Heterologous Cell-free System Research Articles

Cryptic O2- -generating NADPH oxidase in dendritic cells.

All the components of the O(2)(-)-generating NADPH oxidase typically found in neutrophils, namely a membrane-bound low potential flavocytochrome b and oxidase activation factors of cytosolic origin, are immunodetectable in murine dendritic cells (DCs). However, in contrast to neutrophils, DCs challenged with phorbol myristate acetate (PMA) can barely mount a significant respiratory burst. Nevertheless, DCs generate a substantial amount of O(2)(-) in the presence of PMA following preincubation with pro-inflammatory ligands such as lipopolysaccharide and pansorbin, and to a lesser extent with anti-CD40 or polyinosinic polycytidylic acid. We found that the virtual lack of the oxidase response to PMA alone is specifically controlled in DCs. Through the use of homologous and heterologous cell-free systems of oxidase activation, we showed the following: (1) a NADPH oxidase inhibitory factor is located in DC membranes; it exerts its effect on oxidase activation and not on the activated oxidase. (2) The inhibition is relieved by pretreatment of DC membranes with beta-octylglucoside (beta-OG). (3) The beta-OG-extracted inhibitory factor prevents the activation of neutrophil oxidase. (4) The inhibitory activity is lost after treatment of DC membranes with proteinase K or heating, which points to the protein nature of the inhibitory factor. Overall, these data indicate that the O(2)(-)-generating oxidase in DCs is cryptic, owing to the presence of a membrane-bound inhibitor of protein nature that prevents oxidase activation. The inhibition is relieved under specific conditions, including a prolonged contact of DCs with pro-inflammatory ligands from microbial origin, allowing a substantial production of O(2)(-), which may contribute to the response of DCs to a microbial exposure.

Effect of long periods of low temperature exposure on protein synthesis activity in wheat seedlings

Long periods of low temperature exposure induce complex changes in the metabolism of nucleic acids and protein molecules in plants: i.e. new proteins; new tRNA isoacceptors and new mRNAs appear with altered minor nucleotide contents, implying that the components of the protein synthesising system change during cold treatment. To study the effect of changes in the RNA pool on the intensity of protein synthesis, different homologous and heterologous cell-free protein synthesising systems were constructed with polysome fractions and tRNAs isolated from non-treated wheat seedlings and from seedlings cold treated for a long period. The homologous cell-free protein synthesising systems contained polysome fractions from non-treated samples of the wheat cultivar Martonvásári 15 and from samples treated for 1, 5 or 7 weeks together with their own tRNA. Heterologous systems were constructed from the tRNA fractions of cold-treated seedlings with S 23 fractions of non-treated ones and vice versa. Cell-free protein synthesis was carried out at 4 and 30°C. The results demonstrate that independently of the length of the cold period the intensity of protein synthesis in homologous cold-treated systems at 4°C was as high as the intensity of homologous non-treated systems at 30°C. Combinations of cold-treated S 23 fractions with cold-treated tRNAs were about 30% more effective than cold-treated S 23 fractions with non-treated tRNAs at 4°C, while combinations of cold-treated tRNAs with non-treated S 23 fractions resulted in only a slight decrease in activity at 30°C. It can thus be concluded that long-term cold exposure leads to changes in the protein synthesising system, resulting in optimal synthesising capacity under the altered conditions.

A spider tRNA(Ala) requires a far upstream sequence element for expression.

Within the series of timed differential accumulations of small RNAs we have shown to prelude the synthesis of fibroin in the large ampullate glands of Nephila clavipes (Nc), we are currently directing our attention to the alanine tRNAs. This work reports the subcloning of the members of a tRNAAla gene cluster and the optimization of their transcription in a heterologous cell-free system derived from Bombyx mori (Bm) silkglands. Our data show that the heterologous cell-free system supports the faithful and differential transcription of the individual spider alanine tRNA genes. We are thus making use of the extract to characterize the individual genes with respect to flank-contained regulatory elements through cell-free transcription of gene derivatives. The work has been initiated with pNTA3 because of its high transcriptional activity. Interestingly, the transcription of this gene requires a far upstream sequence, an uncommon modality in tRNA genes.

In vitro translation of messenger RNA in a wheat germ extract cell-free system.

