooth muscle cells around tumor. From the results, DOC and heparin were conjugated by bonding carboxyl groups of heparin with amine groups of aminated sodium deoxycholate. Larger antitumor effects of the DOC-heparin VI (8.5 mol of DOC coupled with 1.0 mol heparin) were achieved in animal studies, compared to heparin alone. We confirmed that the conjugated heparin retained its ability to inhibit binding with angiogenic factor, showing a significant decrease in endothelial tubular formation. These results provide new insights into the nontoxic anticancer drug carrier as well as the design of multifunctional bioconjugates for targeted drug delivery.     

The nature of the mesoderm-inducing signal in Xenopus: a transfilter induction study.

A transfilter apparatus is described, which is suitable for neutralization experiments on embryonic induction, and it is used to investigate the sensitivity of the Xenopus mesoderm-inducing signal to various inhibitors. The vegetal (inducing) tissue is placed on one side of a membrane sandwich and the animal (responding) tissue on the other side. The sandwich consists of a nylon gauze in between two Nucleopore filters and enables inhibitors in the solution to have effective access to the gap between the tissues. Control experiments show a high proportion of positive inductions of a ventral character. Using this apparatus, it is shown that the protein follistatin, which effectively inhibits activin A and B in vitro, has little or no effect on the natural signal. Likewise, antibodies to basic fibroblast growth factor, which inhibit in vitro, do not inhibit the natural signal. The two inhibitors together have a slight effect. It is concluded that neither activin nor bFGF are major components of the signal emitted by the vegetal cells of the Xenopus blastula and transmitted across the liquid gap, although they might have some other role to play in the process. Two agents of lower specificity do inhibit the transfilter induction: heparin and suramin. Suramin will also inhibit induction in animal-vegetal combinations with no intervening membranes while heparin does not. This suggests that the heparin inhibition can only occur when there is a liquid gap between the tissues, presumably because it can neutralize the signal in solution but cannot penetrate the explants themselves. The endogenous mesoderm-inducing factor(s) should therefore be sensitive to heparin in vitro.     

The mechanism of heparin stimulation of rat adipocyte lipoprotein lipase

Free fat cells and stromal-vascular cells were prepared from rat adipose tissue by incubation with collagenase. NH(4)OH-NH(4)Cl extracts of acetone-ether powders prepared from fat cells contained lipoprotein lipase activity but extracts of stromal-vascular cells did not. Intact fat cells released lipoprotein lipase activity into incubation medium, but intact stromal-vascular cells did not. The lipoprotein lipase activity of the medium was increased when fat cells were incubated with heparin, and this was accompanied by a corresponding decrease in the activity of subsequently prepared fat cell extracts. Heparin did not release lipoprotein lipase activity from stromal-vascular cells. The lipoprotein lipase activity of NH(4)OH-NH(4)Cl extracts of fat cell acetone powders is increased by the presence of heparin during the assay. This increase is not due to preservation of enzyme activity, but to increased binding of lipoprotein lipase to chylomicrons. Protamine sulfate and sodium chloride have little effect on the binding of lipoprotein lipase to chylomicrons, but they inhibit enzyme activity after binding to substrate has occurred. These inhibitors do, however, inhibit the stimulatory effect of heparin on enzyme-substrate binding.     

Metabolic effects of heparin on rat cervical epithelial cells

The glycosaminoglycan heparin inhibits the growth of a number of different cell types in vitro including smooth muscle cells, mesangial cells, fibroblasts, and rat cervical epithelial cells (RCEC). Studies investigating the antiproliferative effects of heparin on smooth muscle cells have demonstrated the site of the cell cycle block and revealed several metabolic alterations that could be causally associated with growth inhibition. We have investigated these metabolic parameters in RCEC to determine whether they are also associated with the antiproliferative effects of heparin in epithelial cells. Heparin acts rapidly to inhibit RCEC growth with inhibition detectable by autoradiography 7 h after the addition of heparin. Heparin treated RCEC begin to enter S-phase 12 h after the removal of heparin. These findings suggest that heparin blocks RCEC in the early-to-mid G1 phase of the cell cycle rather than late in G1 or early in S-phase as has previously been demonstrated for smooth muscle cells. Unlike smooth muscle cells, the uptake of thymidine and uridine is not inhibited by heparin in RCEC. Treatment of medium with heparin-Sepharose does not reduce the subsequent growth of RCEC; heparin inhibits the growth of RCEC in heparin-Sepharose treated medium in a manner identical to that in nontreated medium. Therefore the growth inhibitory effects of heparin cannot be explained by the inactivation of mitogens present in serum. In contrast to its effects on smooth muscle cells, heparin treatment of RCEC does not result in a reduction in the binding of epidermal growth factor (EGF) to the cells. These results indicate that although heparin inhibits the growth of a variety of cell types, significant differences exist in the responses of the different cells to heparin.     

