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CHARACTERIZATION OF THE INTERACTIONS OF TYPE XII COLLAGEN WITH TWO SMALL PROTEOGLYCANS FROM FETAL BOVINE TENDON, DECORIN AND FIBROMODULIN

In addition to the major collagens, such as type I or type II, connective tissues contain a number of less abundant collagens and proteoglycans, whose association contributes to the different properties of the tissues. Type XII and type XIV collagens have been described in soft connective tissues, and type XIV collagen has been shown to interact specifically with decorin through its glycosaminoglycan chain (Font et al., J. Biol. Chem. 268, 25015-25018, 1993). Interactions between these collagens and the small proteoglycans have been characterized further by studying the binding of type XII collagen to decorin by solid phase assays. Our results show a saturable binding of the proteoglycan through its glycosaminoglycan chain to type XII collagen, which does not seem to involve the large non-collagenous NC3 domain of the molecule. This interaction is strongly inhibited by heparin. Furthermore, we report that another small proteoglycan, fibromodulin, isolated from tendon under non-denaturing conditions, is able to bind to type XII collagen. This interaction has been characterized and, unlike that observed with decorin, type XII collagen-fibromodulin interaction seems to take place with the core protein of the proteoglycan. In addition, we report that type XII-type I collagen interactions are not necessarily mediated by decorin as previously suggested.

Heart mitochondrial creatine kinase solubilization: Effect of mitochondrial swelling and SH group reagents

Abstract The influence of mitochondrial swelling on the binding of creatine kinase to the inner membrane of pig heart mitochondria has been studied. Creatine kinase is not solubilized in isotonic mediums such as 250 m m sucrose or 125 m m KCl which do not induce mitochondrial swelling; however, it is solubilized when mitochondria swell in 100 m m potassium phosphate. This latter effect is suppressed when swelling is prevented by respiratory inhibitors or mersalyl. In hypotonic mediums creatine kinase is strongly released only when permeant anions are present; despite a high degree of swelling creatine kinase dissociation is minimal in 25 m m sucrose or KCl. Solubilization may also be obtained when swelling is induced by P j , accumulation inside mitochondria or when, after an osmotic shock in distilled water, they are resuspended in potassium phosphate or chloride but not in sucrose. Creatine kinase may be strongly released in isotonic KCl but not in sucrose when mitochondria are treated with parahydroxymercuribenzoic acid which induces swelling in the former medium (by allowing Cl − entry). A similar effect was obtained with other organic mercurials (parachloromercuriphenyl sulfonic acid, mersalyl) but not with disulfides (5,5′-dithiobis(2-nitrobenzoic acid), 6,6′-dithiodinicotinic acid, 2,2′-dithiodipyridine). Mercurials are able to solubilize creatine kinase from mitochondria suspended in distilled water so it is apparent that they have a direct effect on the association of the enzyme with the mitochondrial inner membrane. These results show that mitochondrial swelling is a prerequisite but not a sufficient condition for creatine kinase dissociation: the presence of ions or mercurials seems necessary for an efficient creatine kinase Solubilization.

Rabbit heart mitochondrial hexokinase: Solubilization and general properties

In rabbit heart, results show that two isoenzymes of hexokinase (HK) are present. The enzymatic activity associated with mitochondria consists of only one isoenzyme; according to its electrophoretic mobility and its apparent Km for glucose (0.065 mM), it has been identified as type I isoenzyme. The bound HK I exhibits a lower apparent Km for ATPMg than the solubilized enzyme, whereas the apparent Km for glucose is the same for bound and solubilized HK. Detailed studies have been performed to investigate the interactions which take place between the enzyme and the mitochondrial membrane. Neutral salts efficiently solubilize the bound enzyme. Digitonin induces only a partial release of the enzyme bound to mitochondria; this result could be explained by the existence of contacts between the outer and the inner mitochondrial membranes [C. R. Hackenbrock (1968) Proc. Natl. Acad. Sci. USA 61, 598-605]. Furthermore, low concentrations (0.1 mM) of glucose 6-phosphate (G6P) or ATP4- specifically solubilize hexokinase. The solubilizing effect of G6P and ATP4-, which are potent inhibitors of the enzyme, can be prevented by incubation of mitochondria with Pi or Mg2+. In addition, enzyme solubilization by G6P can be reversed by Mg2+ only when the proteolytic treatment of the heart homogenate is omitted during the course of the isolation of mitochondria. These results concerning the interaction of rabbit heart hexokinase with the outer mitochondrial membrane agree with the schematic model proposed by Wilson [(1982) Biophys. J. 37, 18-19] for the brain enzyme. This model involves the existence of two kinds of interactions between HK and mitochondria; a very specific one with the hexokinase-binding protein of the outer mitochondrial membrane, which is suppressed by glucose 6-phosphate, and a less specific, cation-mediated one.

