Production of cartilage-typic proteoglycans in cultures of chondrocytes from elastic cartilage

Chondrocytes from rabbit ear cartilage were isolated and cultured as monolayers in Ham's F-12 medium. The proteoglycans synthesized by short-term cultures formed a high proportion of aggregates and contained chrondroitin-4- and -6-sulfate in a 2:1 proportion. Dermatan sulfate was not present. The average molecular weight of the chondroitin sulfate was about 20,000. Keratan sulfate with an average molecular weight of about 6000 could be isolated from the proteoglycan monomers. Rabbit ear chondrocytes in culture thus produced proteoglycans comparable to those isolated from hyaline cartilage. Culture for longer periods and plating at lower density caused a decrease in the proportion of aggregated proteoglycans. Primary cultures continued to synthesize aggregated proteoglycans for at least 2 weeks, while subdivision of the cultures caused a shift toward the production of small-sized “ubiquitous proteoglycans.” The synthesis of proteoglycan aggregates could, however, be partly restored by transfer of the monolayer cells to a suspension culture.     

Effect of dibutyryl cyclic AMP on the sulphation of proteoglycans by chondrocytes

An intestinal lipodystrophy induced by dietary fat in female Mongolian gerbils (Meriones unguiculatus) was reversed to normal by myo-inositol given in the diet or by injection within 1-4 days. An increase in plasma chylomicron and lipid concentrations was observed before the occurrence of rapid disappearance of accumulated lipids from the intestine. Dietary myo-inositol also caused an increase in triacylglycerol release from everted gut sacs. Thus, myo-inositol might act on the intestine to stimulate the production and secretion of chylomicrons, travelling via the lymphatic pathway into the bloodstream. The activity of pH 9.0 microsomal lipase (EC 3.1.1.3) of gerbil intestine was decreased due to myo-inositol deficiency. The lowered activity could be restored to high levels by feeding of injecting myo-inositol in vivo. A time-course study during intestinal recovery indicates that the increase in microsomal lipase activity correlated with the rapid lipid removal phase of the intestine, but not the initial increase in plasma chylomicron and triacylglycerol concentrations.     

Aggregation of nucleosomes by divalent cations.

Conditions of precipitation of nucleosome core particles (NCP) by divalent cations (Ca(2+) and Mg(2+)) have been explored over a large range of nucleosome and cation concentrations. Precipitation of NCP occurs for a threshold of divalent cation concentration, and redissolution is observed for further addition of salt. The phase diagram looks similar to those obtained with DNA and synthetic polyelectrolytes in the presence of multivalent cations, which supports the idea that NCP/NCP interactions are driven by cation condensation. In the phase separation domain the effective charge of the aggregates was determined by measurements of their electrophoretic mobility. Aggregates formed in the presence of divalent cations (Mg(2+)) remain negatively charged over the whole concentration range. They turn positively charged when aggregation is induced by trivalent (spermidine) or tetravalent (spermine) cations. The higher the valency of the counterions, the more significant is the reversal of the effective charge of the aggregates. The sign of the effective charge has no influence on the aspect of the phase diagram. We discuss the possible reasons for this charge reversal in the light of actual theoretical approaches.     

Effect of divalent cations on the limited proteolysis of prothrombin by thrombin

The inhibitory influence of divalent cations on the ability of bovine alpha-thrombin to hydrolyze prothrombin showed the trend Mn2+ much greater than Ca2+ greater than or equal to Mg2+ greater than Sr2+ much greater than Ba2+. This effect was not due to an inhibition of thrombin's catalytic activity as measured by hydrolysis of a specific synthetic substrate, H-D-Phe-pipecolyl-Arg-p-nitroanilide (D-PhePipArgNA). The presence of divalent cations did not inhibit thrombic proteolysis of gamma-carboxyglutamic acid (Gla)-domainless prothrombin. Prothrombin and Gla-domainless prothrombin were used as competitive inhibitors in the thrombic hydrolysis of D-PhePipArgNA. The apparent Ki value calculated for prothrombin was 18 microM. When either Ca2+ or Mn2+ were present, there was no inhibition. The apparent Ki value determined for Gla-domainless prothrombin was 28 microM in either the absence or presence of Ca2+. Addition of divalent cations to prothrombin, but not to Gla-domainless prothrombin, resulted in an altered protein conformation as measured by high-performance size-exclusion chromatography and ultraviolet difference spectroscopy. These results suggest that a conformational change secondary to the interaction of divalent cations with the Gla-containing domain of prothrombin is required for cation-dependent inhibition of thrombin hydrolysis.     