The wheat germ extract in vitro translation system has been used widely for faithful and efficient translation of viral and eukaryotic messenger RNAs in a heterologous cell-free system (, , , , , , , , ). With respect to the yield of translation products, the wheat germ extract is less efficient than most reticulocyte lysate cell-free systems (see Chapters 29, 30, and 32). There are advantages, however, of using wheat germ extracts: 1 The in vtvo competition of mRNAs for translation IS more accurately represented, making the wheat germ system preferable for studymg regulation of translation (). 2. Particularly low levels of endogenous mRNA and the endogenous nuclease activity () obviate the requirement for treatment with a calcmm-activated nuclease. There IS, therefore, less dlsruption of the in vivo situation and contamination with calcium ions is less harmful The ldentlfication of all sizes of exogenous mRNA-directed translation products IS faclhtated because of the low levels of endogenous mRNA present. 3. There is no posttranslational modification of translation products; primary products are therefore investigated, although processmg may be achieved by the addition of mlcrosomal membranes to the translation reaction. 4. The ionic conditions of the reaction may be altered to optimize the translation of large or small RNAs () (see Note 1)

In vitro translation of messenger RNA in a wheat germ extract cell-free system.

The wheat germ extract in vitro translation system has been used widely for faithful and efficient translation of viral and eukaryotic messenger RNAs in a heterologous cell-free system (, , , , , , , , ). With respect to the yield of translation products, the wheat germ extract is less efficient than most reticulocyte lysate cell-free systems. There are advantages, however, of using wheat germ extracts: 1. The in vivo competition of mRNAs for translation is more accurately represented, making the wheat germ system preferable for studying regulation of translation (). 2. Particularly low levels of endogenous mRNA and the endogenous nuclease activity () obviate the requirement for treatment with a calcium-activated nuclease. There is, therefore, less disruption of the in vivo situation and contamination with calcium ions is less harmful. The identification of all sizes of exogenous mRNA-directed translation products is facilitated because of the low levels of endogenous mRNA present, 3. There is no posttranslational modification of translation products; primary products are therefore investigated, although processing may be achieved by the addition of microsomal membranes to the translation reaction. 4. The ionic conditions of the reaction may be altered to optimize the translation of large or small RNAs () (see Note 1).

The O 2− generating oxidase of B lymphocytes: Epstein-Barr virus-immortalized B lymphocytes as a tool for the identification of defective components of the oxidase in chronic granulamatous disease

The O 2 − generating NADPH oxidase of human Epstein-Barr virus immortalized B lymphocytes (EBV-B lymphocyes) and the NADPH oxidase of human neutrophils were compared. The capacity of the oxidase of EBV-B lymphocytes to generate O 2 − is 100-fold less than that of neutrophils. Like the oxidase of neutrophils, the oxidase of EBV-B lymphocytes is decreased or abolished in chronic granulomatous disease (CGD). Activation of neutrophil oxidase in an heterologous cell-free system, using human neutrophil membranes and EBV-B lymphocyte cytosol from healthy and CGD patients, combined with immunoblotting investigations of the cytosolic activating factors p47 and p67 involved in O 2 − production, suggests that neutrophils and EBV-B lymphocytes possess similar complements of cytosolic factors p47 and p67. Cytochrome b −245, the major membrane redox component of the O 2 − generating oxidase, is only slightly expressed in the membrane of EBV-B lymphocytes. A sensitive and specific immunocytochemical method for detection of the two subunits of cytochrome b −245 is described; it shows that both subunits are virtually absent in EBV-B lymphocytes from CGD patients deficient in the large subunit.

Activation of O2(-)-generating oxidase in an heterologous cell-free system derived from Epstein-Barr-virus-transformed human B lymphocytes and bovine neutrophils. Application to the study of defects in cytosolic factors in chronic granulomatous disease.

