[The role of fibronectin and possible participation of its proteolytic fragments in the changes in cell behavior]. / Rol' fibronektina i vozmozhnoe ychastie ego proteoliticheskikh fragmentov v izmenenii povedeniia kletok.

The paper deals with a potential role of fibronectin proteolysis associated with plasma cell membrane receptors in the control of cell behaviour. The molecule of fibronectin contains at least 5 adhesive domains providing its interaction with cell receptors and at least 2 domains interacting with other molecules of an extracellular matrix (ECM). Different cells in various states (steady state, motion, proliferation) interact with all or some of the adhesive domains of fibronectin. Limited fibronectin proteolysis as a linking between the cell and ECM results in a change in the cell status. Limited proteolysis of cell-bound fibronectin may occur with several proteinases: 1) uPA having a receptor in the focal contact of a cell; 2) plasmin resulted from plasminogen under the action of uPA; 3) stromelysin whose synthesis is induced by fibronectin proteolytic fragments; 4) metalloproteinases secreted by some cells and involving in the hapatotactic motion of a cell over fibronectin. Proteolysis of fibronectin and other ECM molecules may be inhibited itself due to proteolysis-induced release of inhibitors via binding to fibronectin (proteasonexin) and via binding to other ECM molecules (PAI-1). The fact that there is a direct and inverse correlation in the proteolytic process associated with a fibronectin cell (and other ECM molecules) indicate that the behavior of a cell can be controlled by the mechanism of proteolytic impairment of the cell-EMC and cell-cell bonds.

[Isolation of fibronectin peptide fragments of various sizes and biological activity using affinity chromatography on an immobilized plasmin]. / Vydelenie razlichaiushchikhsia po razmeram i biologicheskoi aktivnosti peptidnykh fragmentov fibronektina s pomoshch'iu affinnoi khromatografii na immobilizovannom plazmine.

Plasmin, immobilized on Sepharose, was used for isolation of human blood plasma fibronectin fragments obtained after proteolysis. Under definite conditions the major part of the fibronectin fragments, liberated during proteolysis, remained to be bound to plasmin-Sepharose. As shown by electrophoretic analysis, the fraction of fragments bound to plasmin-Sepharose constituted mainly "heavy" (greater than or equal to 120 kD) peptides and one "light" (29 kD) peptide, while only "light" fragments (less than or equal to 45 kD) were detected in the free unbound fraction. These unbound to plasmin-Sepharose fibronectin fragments were found to stimulate proliferation of human embryonal fibroblasts in cell culture, whereas the plasmin-Sepharose bound peptides did not exhibit any effects on proliferation.

[The effect of fibronectin fragments on proliferative activity of fibroblasts]. / Vliianie fragmentov fibronektina na proliferativnuiu aktivnost' fibroblastov.

The effects of fibronectin and fragments of its limited proteolysis by plasmin on the proliferative activity of human embryo fibroblasts in culture were studied. It was found that native fibronectin and its fragments with Mr greater than or equal to 120 kD do not exert either a stimulating or inhibiting influence, whereas the 15-43 kD fragments significantly stimulate cell proliferation. The stimulating effect increases with a rise in the fragment concentration, reaching a maximum at 12-25 micrograms/ml and decreases at their higher concentrations. The preparation of proliferation-stimulating fragments contains no proteinases admixtures that are active at neutral pH and does not possess any intrinsic proteolytic activity. The proliferation-stimulating activity does not change after removal of collagen-binding fragments.

[Fibronectin fragmentation unmasks the activity stimulating DNA and RNA biosynthesis in granulation tissue cells in vitro]. / Fragmentirovanie fibronektina demaskiruet aktivnost', stimuliruiushchuiu sintez DNK i RNK v kletkakh granulatsionnoi tkani in vitro.