The regulation of integrin function by divalent cations

Integrins are a family of α/β heterodimeric adhesion metalloprotein receptors and their functions are highly dependent on and regulated by different divalent cations. Recently advanced studies have revolutionized our perception of integrin metal ion-binding sites and their specific functions. Ligand binding to integrins is bridged by a divalent cation bound at the MIDAS motif on top of either α I domain in I domain-containing integrins or β I domain in α I domain-less integrins. The MIDAS motif in β I domain is flanked by ADMIDAS and SyMBS, the other two crucial metal ion binding sites playing pivotal roles in the regulation of integrin affinity and bidirectional signaling across the plasma membrane. The β-propeller domain of α subunit contains three or four β-hairpin loop-like Ca²⁺-binding motifs that have essential roles in integrin biogenesis. The function of another Ca²⁺-binding motif located at the genu of α subunit remains elusive. Here, we provide an overview of the integrin metal ion-binding sites and discuss their roles in the regulation of integrin functions.     

The regulation of integrin function by divalent cations

Integrins are a family of α/β heterodimeric adhesion metalloprotein receptors and their functions are highly dependent on and regulated by different divalent cations. Recently advanced studies have revolutionized our perception of integrin metal ion-binding sites and their specific functions. Ligand binding to integrins is bridged by a divalent cation bound at the MIDAS motif on top of either α I domain in I domain-containing integrins or β I domain in α I domain-less integrins. The MIDAS motif in β I domain is flanked by ADMIDAS and SyMBS, the other two crucial metal ion binding sites playing pivotal roles in the regulation of integrin affinity and bidirectional signaling across the plasma membrane. The β-propeller domain of α subunit contains three or four β-hairpin loop-like Ca²⁺-binding motifs that have essential roles in integrin biogenesis. The function of another Ca²⁺-binding motif located at the genu of α subunit remains elusive. Here, we provide an overview of the integrin metal ion-binding sites and discuss their roles in the regulation of integrin functions.     

Regulation of smooth muscle phosphatase-II by divalent cations

Summary Smooth Muscle Phosphatases II (SMP-I1) which has been purified from turkey gizzards and previously classified as protein phosphatase 2C, is inactive in the absence of divalent cations. Study of the activation of SMP-II by Mg²⁺ and Mn²⁺ revealed differences in the modes of activation by these cations. The maximal activation elicited by Mg²⁺ is 1.5–2.5-fold higher than the maximal Mn²⁺ activation. However, the latter is achieved at a lower concentration than the maximal Mg²⁺-activation. Furthermore, at low cation concentrations ( 2 mM), the Mn²⁺-activated activity is higher than the Mg²⁺-activated activity. In the presence of both cations, the effect of Mn²⁺ predominates suggesting that the affinity of the enzyme for Mn²⁺ is greater than for Mg²⁺. In contrast to Mg²⁺ and Mn²⁺, Ca²⁺ does not activate SMP-II but it was observed to antagonize the effects of Mg²⁺ and Mn²⁺. Ca²⁺ acts as a competitive inhibitor of Mg²⁺. However, the inhibitory effect at high Ca²⁺ concentrations is not completely reversed by increasing the Mg²⁺ concentration. Mn²⁺ activation is also inhibited by Ca²⁺ but to a lesser extent. Ca²⁺ cannot completely inhibit Mn²⁺-activation suggesting that SMP-I1 has greater affinity for Mn²⁺ than for Ca²⁺. The finding that Ca²⁺ inhibits the activation of SMP-II raises the possibility that Ca²⁺ may be a regulator of SMP-II in vivo.Abbreviations SMP-II Smooth Muscle Phosphatase-II - MOPS 3-[N-Morpholine]propane Sulfonic Acid - PLC Phosphorylated Myosin Light Chains     

Effect of ascorbic acid on secreted proteoglycans from rabbit articular chondrocytes

Addition of ascorbic acid (25, 50 100 micrograms/ml) to cultures of rabbit articular chondrocytes did not change the total amount of proteoglycans produced. However, it induced an increased retention of these macromolecules in the pericellular fraction. The size of the proteoglycan subunits and the length of glycosaminoglycan chains, released in the medium, were not modified on exposure to ascorbic acid (25 micrograms/ml). On the other hand, the rate of non-sulfated chondroitin was increased 2.5-fold, whereas chondroitin-4-sulfate was depressed 1.5-fold.     