Epstein-Barr-virus-transformed human B lymphocytes (EBV B lymphocytes) stimulated by 4 beta-phorbol 12-myristate 13-acetate exhibit an NADPH-dependent oxidase activity capable of generating the superoxide anion O2-, similar to, but less efficient than that of activated neutrophils. A cell-free system of oxidase activation consisting of a membrane fraction and cytosol from EBV B lymphocyte homogenate supplemented with guanosine 5'-[gamma-thio]triphosphate (GTP[S]), arachidonic acid and Mg2+ was found to be competent in the production of O2-, assessed by the superoxide-dismutase-sensitive reduction of cytochrome c in the presence of NADPH. However, cytochrome c reduction was slow and largely insensitive both to superoxide dismutase, and to iodonium biphenyl, a powerful inhibitor of the oxidase activity in neutrophils. A markedly faster reduction of cytochrome c in the presence of NADPH was obtained with a heterologous system consisting of cytosol from EBV B lymphocytes and bovine neutrophil membranes, GTP[S], arachidonic acid and Mg2+; in this system, reduction of cytochrome c was totally inhibited by superoxide dismutase and iodonium biphenyl. These results show that EBV B lymphocytes contain a substantial amount of cytosolic factors of oxidase activation, and that the limiting factors for O2- production in B lymphocytes are the membrane components of the oxidase complex. The heterologous system of EBV B lymphocyte cytosol and bovine neutrophil membranes provided a rapid and convenient method to diagnose cytosolic defects in autosomal forms of chronic granulomatous disease. In addition, it might be a useful tool to explore the mechanism of action of the cytosolic factors in oxidase activation.

Efficient translation of the UAG termination codon in Candida species

Clinical isolates of the dimorphic fungus Candida albicans encode a tRNA that, in a cell-free translation system prepared from the yeast Saccharomyces cerevisiae, efficiently translates the amber (UAG) termination codon. Unusually, the efficiency of this UAG read-through in the heterologous cell-free system is not further enhanced by polyamines. The suppressor tRNA is also able to efficiently translate the UAG codon in the rabbit reticulocyte cell-free system and with efficiencies approaching 100% in a homologous (C. albicans) cell-free system. That the suppressor tRNA is nuclear-encoded is demonstrated by the lack of activity in purified C. albicans mitochondrial tRNAs. Finally, UAG suppressor tRNA activity is also demonstrated in three other pathogenic Candida species, C. parapsilosis, C. guillermondii and C. tropicalis. These results suggest that some, but not all, Candida species have evolved an unusual nuclear genetic code in which UAG is used as a sense codon.

The role of cytosolic and membrane factors in processing of the human beta-2 adrenergic receptor following translocation and glycosylation in a cell-free system.

The beta-2 adrenergic receptor has been proposed to have seven membrane-spanning domains. Expression of functional beta-2 adrenergic receptor was achieved in a heterologous cell-free system composed of rabbit reticulocyte lysate and microsomal membranes from Xenopus laevis oocytes. The functional state of the receptor protein can be determined by ligand-binding assays and by the ability of ligands to alter the susceptibility of the receptor to proteinase K digestion. The process by which functional receptor is made was studied. The receptor protein remains nonfunctional immediately following translocation and glycosylation, and additional processing steps are needed before the receptor is able to interact with ligands. These processing steps require intact microsomal membranes as well as several cytosolic factors including ATP and one or more high molecular mass (greater than 30 kDa) factors but do not require receptor glycosylation and are not inhibited by nonhydrolyzable GTP analogues.

Assembly kinetics and identification of precursor proteins of complex I from Neurospora crassa

Complex I from Neurospora crassa was fractionated using chaotropic agents and various chromatographic techniques. Several subunits were isolated. Polyclonal antibodies directed against the holocomplex or individual subunits were raised in rabbits, and employed to analyse the composition and assembly of this respiratory chain enzyme in vivo. N. crassa cells were pulse-labelled with radioactive amino acids. The time course of incorporation of radioactivity into complex-I polypeptides was studied by immunoprecipitation. The labelling kinetics of whole complex I was found to be similar to that of cytochrome oxidase, displaying a half-maximal labelling time of 10 min. Newly synthesized individual polypeptide subunits (about 23 species) assembled into the holoenzyme at markedly different rates. Two mitochondrially synthesized proteins, a 29-kDa polypeptide (the ND-1 gene product) and a 12-kDa polypeptide were the fastest components to appear in the enzyme. We estimate that the precursor pool sizes of all components range between 1-25% of the amounts present in the final complex. Precursors of polypeptides of complex I were synthesized in an heterologous cell-free system and immunoprecipitated with subunit specific antibodies. Six isolated precursors were compared with the corresponding mature proteins. It appears that four subunits (apparent molecular masses of 22, 25, 31 and 33 kDa) are initially synthesized as larger-molecular-mass precursors. Two subunits (apparent molecular masses of 12.5 and 14 kDa) are made with the same size as their mature forms.