Human blood plasma fibronectin decreased slightly the incorporation of precursors into nucleic acids of granulation tissue culture cells. A slight fragmentation of fibronectin, where the fragments with 180-200 kD molecular mass were developed, led to occurrence of the activity 2-fold stimulating the DNA synthesis. After more effective proteolysis using plasmin and trypsin the stimulating effect of fibronectin fragments on synthesis of nucleic acids maintained and constituted 165 +/- 12% and 127 +/- 7% for DNA and RNA, respectively.

[Comparative study of the proteolytic products of fibronectins in the serum of adult and embryonic animals formed under the effects of proteinases of the spleen]. / Sravnitel'noe issledovanie produktov proteoliza fibronektinov syvorotki vzroslykh i émbrional'nykh zhivotnykh, obrazuiushchikhsia pri deistvii proteinaz selezenki.

The effects of two spleen proteinases, cathepsin D and thiol proteinase I active in neutral media--on the structural properties of fibronectins from blood plasma on adult animals and their embryos were investigated. Proteinase I and cathepsin D caused rapid fragmentation of all fibronectins under study. Fibronectin from calf embryonic serum was more sensitive to proteinase I than that from adult animal serum. The molecular weight and the correlation between the proteolytic products formed under the influence of each enzyme, on the embryonic and "adult" fibronectins, are very similar but not identical. Similar results were obtained in experiments with proteolytic products of chicken serum and embryo fibronectins. Fragmentation of embryonic fibronectin occurs more rapidly than that of chicken fibronectin; the fibronectin proteolytic products differ both qualitatively and quantitatively. However, the determination of structural differences between these fibronectins is considerably hampered by the presence of protein contaminations in chicken fibronectin preparations.

[Intracellular localization of fibronectin-synthesizing polyribosomes using I125 collagen]. / Izuchenie vnutrikletochnoi lokalizatsii fibronektinsinteziruiushchikh poliribosom pri pomoshchi [125I]kollagena.

Interaction of 125I-collagen with fibronectin-synthesizing polyribosomes, isolated from 9 days old chicken embryos, was studied. The interaction was maximal at the ratio of 0.1 microgram 125I-collagen and 5 ou of polyribosomes. Specific reaction of 125I-collagen with fibronectin polypeptides growing on polyribosomes was estimated by means of antibodies to fibronectin inhibiting the complex formation of polyribosomes-125I-collagen. The antibodies were shown to decrease the reaction rate of total polyribosomes (free and membrane-bound) with 125I-collagen by 25.4% and of free polyribosomes - by 6.7%. The data obtained suggest that fibronectin is synthesized mainly on polyribosomes, bound with the membranes of endoplasmic reticulum.

[Detection of an immunoglobulin-like serum protein possessing a high affinity for collagen]. / Obnaruzhenie syvorotochnogo belka immunoglobulinovoi prirody, obladaiushchego vysokim srodstvom k kollagenu.

Fibronectin isolated from bovine serum by affinity chromatography on collagen-Sepharose was found to contain a great number of concomitant proteins. Polyacrylamide gel electrophoresis of experimental samples pretreated with beta-mercaptoethanol under denaturation conditions resulted in the polypeptide fractions with Mr of 25, 54 and 82 KD, while the non-treated samples contained only one protein of non-fibronectin type (Mr = = 180-190 KD). This protein was isolated from the total preparations of collagen-binding proteins by the procedures generally employed for the isolation of purified preparations of immunoglobulins G; this protein was also isolated from purified immunoglobulins G using affinity chromatography on collagen-Sepharose. In terms of its molecular weight, subunit composition and immunological and chromatographical behaviour this protein can be related to immunoglobulins. The immunoglobulin-like protein isolated together with fibronectin revealed an affinity for denatured collagen, but not for fibronectin or Sepharose. The content of immunoglobulin with an affinity for denatured collagen in the total fraction of immunoglobulins G is 0.3-0.5%.

[A simple and effective step in the purification of bovine blood fibronectin]. / Prostoi i éffektivnyi dopolnitel'nyi étap ochistki fibronektina syvorotki byka.