肝素在肿瘤治疗中的应用

肝素作为传统抗凝剂已众所周知。研究发现,肝素尚具有多种生物学活性,特别是抗肿瘤作用备受学者关注。尽管临床上并未将肝素疗法作为一种常规抗肿瘤手段,但是许多研究已经证明了肝素能够抑制肿瘤细胞的侵袭与转移。本文综述肝素治疗肿瘤的主要机制以及肝素结构修饰在抗癌方面的应用。     

Inhibition of the beta-adrenergic receptor kinase by polyanions

The beta-adrenergic receptor kinase, which specifically phosphorylates the agonist-occupied beta-adrenergic receptor, is strongly inhibited by polyanions. Heparin and dextran sulfate inhibit the enzyme with an IC50 of approximately 0.15 microM. De-N-sulfated heparin is approximately 8-fold less potent. Other acid mucopolysaccharides such as heparan sulfate and chondroitin sulfates B and C are also less effective. Polyaspartic and polyglutamic acid also inhibit with IC50 values of 1.3-2 microM. Inositol hexasulfate, with an IC50 of 13 microM is approximately 270-fold more potent than inositol hexaphosphate implicating the sulfate group as a major determinant of the inhibition. The inhibition by heparin is competitive with substrate and of mixed type with respect to ATP. Polycations also inhibit receptor phosphorylation by beta-adrenergic receptor kinase. Polylysine is more effective with an IC50 of 69 microM, while spermine (990 microM) and spermidine (2570 microM) are less potent. Polylysine, spermine, and spermidine are also able to block effectively the inhibition by heparin. The identification of compounds which specifically inhibit beta-adrenergic receptor kinase should prove useful in further defining the biological role of this enzyme.     

肝素6位羧基修饰对抑制P?鄄选择素介导的A375细胞粘附活性的影响

已有的研究表明,肝素可以作为P-选择素的配体,显著抑制肿瘤转移过程中P-选择素介导的肿瘤细胞与血小板间的粘附.但是,肝素被P-选择素识别所必需的确切寡糖结构信息仍很缺乏.通过选择性化学修饰方法制备了2种低抗凝血肝素衍生物,即羧基还原肝素(CR-肝素)和羧基还原后再硫酸化肝素(SCR-肝素),系统地研究了它们对P-选择素介导的A375细胞粘附的抑制.研究结果表明,显著失去抗凝血活性的CR-肝素仍能有效地抑制P-选择素介导的A375细胞粘附,说明肝素的C6羧基并不是被P-选择素识别所必需的.而SCR-肝素所发生的C6羧基向羟甲硫酸酯基的转化却显著降低了抗粘附活性,说明P-选择素对肝素的识别并不只依赖于肝素的电荷密度.研究结果为深入阐明拮抗P-选择素介导的肿瘤细胞粘附的分子机制提供了有价值的实验基础.     

Heparin Oligosaccharides that Pass the Blood-Brain Barrier Inhibit β-Amyloid Precursor Protein Secretion and Heparin Binding to β-Amyloid Peptide

Abstract: We have previously demonstrated that full-length heparin stimulates the synthesis and secretion of β-amyloid precursor protein (APP) through an amyloidogenic pathway in neuroblastoma cells. In the present study, heparin was chemically depolymerized, and the effect of low-molecular-weight (LMW) heparin on APP secretion was investigated. In contrast to full-length heparin, LMW heparin had no significant effect on APP secretion. However, LMW heparin fragments, especially heparin disaccharides, were able to inhibit efficiently the stimulatory effect of heparin on APP secretion. LMW heparin derivatives also inhibit the binding of heparin to the β-amyloid peptide (1–28). Using an in vitro model, we further demonstrated the passage of LMW heparin derivatives through the blood-brain barrier. This study suggests that LMW heparin derivatives or analogues may be effective as therapeutic agents to prevent or slow the process of amyloidogenesis in Alzheimer's disease.     