Post-translational translocation of polypeptides across the mammalian endoplasmic reticulum membrane is size and ribosome dependent.

The translation and translocation of two yeast glycoproteins, invertase and carboxypeptidase Y, were studied in a heterologous cell-free translation system from reticulocytes supplemented with dog pancreas microsomes. Using in vitro synthesized mRNA transcripts, encoding complete or truncated invertase forms, the influence of polypeptide size and ribosome dependence was studied. It was found that C-terminal truncated fragments of 25 kDa, i.e. a size larger than the average size of a domain structure, are translocated and processed post-translationally with a similar efficiency to the cotranslational events. Post-translational import decreases with increasing peptide chain, mature polypeptide (60 kDa) being no longer translocated. Post-translational competence is only maintained as long as the peptide remains associated with ribosomes. Translocation of invertase depends on the presence of the leader peptide and requires energy independent of protein synthesis. Size dependence of post-translational import could also be demonstrated for carboxypeptidase Y.

In vitro translation of mRNA from Nostoc commune (Cyanobacteria)

RNA pools were extracted from cells of Nostoc commune UTEX 584 in exponential growth (liquid cultures) and from cells which had been immobilized and dried rapidly at -99.5 MPa. Levels of incorporation of 35S-methionine, five- to sixfold higher than the endogenous level, were obtained after in vitro translation of the RNA preparations in a heterologous S30 cell-free system purified from Escherichia coli Q13. The levels of incorporation, obtained with a homologous N. commune UTEX 584 S30 system, were much lower. The requirement for magnesium in the heterologous system was 15–21 mM, translation of N. commune UTEX 584 RNA was inhibited when the RNA concentration was greater than 0.3 mg ml−1, and translation was stimulated significantly by the presence of ammonium chloride. Few qualitative differences were observed between the pattern of proteins (SDS-PAGE) obtained after translation of the RNA pools from cells in exponential growth, and from those cells subjected to immobilization and rapid drying. The data suggest that short-term desiccation of N. commune UTEX 584 does not have a marked selective effect on the composition of the mRNA pool. In contrast, preparations of RNA from field materials of Nostoc commune HUN (desiccated for 5 years) were unable to drive high rates of translation in any of the systems tested and optimized for use in this study.

Fertilization triggers unmasking of maternal mRNAs in sea urchin eggs.

Fertilization of sea urchin eggs results in a large increase in the rate of protein synthesis which is mediated by the translation of stored maternal mRNA. The masked message hypothesis suggests that messenger ribonucleoprotein particles (mRNPs) from unfertilized eggs are translationally inactive and that fertilization results in alterations of the mRNPs such that they become translationally active. Previous workers have isolated egg mRNPs by sucrose gradient centrifugation and have assayed their translational activity in heterologous cell-free systems. The conflicting results they obtained are probably due to the sensitivity of mRNPs to artifactual activation and inactivation. Previously, we demonstrated that unfractionated mRNPs in a sea urchin cell-free translation system were translationally inactive. Now, using large-pore gel filtration chromatography, we partially purified egg mRNPs while retaining their translationally repressed state. Polysomal mRNPs from fertilized eggs isolated under the same conditions were translationally active. The changes in the pattern of proteins synthesized by fractionated unfertilized and fertilized mRNPs in vitro were similar to those changes observed in vivo. Treatment of egg mRNPs with buffers containing high salt and EDTA, followed by rechromatography, resulted in the activation of the mRNPs and the release of an inhibitor of translation from the mRNPs. Analysis of the inhibitory fraction on one-dimensional sodium dodecyl sulfate gels indicated that this fraction contains a complex set of proteins, several of which were released from high-salt-EDTA-activated mRNPs and not from inactive low-salt control mRNPs. One of the released proteins may be responsible for the repression of egg mRNPs in vitro and be involved in the unmasking of mRNPs at fertilization.