Dissociation and reassociation of creatine kinase with heart mitochondria; pH and phosphate dependence.

Abstract Experimental evidence is given of mitochondrial creatine kinase ability to dissociate from or reassociate with mitochondrial membrane as compared to the behaviour of adenylate kinase. CK release occurs for P i concentrations higher than 5 mM and is strongly pH-dependant. Solubilized CK is able to reassociate with mitochondrial inner membrane when either P i concentration or pH are decreased. The possible physiological effects of events, such as ischemia, which modify the intracellular pH or P i concentration are discussed, in view of the special role which has been attributed to mitochondrial CK in the transfer of energy in heart cells.

Interaction of creatine kinase with phosphorylating rabbit heart mitochondria and mitoplasts

This paper demonstrates that the mitochondrial isoenzyme of creatine kinase (CKm) can be solubilized from rabbit heart mitochondria, the outer membrane of which has been removed or at least broken by a digitonin treatment or a short hypotonic exposure, but which has retained an important part of the capacity to phosphorylate ADP. Phosphate, ADP, or ATP, at concentrations which are used to study oxidative phosphorylation and creatine phosphate synthesis, solubilize CKm; the same is true with MgCl2 and KCl. The effect of adenine nucleotides does not seem to be due to their interaction with the adenine nucleotide translocase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that CKm is the main protein released in the described conditions; however, it does not amount to more than 1% of the total protein content of the mitoplasts. When the apparent Km for ATP of CKm was estimated by measuring creatine phosphate synthesis, the values obtained using water-treated mitochondria (0.21 mM) were slightly higher than those of intact mitochondria (0.12 mM) but the difference was not significant. In the former preparation 77% of CKm was in a soluble state. If we can extrapolate these results to intact mitochondria and suppose that in this case a fraction of CKm is also soluble in the intermembrane space, this does not support the theory of functional association between CKm and the adenine nucleotide translocase.

Effects of SH group reagents on creatine kinase interaction with the mitochondrial membrane

Solubilization of the specific mitochondrial isoenzyme of creatine kinase (CKm) from rabbit heart mitochondria by treatment with SH group reagents has been studied. From the various compounds tested only the negatively charged organomercurials are able to induce an extensive solubilization of the enzyme. This effect is fully reversible since the solubilized enzyme readily reassociates with the membrane when the bound organomercurial is removed by treatment of the homogenate by an excess of dithiothreitol. Solubilization by negatively charged organomercurials can be partly prevented by pretreatment of mitochondria with either disulfide or uncharged organomercurials. No clear-cut relationship has been pointed out when the amount of SH titrated by various reagents has been compared with the extent of CKm solubilization. More detailed studies with para-chloromercuribenzoate (pCMB) show that extensive CKm solubilization (about 75%) occurs for pCMB concentration as low as 25 microM, whereas pronounced inhibition of the enzyme is observed only for concentrations greater than 200 microM. By cross-reassociation of enzyme solubilized either by para-hydroxymercuribenzoate (pHMB) or by 20 mM sodium phosphate (NaPi) with mitochondria depleted of CKm by pHMB or by NaPi treatment, SH groups whose titration impedes CKm reassociation with the mitochondrial membrane have been tentatively located on the enzyme. Thus, negatively charged organomercurials, could induce a reversible conformational modification of the enzyme which is no longer able to bind on the inner mitochondrial membrane. Furthermore, our results show that the binding of an excess of mitochondrial CK, which has been previously reported, could reflect unspecific binding since it occurs only on mitoplasts incubated in very hypotonic medium, but not in isotonic medium.

Intracellular and submitochondrial localization of pig heart hexokinase.