Effect of divalent cations on malformin-induced efflux and biological activity

The effect of malformin on permeability was determined by efflux.Malformin increased membrane permeability to various organicand inorganic compounds or ions and was inhibited by severaldivalent cations. From the time required to increase permeabilityand the amount of material effluxed malformin primarily increasedtonoplast permeability. Calcium inhibited malformin-inducedstem collapse, abscission, and to a slight extent, inhibitionof primary leaf expansion. Although calcium inhibited malformin-inducedroot curvatures, malformin did not enhance permeability of rootslices at concentrations optimal for the curvature response. (Received May 6, 1975; )     

Modulation of potassium channel gating by external divalent cations

We have examined the actions of Zn2+ ions on Shaker K channels. We found that low (100 microM) concentrations of Zn2+ produced a substantial (approximately three-fold) slowing of the kinetics of macroscopic activation and inactivation. Channel deactivation was much less affected. These results were obtained in the presence of 5 mM Mg2+ and 4 mM Ca2+ in the external solution and so are unlikely to be due to modification of membrane surface charges. Furthermore, the action of 100 microM Zn2+ on activation was equivalent to a 70-mV reduction of a negative surface potential whereas the effects on deactivation would require a 15-mV increase in surface potential. External H+ ions reduced the Zn-induced slowing of macroscopic activation with an apparent pK of 7.3. Treatment of Shaker K channels with the amino group reagent, trinitrobenzene sulfonic acid (TNBS), substantially reduced the effects of Zn2+. All these results are qualitatively similar to the actions of Zn2+ on squid K channels, indicating that the binding site may be a common motif in potassium channels. Studies of single Shaker channel properties showed that Zn2+ ions had little or no effect on the open channel current level or on the open channel lifetime. Rather, Zn2+ substantially delayed the time to first channel opening. Thus, K channels appear to contain a site to which divalent cations bind and in so doing act to slow one or more of the rate constants controlling transitions among closed conformational states of the channel.     

Regulation of the fibronectin receptor affinity by divalent cations

The cell surface receptor for fibronectin is a heterodimeric membrane protein that recognizes an Arg-Gly-Asp sequence in fibronectin and that requires cations such as Mg2+ or Ca2+ for binding to fibronectin. The divalent cation requirements of this receptor were analyzed by measuring attachment of receptor liposomes to ligand-coated surfaces in the presence of different cations. The most striking effect observed was produced by Mn2+, which increased the binding of the receptor liposomes to fibronectin 2-3-fold over their binding in buffers containing Ca2+ and Mg2+. The binding activities of two related adhesion receptors, the vitronectin receptor and platelet GP IIb-IIIa, were supported but not enhanced by Mn2+. Two observations suggest that Mn2+ can compete with Ca2+ for the same cation-binding sites of the receptor. First, Mn2+ could still enhance fibronectin receptor binding activity even in the presence of 10-fold higher concentrations of Ca2+ or Mg2+. Second, Mn2+ inhibited the binding of radioactive Ca2+ to the alpha subunit of the receptor. The increased fibronectin receptor activity in the presence of Mn2+ appeared to be due to an increase in the affinity of the receptor for the Arg-Gly-Asp sequence because a 110-kDa cell attachment fragment and a synthetic hexapeptide containing the Arg-Gly-Asp sequence inhibited liposome binding more effectively in the presence of Mn2+ than in the presence of Ca2+/Mg2+. The affinity for the peptide was affected more than the affinity for the fragment, indicating that Mn2+ also induces a change in receptor specificity. Increased receptor binding in the presence of Mn2+ was also apparent in affinity chromatography of the fibronectin receptor on the 110-kDa fibronectin fragment; Mn2+ improved the yield of the receptor 4-fold. Mn2+ similarly increased the number of receptor-fibronectin complexes in preparations analyzed by electron microscopy. These results show that exogenous influences can modulate the affinity and specificity with which the fibronectin receptor binds to its ligands.