Fertilization Triggers Unmasking of Maternal mRNAs in Sea Urchin Eggs

Fertilization of sea urchin eggs results in a large increase in the rate of protein synthesis which is mediated by the translation of stored maternal mRNA. The masked message hypothesis suggests that messenger ribonucleoprotein particles (mRNPs) from unfertilized eggs are translationally inactive and that fertilization results in alterations of the mRNPs such that they become translationally active. Previous workers have isolated egg mRNPs by sucrose gradient centrifugation and have assayed their translational activity in heterologous cell-free systems. The conflicting results they obtained are probably due to the sensitivity of mRNPs to artifactual activation and inactivation. Previously, we demonstrated that unfractionated mRNPs in a sea urchin cell-free translation system were translationally inactive. Now, using large-pore gel filtration chromatography, we partially purified egg mRNPs while retaining their translationally repressed state. Polysomal mRNPs from fertilized eggs isolated under the same conditions were translationally active. The changes in the pattern of proteins synthesized by fractionated unfertilized and fertilized mRNPs in vitro were similar to those changes observed in vivo. Treatment of egg mRNPs with buffers containing high salt and EDTA, followed by rechromatography, resulted in the activation of the mRNPs and the release of an inhibitor of translation from the mRNPs. Analysis of the inhibitory fraction on one-dimensional sodium dodecyl sulfate gels indicated that this fraction contains a complex set of proteins, several of which were released from high-salt-EDTA-activated mRNPs and not from inactive low-salt control mRNPs. One of the released proteins may be responsible for the repression of egg mRNPs in vitro and be involved in the unmasking of mRNPs at fertilization.

Effects of cortisol treatment on protein synthesis in septum and hippocampus of rat brain.

The effects of cortisol on protein synthesis in distinct brain regions were studied. Hormone administration produced increases in tubulin and actin content in the soluble fraction isolated from septum and hippocampus. Therefore, the poly(A)RNAs were isolated from the same brain regions and their translational capacity was tested in a heterologous cell-free system. Poly(A)RNAs from cortisol-treated animals, as compared to control animals, had a stimulatory effect on 35S-methionine incorporation into tubulin and actin. Thus, our results suggest that cortisol treatment increases the amount of mRNAs coding for tubulin and actin. More importantly, this study indicates that the molecular mechanism of glucocorticoid action in the brain involves modulation of protein synthesis.

Attachment of the 5'-terminal portion of globin mRNAs to 5S-RNA X L5-protein in the 80S initiation complex.

An 80S initiation complex was formed by incubating a heterologous cell-free system with 125I-labeled globin mRNAs in the presence of sparsomycin. The 80S initiation complex was then digested with micrococcal nuclease. The ribosomal 5S-RNA X L5-protein (5S RNP) fraction, released by EDTA treatment, contained 125I-labeled mRNA fragments. The attachment of labeled mRNA fragments to 5S RNP was shown by (a) CsCl isopycnic centrifugation, (b) recentrifugation through a sucrose density gradient and (c) acrylamide gel electrophoresis of 5S RNP purified by (b). Labeled fragments were released from 5S RNP by treatment with sodium dodecyl sulfate or pronase, indicating the participation of protein L5 in the attachment. The attached mRNA fragments were 23-25 nucleotides in length. Hybridization experiments, using restriction fragments of cDNA for rabbit beta globin mRNA, showed that the attached mRNA fragments were derived from the 5' portion of globin mRNAs. The attachment of 125I-labeled mRNA fragments to 5S RNP was also observed in the 80S initiation complex formed by incubation of reticulocyte lysate with 125I-labeled globin mRNA, but not in labeled polysomal fractions. These findings may indicate that 5S RNP interacted with the 5' portion of globin mRNA, containing the translation initiation codon of globin mRNA in the 80S initiation complex. The biochemical significance of these results is discussed.

Translation in Xenopus oocytes of messenger RNA from A431 cells for human epidermal growth factor receptor proteins.