Heparin activation of protein Z-dependent protease inhibitor (ZPI) allosterically blocks protein Z activation through an extended heparin-binding site

Protein Z (PZ)-dependent protease inhibitor (ZPI) is a plasma anticoagulant protein of the serpin superfamily, which is activated by its cofactor, PZ, to rapidly inhibit activated factor X (FXa) on a procoagulant membrane surface. ZPI is also activated by heparin to inhibit free FXa at a physiologically significant rate. Here, we show that heparin binding to ZPI antagonizes PZ binding to and activation of ZPI. Virtual docking of heparin to ZPI showed that a heparin-binding site near helix H close to the PZ-binding site as well as a previously mapped site in helix C was both favored. Alanine scanning mutagenesis of the helix H and helix C sites demonstrated that both sites were critical for heparin activation. The binding of heparin chains 72 to 5-saccharides in length to ZPI was similarly effective in antagonizing PZ binding and in inducing tryptophan fluorescence changes in ZPI. Heparin binding to variant ZPIs with either the helix C sites or the helix H sites mutated showed that heparin interaction with the higher affinity helix C site most distant from the PZ-binding site was sufficient to induce these tryptophan fluorescence changes. Together, these findings suggest that heparin binding to a site on ZPI extending from helix C to helix H promotes ZPI inhibition of FXa and allosterically antagonizes PZ binding to ZPI through long-range conformational changes. Heparin antagonism of PZ binding to ZPI may serve to spare limiting PZ and allow PZ and heparin cofactors to target FXa at different sites of action.     

Enhanced ability of heparin-carrying polystyrene (HCPS) to bind to heparin-binding growth factors and to inhibit growth factor-induced endothelial cell growth

Heparin-carrying polystyrene (HCPS) consists of low-molecular-weight heparin chains enriched in trisulfated disaccharide structures linked to a polystyrene core. In this study, the interactions between HCPSs of various molecular weights and heparin-binding growth factors, VEGF(165), FGF-2, and HGF, were compared to the interactions of the same factors with native heparin, periodate-oxidized heparin (IO(4)-heparin) and periodate-oxidized alkaline-degraded heparin (IO(4)-LMW-heparin). The binding of each growth factor to heparin-agarose beads (heparin-beads) was more strongly inhibited by HCPSs in a molecular weight-dependent manner than by native heparin or the modified heparins, indicating a stronger interaction between HCPS and these growth factors. HCPSs also inhibit heparin-binding growth factor-induced endothelial cell growth in a molecular weight-dependent manner much more strongly than the native or modified heparins. However, HCPSs did not inhibit the mitogenic activity of VEGF(121), which has a non-heparin-binding nature. Thus, HCPSs exhibit enhanced abilities to interact with each of the heparin-binding growth factors studied and to inhibit heparin-binding growth factor-induced endothelial cell proliferation in a molecular weight-dependent manner. These effects might be ascribed to the heparin-clustering effect of HCPSs.     

RhoA-derived peptide dimers share mechanistic properties with other polyanionic inhibitors of respiratory syncytial virus (RSV), including disruption of viral attachment and dependence on RSV G