In the cardiac muscle, during aerobiosis, the regulation of energetic metabolism is mainly linked to the availability of ADP which stimulates oxidative phosphorylation. Several mitochondrial enzymes provide ADP, among them creatine kinase and adenylate kinase which have been studied in our laboratory, seem to have a very important role in ADP supply and in energy transfer. Bessman [l] has suggested that hexokinase could be an important parameter in the regulation of oxidative phosphorylation because of its acceptor effect, esnecially if it is associated with ‘mitochondria. In the present paper, the subcellular distribution of hexokinase in pig heart muscle was studied by fractional extraction and differential centrifugation. It was proved that a significant fraction is firmly bound to mitochondria. The intramitochondrial localization has also been determined. During the purification of submitochondrial fractions, the specific activity of hexokinase increased by 1 S-fold in purified outer membranes and did not increase in other fractions. The De Duve’s plot [2] and comparisons with specific activities of other marker enzymes prove the association of hexokinase with outer membranes. Kinetic parameters of this mitochondrial hexokinase were determined.

Procollagen C-proteinase enhancer-1 (PCPE-1) interacts with β2-microglobulin (β2-m) and may help initiate β2-m amyloid fibril formation in connective tissues

Dialysis related amyloidosis (DRA) is a progressive and serious complication in patients under long-term hemodialysis and mainly leads to osteo-articular diseases. Although beta(2)-microglobulin (beta2-m) is the major structural component of beta2-m amyloid fibrils, the initiation of amyloid formation is not clearly understood. Here, we have identified procollagen C-proteinase enhancer-1 (PCPE-1) as a new interacting protein with beta2-m by screening a human synovium cDNA library. The interaction of beta2-m with full-length PCPE-1 was confirmed by immunoprecipitation, solid-phase binding and pull-down assays. By yeast two-hybrid analysis and pull-down assay, beta2-m appeared to interact with PCPE-1 via the NTR (netrin-like) domain and not via the CUB (C1r/C1s, Uegf and BMP-1) domain region. In synovial tissues derived from hemodialysis patients with DRA, beta2-m co-localized and formed a complex with PCPE-1. beta2-m did not alter the basal activity of bone morphogenetic protein-1/procollagen C-proteinase (BMP-1/PCP) nor BMP-1/PCP activity enhanced by PCPE-1. PCPE-1 did not stimulate beta2-m amyloid fibril formation from monomeric beta2-m in vitro under acidic and neutral conditions as revealed by thioflavin T fluorescence spectroscopy and electron microscopy. Since PCPE-1 is abundantly expressed in connective tissues rich in type I collagen, it may be involved in the initial accumulation of beta2-m in selected tissues such as tendon, synovium and bone. Furthermore, since such preferential deposition of beta2-m may be linked to subsequent beta2-m amyloid fibril formation, the disruption of the interaction between beta2-m and PCPE-1 may prevent beta2-m amyloid fibril formation and therefore PCPE-1 could be a new target for the treatment of DRA.

A 28 kDa mitochondrial protein is radiolabelled by crosslinking with a 123I-labelled presequence

A 13‐residue peptide containing the first 12 amino acids of the N‐terminal part of the signal sequence of yeast cytochrome ???oxidase subunit IV is shown by chemical crosslinking to interact with a mitochondrial protein. This result is obtained with mitochondria from four different origins. Submitochondrial localization experiments suggest that the 28 kDa labelled component is present on the outer face of the inner membrane. Since such addressing peptides are imported into mitochondria through the same machinery as protein precursors, the 28 kDa protein might be a component of the translocation apparatus.

Modified properties of hexokinase from heart mitochondria prepared using proteolytic enzyme

SummaryIsolation of muscle mitochondria is made easier by using proteolytic treatment of the tissue before homogenization. Normally, the proteolytic enzyme is discarded with the supernatant of the first centrifugation. However, our results show that a fraction of enzyme activity remains associated with mitochondria. As shown in experiments described in this paper, mitochondrial hexokinase from tissue treated or not with the proteolytic enzyme exhibits similar properties except that the solubilized enzyme from protease treated tissue is no longer able to rebind to mitochondrial membrane. This modification of the binding ability of the enzyme results from a partial hydrolysis of hexokinase during solubilization experiments by the proteolytic enzyme.Since, as pointed out here, proteolytic enzyme can remain associated with mitochondria, [either adsorbed on mitochondrial membrane or included in the mitochondrial pellet] its use for the isolation of muscle mitochondria should be avoided.