Preparations of fibronectin from bovine blood serum, obtained by means of affinity chromatography on collagen-Sepharose, contained immunoglobulins and other proteins, concentration of which constituted 48 +/- 5%. Differential salting out of fibronectin and other non-fibronectin proteins, using 0.8-2.0 M ammonium sulfate at pH 5.0, demonstrated that precipitation of fibronectin occurred more effectively as compared with non-fibronectin proteins at all the salt concentrations studied. If 0.8 M or 1.0 M ammonium sulfate concentrations were used, the fibronectin preparations contained less than 10% of other proteins and fibronectin loss was about 20%. Salting out of fibronectin is an effective additional step of its purification.

[Reaction between 125I-collagen and growing fibronectin polypeptides of chick embryo polyribosomes]. / Vzaimodeistvie 125I-kollagena s rastushchimi polipeptidami fibronektina polirobosm kurinykh émbrionov.

Polyribosomes from chicken embryos and 125I-collagen were shown to form complexes, which might be isolated from free 125I-collagen by means of centrifugation. The fraction of rapidly sedimenting polyribosomes was the most active in formation of complexes with 125I-collagen. The complexes ribosomes-125I-collagen appeared after destruction of polyribosomes-125I-collagen complexes by means of RNAase. Treatment of the complexes with puromycine led to liberation of about 40% of 125I-collagen. Antibodies against fibronectin inhibited the complex formation by about 30%. The data obtained suggest that the growing fibronectin polypeptides participate in formation of at least 30% of the complexes.

[Mechanical destruction of collagen]. / Mekhanicheskaia destruktsiia kollagena.

Mechanical destruction of collagen was studied by means of electrophoresis in polyacrylamid gel and ESR-spectroscopy. It has been stated that the rates of the breakage of polypeptide chains measured by these two methods are in a good agreement. An analysis of ESR spectra of free radicals in collagen suggests that mechanical action induces the rupture of C alpha-N-bonds.

[The effect of cycloheximide on incorporation of newly formed mRNA into membrane-bound polyribosomes in rat liver cells]. / Vliianie tsiklogeksimida na vkliuchenie novoobrazovannoi iRNK v sviazannye s membranami poliribosomy v kletkakh pecheni krysy

Incorporation kinetics of new synthesized mRNA into free and endoplasmic membrane-bound polyribosomes in the absence of normal translation (when protein synthesis in inhibited by 98% with cycloheximide) is studied. mRNA is found to incorporate into both free and bound polyribosomes. Relative content of new synthesized membrane-bound polyribosomes in the presence of cycloheximide within 2.5-4.5 hours is by 30-40% lower as compared with the control. This fact can be explained either by the absence of a growing peptide of a sufficient length, which is necessary for the formation of a part of membrane-bound polyribosomes, or by a restricted number of attachment sites on membranes as a result of delayed translation of mRNA in pre-existed polyribosomes. It is suggested that 1) the growing peptide in liver cells is responsible for the recognition of a membrane only under the formation of only one type of membrane-bound polyribosomes, or 2) the formation of all bound polyribosomes has a single mechanism and the growing peptide does not participates in the membrane recognition.

[The role of ribosomal proteins in in vitro ribosome-membrane interactions]. / Izuchenie roli ribosomnykh belkov v ribosomno-membrannom vzaimodeistvii in vitro

The in vitro binding of total ribosomal proteins with rough endoplasmic membranes, from which 70% of ribosomes are eliminated by EDTA (ME) is studied. It is found that in conditions of specific interaction of ribosomes with membranes about 75% of total ribosomal proteins are bound with ME. Membranes, heterogenous in their content (different protein/lipid ratio), became homogenous in their buyoant density after the binding with proteins. The ability of membrane-ribosomal protein complex to bind ribosomes is not decreased, as it can be expected, but is considerablly increased, thus indicating on a non-specific character of ribosome binding. Ribosomal subunits lacking about half of structural protein are capable to bind with ribosome-binding membrane receptors and with some additional sites. This binding is also non-specific, because the binding efficiency of large and small subunits is the same.