Large polyanionic molecules, such as sulfated polysaccharides (including soluble heparin and dextran sulfate), synthetic polyanionic polymers, and negatively charged proteins, have been shown to broadly inhibit several enveloped viruses. We recently reported the antiviral activity of a peptide derived from amino acids 77 to 95 of a potential binding partner of respiratory syncytial virus F protein (RSV F), the GTPase RhoA. A subsequent study with a truncated peptide (amino acids 80 to 94) revealed that optimal antiviral activity required dimerization via intermolecular disulfide bonds. We report here that the net negative charge of this peptide is also a determining factor for its antiviral activity and that it, like other polyanions, inhibits virus attachment. In a flow cytometry-based binding assay, peptide 80-94, heparin, and dextran sulfate inhibited the attachment of virus to cells at 4 degrees C at the same effective concentrations at which they prevent viral infectivity. Interestingly, time-of-addition experiments revealed that peptide 80-94 and soluble heparin were also able to inhibit the infectivity of a virus that had been prebound to cells at 4 degrees C, as had previously been shown for dextran sulfate, suggesting a potential role for postattachment effects of polyanions on RSV entry. Neutralization experiments with recombinant viruses showed that the antiviral activities of peptide 80-94 and dextran sulfate were diminished in the absence of the RSV attachment glycoprotein (G). Taken together, these data indicate that the antiviral activity of RhoA-derived peptides is functionally similar to that of other polyanions, is dependent on RSV G, and does not specifically relate to a protein-protein interaction between F and RhoA.     

Hexasaccharides from the histamine-modified depolymerization of porcine intestinal mucosal heparin

Specific sequences in heparin are responsible for its modulation of the biological activity of proteins. As part of a program to characterize heparin-peptide and heparin-protein binding, we are studying the interaction of chemically discrete heparin-derived oligosaccharides with peptides and proteins. We report here the isolation and characterization, by one- and two-dimensional 1H NMR spectroscopies, of ten hexasaccharides, one pentasaccharide, and one octasaccharide serine that were isolated from depolymerized porcine intestinal mucosal heparin. Hexasaccharides were chosen for study because they fall within the size range, typically tetra- to decasaccharide in length, of heparin sequences that modulate the activity of proteins. The depolymerization reaction was catalyzed by heparinase I (EC 4.2.2.7) in the presence of histamine, which binds site specifically to heparin. Histamine increases both the rate and extent of heparinase I-catalyzed depolymerization of heparin. It is proposed that oligosaccharides produced by heparinase I-catalyzed depolymerization can inhibit the enzyme by binding to the imidazolium group of histidine-203, which together with cysteine-135 forms the catalytic domain of heparinase I. The increased rate and extent of depolymerization are attributed to competitive binding of the oligosaccharides by histamine.     

Mechanisms of inhibition by heparin of PDGF stimulated MAP kinase activation in vascular smooth muscle cells

Heparin and heparan are potent inhibitors of vascular smooth muscle cell (VSMC) proliferation. To investigate the mechanisms by which heparin suppresses growth factor stimulated mitogenesis, the present experiments investigated the effects of heparin on platelet-derived growth factor (PDGF) stimulated signal transduction pathways. Heparin treatment substantially inhibited PDGF-BB stimulated rat VSMC growth. Western analysis showed a 30 min PDGF-BB treatment of VSMC induced the tyrosine phosphorylation of multiple protein bands; cotreatment with heparin inhibited mitogen-activated protein (MAP) kinase tyrosine phosphorylation but had little effect on PDGF receptor tyrosine phosphorylation. In-gel kinase assays demonstrated that heparin inhibited PDGF-BB stimulated MAP kinase activity at late (25 min) but not early (10 min) time points. These data indicate that heparin does not inhibit the initial signalling events after PDGF-BB binding but instead acts through an alternate mechanism to inhibit MAP kinase. To investigate if heparin directly stimulates tyrosine phosphatase-mediated suppression of MAP kinase, we treated VSMC with orthovanadate, a tyrosine phosphatase inhibitor. Heparin inhibited MAP kinase tyrosine phosphorylation after orthovanadate treatment, indicating that heparin does not suppress MAP kinase by enlistment of a tyrosine phosphatase. Experiments were performed to investigate signalling pathways upstream of MAP kinase. To determine if protein kinase C (PKC) mediates PDGF-BB, serum, and EGF stimulation of MAP kinase, we treated VSMC overnight with phorbol ester (PMA) to downregulate PKC. Abolition of conventional and novel PKC activity significantly suppressed both serum and PDGF-BB induced MAP kinase activation, indicating protein kinase C is an important mediator for these mitogens. In contrast, downregulation of these PKC isoforms had little effect on EGF stimulation of MAP kinase. As heparin inhibits PDGF and serum but not EGF stimulation of MAP kinase, these data precisely correlate heparin inhibition of MAP kinase with activation through PKC-dependent pathways. Immunoprecipitation analysis found that heparin inhibited serum, PMA, and PDGF but not EGF induced raf-1 phosphorylation. These studies demonstrate that heparin did not block PDGF-BB receptor activation, which initiates the mitogenic signalling cascade. Heparin did inhibit specific postreceptor second messenger signals, such as the late phase activation of MAP kinase, which may be mediated by suppression of PKC-dependent pathways. J. Cell. Physiol. 172:69–78, 1997. © 1997 Wiley-Liss, Inc.     

