Effects of starvation and different culture conditions on the phospholipid content of isolated pancreatic islets

Three forms of the normal human plasma fibrinogen gamma-chain which differ in molecular weight have been purified. Plasma fibrinogen was separated by ion exchange chromatography on DEAE-Sephacel into three populations of molecules, each with a unique gamma-chain composition. Following reduction and S-carboxymethylation, the fibrinogen polypeptide chains in each chromatographic peak were separated by ion exchange chromatography on DEAE-Sephacel and identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The A alpha, B beta and smallest gamma-chain (gamma 50) eluted at progressively higher ionic strengths, but the elution positions of A alpha, B beta and gamma 50 chains were identical for fibrinogen from each of the three different chromatographic fractions. The unique gamma chain of fibrinogen in the second chromatographic peak (gamma 55) eluted at an ionic strength higher than that of the gamma 50 chain, while the largest gamma-chain (gamma 57.5), which was contained only in the third chromatographic peak of fibrinogen, eluted at the highest ionic strength. The higher ionic strengths needed to elute fibrinogen in the second and third peaks was paralleled by the higher ionic strengths needed to elute the gamma-chains unique to them, suggesting that the gamma-chain composition of the three fibrinogen fractions accounted for their differential binding to the ion exchange resin. Following desialation with neuraminidase, the differences in electrophoretic mobilities between the three gamma-chain forms was maintained, indicating that differential migration on SDS-polyacrylamide gel electrophoresis was not due to variation in sialic acid content.     

Isolation and characterization of pepsin-treated type III collagen from calf skin.

Calf skin collagen was solubilized by incubating acid-extracted calf skin with pepsin at pH 2.0 and 25 degrees C, conditions that did not cause degradation of the triple helical region of collagen. Type III collagen was separated from type I collagen by differential salt precipitation at pH 7.5. The isolated type III collagen contained mainly gamma and higher molecular weight components cross-linked by reducible and/or non-reducible bonds. The isolated alpha1 (III) chains had an amino acid composition characteristic of type III collagen. Denatured but unreduced type III collagen, chromatographed on carboxymethyl-cellulose, eluted in the alpha 2 region, while after reduction and alkylation the alpha1 (III) chains eluted between the positions of alpha1 (I) and alpha2. The mid-point melting temperature temperature (tm) of type III collagen (35.1 degrees C) in a citrate buffer at pH 3.7 was somewhat lower than that of type I collagen (35.9 degrees C). Renaturation experiments at 25 degrees C showed that denatured type III collagen molecules with intact intramolecular disulfide bridges (gamma components) reform the triple helical structure of collagen much faster than reduced and carboxymethylated alpha1 (III) chains.     

Properties of the collagenous domain of the alpha 3(IV) chain, the Goodpasture antigen, of lens basement membrane collagen. Selective cleavage of alpha (IV) chains with retention of their triple helical structure and noncollagenous domain

A third chain, alpha 3(IV), of basement membrane collagen was recently discovered and was identified as the primary target for the autoantibodies of patients with Goodpasture syndrome (Saus, J., Wieslander, J., Langeveld, J. P. M., Quinones, S., and Hudson, B. G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, this chain was excised in the form of a truncated promoter by cleavage of basement membrane with Pseudomonas aeruginosa elastase and characterized. The triple helical structure and NC1 domain were retained. Elastase selectively cleaved at a site within the triple helical domain of the alpha 3 chain that is distinct from the cleavage site of the alpha 1 and alpha 2 chains. The truncated alpha 3 chain was found to contain 1460 residues, of which 1225 comprise the collagenous domain, and is cross-linked within this domain by disulfide bonds, forming a high Mr complex (greater than 300,000). Truncated protomers with a length of 340 nm corresponding to the theoretical length for the truncated alpha 3 chain were observed by electron microscopy as suprastructures in which the triple helical domains of three protomers were interwined. These protomers were also connected to each other and to the 140-nm protomers that appear to be comprised of the alpha 1 and alpha 2 chains. These results extended the known length of the alpha 3 chain by about 1000 residues and suggested that protomers of this chain self-associate through interactions between their triple helical domains and between their NC1 domains.     