Translation of messenger RNAs (mRNAs) for human epidermal growth factor receptor (EGF-R) proteins was accomplished in a Xenopus oocyte translation system. Translation of these mRNAs in rabbit reticulocyte lysates and wheat germ extracts were unsuccessful, in accordance with the general difficulty in translating mRNAs for membrane-bound glycoproteins in conventional heterologous cell-free systems. Total poly(A)+RNA from a human epidermoid carcinoma cell line A431 was injected into oocytes from Xenopus laevis. After incubation in the presence of [35S]methionine, the oocytes were homogenized and the supernatant incubated with several different polyclonal antibodies (IgGs) against the human EGF-R. Immunocomplexes were bound to protein A-Sepharose, washed extensively, and electrophoresed in sodium dodecyl sulfate-polyacrylamide gels. A protein of Mr = 160,000, corresponding to the EGF-R glycoprotein, was specifically precipitated from RNA-injected oocytes by each of the anti-EGF-R IgGs. This protein was not detectable in the immunoprecipitates from uninjected oocytes nor was it precipitated from RNA-injected oocytes by IgG from normal rabbit serum or IgG to an unrelated protein. A second protein of Mr = 100,000 was also immunoprecipitated from the homogenates of RNA-injected oocytes. This product corresponds to the EGF-R-related protein, present in A431 cells, which is structurally similar to the EGF-R (Weber et al., 1984). Thus, frog oocytes provide an efficient and generally applicable heterologous system for translation of mRNAs for large glycoproteins such as growth factor receptors. In addition, successful immunoprecipitation of translated products in this system provides a method for assay of mRNAs where biological activity of the proteins cannot be monitored.

Amino terminal acetylation and protein synthesis in eukaryotes

The amino terminal residue in many proteins is N-acetylated. Available evidence indicates that acetylation occurs during translation whilst the nascent polypeptide is still attached to the ribosome; with some proteins, like the actins, an additional acetylation step occurs which may be catalysed by cytoplasmic enzymes. Studies of protein synthesis in heterologous cell-free systems show that the enzymes which catalyse amino terminal acetylation are ubiquitous and such studies have provided insights into the effects of limitations in the supply of acetyl CoA in vitro. Evidence that availability of acetyl groups in vivo may in part determine the cellular concentration of N-acetylated proteins is examined in terms of the relative amounts of the aceylated fraction of foetal haemoglobin and carbonic anhydrase I in human erythrocytes. Tissue and developmental differences in the concentration of the muscle isoenzyme, carbonic anhydrase III, are considered in terms of changes in the activity and distribution of mitochondria in muscle. Finally, the possibility is discussed that differential effects of thyroid hormone on protein concentration might be partly due to an effect of the hormone on acetyl CoA availability via its action on mitochondria.

Synthesis of pigment-binding protein in toluene-treated Rhodopseudomonas capsulata and in cell-free systems.

Pigment-binding protein of the facultatively phototrophic bacterium Rhodospeudomonas capsulata could be selectively synthesized in toluene-treated cells as well as in homologous and heterologous cell-free translation systems by isolated polysomes. It is shown that the pigment-binding polypeptides of the light-harvesting complexes are encoded by messenger RNA of extreme longevity. The dependence of their synthesis on the concomitant synthesis of tetrapyrroles was demonstrated in the toluene-treated cells. The large Mr-28 000 polypeptide of the reaction center and the Mr-10 000 pigment-binding polypeptide of the light-harvesting complex II were found to be synthesized by free (water-soluble) polysomes without a cleavable 'leader' or 'signal' peptide [reviewed by W. Wickner (1979) Annu. Rev. Biochem. 48, 23-45]. The Mr-10 000 polypeptide, as synthesized in vitro, was studied in more detail. Unlike the membrane-assembled polypeptide in vivo it was insoluble in an organic solvent mixture (chloroform/methanol 1:1, v/v). After detergent denaturation in the presence of membrane isolated from the organism it became organic-solvent-soluble. Obviously the polypeptide could be induced to assume alternative conformations in which its apolar residues were either exposed to the solvent or buried within. These findings, in agreement with Wickner's hypothesis, indicate that the Mr-10 000 polypeptide may enter the lipid bilayer by a 'membrane-triggered' conformational change.

Construction of a cDNA clone corresponding to mouse alpha 1(IV) procollagen.

A new procedure for the synthesis of double stranded cDNA, based upon release of mRNA by "in vitro" translation, was used to clone type IV collagen. Collagen synthesizing polysomes selectively isolated from a mouse parietal yolk sac carcinoma (PYS-2) were used for translation in an heterologous cell-free system. Translation products were collagenase-sensitive and displayed an electrophoretic mobility correspondent to type IV collagen. Translation released mRNA was employed to construct a 100 base pairs long cDNA clone which hybridized to a 7,800 nucleotides long mRNA. Peptides synthesized by "in vitro" translation of hybrid selected mRNA displayed an electrophoretic mobility compatible with that of alpha 1 (IV) collagen, were sensitive to collagenase and were immunoprecipitated by anti-type IV collagen antibody.