Heparin suppresses specific second messenger pathways for protooncogene expression in rat vascular smooth muscle cells.

We investigated the molecular mechanisms underlying the ability of heparin to inhibit vascular smooth muscle cell (VSMC) growth. Previous experiments have shown that heparin inhibits induction of c-fos and c-myc protooncogene mRNA in rat VSMC stimulated by phorbol 12-myristate 13-acetate (PMA) but not when stimulated by epidermal growth factor (EGF) (Pukac, L. A., Castellot, J. J., Wright, T. C., Caleb, B. L., and Karnovsky, M. J. (1990) Cell Regul. 1, 435-443). The present experiments show that these mitogens activate distinct second messenger pathways in VSMC, because PMA but not EGF induction of c-fos and c-myc mRNA was suppressed in protein kinase C (PKC) down-regulated VSMC; this suggests that EGF does not act through a PKC-dependent pathway for induction of these genes. Heparin inhibited serum stimulation of c-fos mRNA in control VSMC, but heparin did not inhibit the smaller but significant serum stimulation of c-fos mRNA in PKC down-regulated VSMC, indicating that heparin may selectively inhibit PKC-dependent, but not PKC-independent, stimulation of gene expression. To further determine if heparin inhibits non-PKC pathways, VSMC were treated with dibutyryl cAMP, 3-isobutyl-1-methyl-xanthine, and Ca2+ ionophore A23187; stimulation of c-fos mRNA by this treatment was not inhibited by heparin. DNA synthesis and cell proliferation were inhibited in rat VSMC exposed briefly to heparin during the G0/G1 phase of the cell cycle. These experiments indicate heparin can act early in the cell cycle and suggest PKC-dependent but not PKC-independent signaling pathways for gene expression are selectively sensitive to heparin inhibition.     

Heparin inhibition of smooth muscle cell proliferation: a cellular site of action

The potential of a given amount of heparin to inhibit smooth muscle cell (SMC) proliferation can be increased more than 13 fold if quiescent cultures are pretreated with this mucopolysaccharide for 48 h. The large increase in antiproliferative activity was attributable to a 74% inhibition of the first cell cycle traverse of SMC after serum addition. If the mucopolysaccharide was added to SMC coincident with serum, the initial cell cycle traverse was only suppressed by 27%. In both heparin pretreated and nonpretreated SMC cultures, 48 to 72 h elapsed before substantial inhibition was observed. The inhibitory effects of heparin were reversible and inversely proportional to the starting cell density of the cultures. The effects of known heparin binding proteins on the inhibitory capability of heparin were examined. Neither platelet-derived growth factor (PDGF), low density lipoprotein (LDL), nor platelet factor 4 (PF4) were able to reduce the antiproliferative effects. Heparin retained full biological activity in medium containing serum depleted of all heparin binding proteins by heparin-Sepharose chromatography. These results indicate that heparin does not inhibit growth by preventing serum mitogens or nutrients from interacting with SMC. Rather, our data suggest that heparin is slowly internalized by SMC following binding to specific, non-PF4 dissociable sites. Heparin may accumulate intracellularly and block a crucial point in the proliferative machinery of SMC.     

Effect of heparin, spermidine and Be2+ ions on the phosphatase and RNAse activity of rat liver cell nuclei]

Effects of heparin, spermidine, and Be2+ ions on the ATPase and beta-glycerophosphatase and RNA-ase activities of the rat liver cell nuclei were studied. Be2+ was shown to inhibit the ATPase activity and, to a lesser extent, beta-glycerophosphatase activities. Physiological concentrations of heparin and spermidine also lowered the mentioned two activities, as well as the RNAase activity of the nuclei. Evidence is presented for the inhibitory effect of heparin and spermidine on endonucleases.