Chemical structure and properties of duck and goose fibrinogen

Duck and goose fibrinogen were isolated from fresh pooled plasma by three different methods. To minimize proteolytic activity, epsilon-aminocaproic acid and trasylol were used throughout the preparation procedures. Amino acid composition of fibrinogens and carbohydrate content (hexose, hexosamine, sialic acid) as well as phosphorus were analysed. Intact preparations showed single band on SDS-polyacrylamide gel electrophoresis. After reduction and modification of the thiol groups, the material could be separated by SDS-polyacrylamide gel electrophoresis into four bands corresponding to the gamma, partially degraded A alpha, B beta and intact A alpha chain. Intact polypeptide subunits were separated by ion-exchange chromatography or preparative SDS-polyacrylamide gel electrophoresis and their amino acid compositions were determined. Evidences supporting the view that bird fibrinogen is very sensitive to proteolytic degradation and that a partial degradation of the A alpha chain takes place even when inhibitors are used in all steps of the purification procedures are presented.     

Separation of the alpha chains of type I and III collagens by SDS-polyacrylamide gel electrophoresis.

A method for the separation of type III collagen from type I collagen by SDS-polyacrylamide gel electrophoresis has been developed. This is based on the observation that the presence of 3-4 M urea decreases the mobility of the alpha 1 [III] chain to a greater extent than those of the alpha 1[I] and alpha 2 chains, although the alpha 1[I] and alpha 1[III] chains move at the same rate in the absence of urea. An attempt to separate the alpha 1[II] chain of type II collagen from the alpha 1[I] chain was unsuccessful under the experimental conditions employed.     

Conformational analysis and stability of collagen peptides by CD and by 1H- and 13C-NMR spectroscopies

Four small type I collagen CNBr peptides containing complete natural sequences were purified from bovine skin and investigated by CD and 1H- and 13C-nmr spectroscopies to obtain information concerning their conformation and thermal stability. CD showed that a triple helix was formed at 10 degrees C in acidic aqueous solution by peptide alpha l(I) CB2 only, and to lesser extent, by alpha 1(I) CB4, whereas peptides alpha 1(I) CB5 and alpha 2(I) CB2 remained unstructured. Analytical gel filtration confirmed that peptides alpha 1(I) CB2 and alpha 1(I) CB4 only were able to form trimeric species at temperature between 14 and 20 degrees C, and indicated that the monomer = trimer equilibrium was influenced by the chaotropic nature of the salt present in the eluent, by its concentration, and by temperature variations. CD measurements at increasing temperatures showed that alpha 1(I) CB2 was less stable than its synthetic counterpart due to incomplete prolyl hydroxylation of the preparation from the natural source. 1H- and 13C-nmr spectra acquired in the temperature range 0-47 and 0-27 degrees C, respectively, indicated that with decreasing temperature the most abundant from of alpha 1(I) CB2 was in slow exchange with an assembled form, characterized by broad lines, as expected for the triple-helical conformation. A large number of trimer cross peaks was observed both in the proton and carbon spectra, and these were most likely due to the nonequivalence of the environments of the three chains in the triple helix. This nonequivalence may have implications for the aggregation of collagen molecules and for collagen binding to other molecules. The thermal transition from trimer to monomer was also monitored by 1H-nmr following the change in area of the signal belonging to one of the two beta protons of the C-terminal homoserine. The unfolding process was found to be fully reversible with a melting temperature of 13.4 degrees C, in agreement with CD results. The qualitative superposition of the melting curves obtained by CD for the peptide bond characteristics and by nmr for a side chain suggests that triple-helical backbone and side chains constitute a single unit.     

The solubilization of bone and dentin collagens by pepsin. Effect of cross-linkages and non-collagen components.

Bone and dentin collagen are less susceptible to solubilization by pepsin digestion then is skin collagen. Digestion at 4 degrees C for 72 h solubilized only 35.3% of bovine cortical bone and 5.6% of bovine dentin compared with nearly 100% dissolution of bovine skin. Sodium dodecyl sulfate-acrylamide gel electrophoresis and molecular sieve chromatography showed that, for bone and dentin, intact alpha chains and cross-linked aggregates of beta, gamma and higher weight remained intact after pepsin solubilization but lower molecular weight fragments also were prevalent indicating chain scission in helical regions. Electron microscopic examination of segment long spacing precipitates of the soluble collagens confirmed the presence of solubilized polymerized collagen. The principal reducible cross-link in both bone and dentin was the precursor of dihydroxylsinonorleucine and this cross-link was also present in the solubilized collagens. Small amounts of non-collagenous proteins and glycosaminoglycans of different compositions in dentin and bone resisted extraction before pepsin digestion. However, the differences in solubilization of the collagens have been related to differences in cross-linkage placement.     

Identification of P-57, a serine proteinase, from human erythrocyte membranes, which cleaves both chains of human third component (C3) of complement

A proteinase, which cleaves human third component of complement, was solubilized from erythrocyte membranes then purified by gel filtration chromatography, fluid phase electrophoresis, and hydroxylapatite chromatography. Labeling of the purified material by 125I or 3H-DFP and measurement of proteolytic activity subsequently isolated by SDS-polyacrylamide gel electrophoresis allowed to identify a 57 kDa single band, in non reducing conditions. Inhibition of this activity by PMSF supports covalent modification of an active serine residue. This membrane serine proteinase cleaved alpha and beta chains of human third component of complement, suggesting that p-57 is distinct from plasma serine proteinases.     

Localization of a structural defect in type I procollagen in a patient affected with the severe non-lethal form of Osteogenesis imperfecta

A case of severe non-lethal Osteogenesis imperfecta was studied. The patient's cultured skin fibroblasts synthesised a mixed population of type I collagen chains some of which showed abnormal behaviour on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Further analysis revealed that two types of alpha 1(I) chains were synthesised, both an abnormal, slower migrating and a normal species. A small defect in one allele of one of the type I procollagen chains could lead to the larger size of the abnormal chains, probably caused by overmodifications of the triple helical region. CNBr peptide mapping allowed us to localise the defect midway along the triple helix: the defect site could be assigned to the region between the alpha 1(I)CB-3 and CB-7 peptides. The abnormal alpha 1(I) chains synthesised by the patient's cells had a melting temperature which was about 2 degrees C lower than normal chains. The results appear to be in agreement with the defect localisation and the phenotype.     

Functional heterogeneity of beta gamma-subunit of frog transducin

1. Transducin subunits (T alpha and T beta gamma) were purified from freshly dissected frog (Rana catesbeiana) retinas. It was found that purified T beta gamma is composed of three components which can be separated from each other by an anion exchange column chromatography under nondenaturing conditions. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of these three components demonstrated that each contains T beta (mol. wt 35,000) and T gamma (mol. wt approximately 8000). 3. Only one of the three components retained an ability to enhance the binding of GppNHp to T alpha in the presence of a photobleaching intermediate of rhodopsin, while the others showed very low abilities to enhance the binding. 4. These observations, together with the similar findings on bovine T beta gamma, strongly suggest that the functional heterogeneity of T beta gamma is conserved in vertebrate photoreceptor cells.     

Crystal structure of NC1 domains. Structural basis for type IV collagen assembly in basement membranes

Type IV collagen, which is present in all metazoan, exists as a family of six homologous alpha(IV) chains, alpha1-alpha6, in mammals. The six chains assemble into three different triple helical protomers and self-associate as three distinct networks. The network underlies all epithelia as a component of basement membranes, which play important roles in cell adhesion, growth, differentiation, tissue repair and molecular ultrafiltration. The specificity of both protomer and network assembly is governed by amino acid sequences of the C-terminal noncollagenous (NC1) domain of each chain. In this study, the structural basis for protomer and network assembly was investigated by determining the crystal structure of the ubiquitous [(alpha1)(2).alpha2](2) NC1 hexamer of bovine lens capsule basement membrane at 2.0 A resolution. The NC1 monomer folds into a novel tertiary structure. The (alpha1)(2).alpha2 trimer is organized through the unique three-dimensional domain swapping interactions. The differences in the primary sequences of the hypervariable region manifest in different secondary structures, which determine the chain specificity at the monomer-monomer interfaces. The trimer-trimer interface is stabilized by the extensive hydrophobic and hydrophilic interactions without a need for disulfide cross-linking.     

Incorporation of type I collagen molecules that contain a mutant alpha 2(I) chain (Gly580-->Asp) into bone matrix in a lethal case of osteogenesis imperfecta.

To understand more directly the tissue defect in osteogenesis imperfecta (OI), bone matrix was analyzed from an infant with lethal OI (type II) of defined mutation (collagen alpha 2(I)Gly580-->Asp). Pepsin-solubilized alpha 1(I) and alpha 2(I) chains and derived CNBr-peptides migrated more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis compared with normal human controls. The peptide alpha 2(I)CB3,5, predicted to contain the mutation site, ran as a retarded doublet band and was purified by high performance liquid chromatography and digested with V8 protease. Two peptides with amino-terminal sequences beginning at residue 576 of the alpha 2(I) chain were isolated. One had the normal sequence. The other differed in that aspartic acid replaced glycine at residue 580 as predicted from cDNA analysis, and in having an unhydroxylated proline at residue 579. From yields on microsequencing and the relative intensities of the two forms of alpha 2(I)CB3,5 on SDS-polyacrylamide gel electrophoresis, the ratio of mutant to normal alpha 2(I) chains in the infant's bone matrix was 0.7/1. Although the effects of an efficient incorporation of mutant chains on the properties of the bone matrix are unknown, it may be that in this OI case the tissue abnormalities result more from the presence of mutant protein than from an underexpression of matrix.     

The structural organization of type IV collagen. Identification of three NC1 populations in the glomerular basement membrane.

Type IV collagen, which has long been assumed to contain two alpha 1(IV) and one alpha 2(IV) chains, also contains alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains. Stoichiometry of collagenous alpha(IV) chains differs among tissues, suggesting the existence of subclasses of type IV collagen, each with a unique chain composition. This study seeks to define, by characterization of subunit compositions of NC1 domain populations, the structural organization of type IV collagen from bovine glomerular basement membrane. NC1 hexamers from type IV collagen were separated on two affinity chromatography columns, one containing monoclonal antibodies to the alpha 3 chain, and another, to the alpha 1 chain. SDS-polyacrylamide gel electrophoresis, immunoblotting, reversed phase high-performance liquid chromatography, and enzyme-linked immunosorbent assay identified three NC1 hexamer populations: 1) a hexamer composed of (alpha 1)2 and (alpha 2)2 homodimers; 2) a hexamer composed of (alpha 3)2 and (alpha 4)2 homodimers; 3) a hexamer containing all four alpha chains connected in heterodimers, alpha 1-alpha 3 and alpha 2-alpha 4. Results suggest that there are two distinct type IV collagen molecules, one composed of alpha 1(IV) and alpha 2(IV) chains and another composed of alpha 3(IV) and alpha 4(IV) chains. Furthermore, polymerization occurs between molecules with the same chain composition and between molecules with different chain composition. Moreover, crosslinking between different alpha chains is restricted, thus limiting the number of possible macromolecular structures.     

Bethlem myopathy and engineered collagen VI triple helical deletions prevent intracellular multimer assembly and protein secretion.

Mutations in the genes that code for collagen VI subunits, COL6A1, COL6A2, and COL6A3, are the cause of the autosomal dominant disorder, Bethlem myopathy. Although three different collagen VI structural mutations have previously been reported, the effect of these mutations on collagen VI assembly, structure, and function is currently unknown. We have characterized a new Bethlem myopathy mutation that results in skipping of COL6A1 exon 14 during pre-mRNA splicing and the deletion of 18 amino acids from the triple helical domain of the alpha1(VI) chain. Sequencing of genomic DNA identified a G to A transition in the +1 position of the splice donor site of intron 14 in one allele. The mutant alpha1(VI) chains associated intracellularly with alpha2(VI) and alpha3(VI) to form disulfide-bonded monomers, but further assembly into dimers and tetramers was prevented, and molecules containing the mutant chain were not secreted. This triple helical deletion thus resulted in production of half the normal amount of collagen VI. To further explore the biosynthetic consequences of collagen VI triple helical deletions, an alpha3(VI) cDNA expression construct containing a 202-amino acid deletion within the triple helix was produced and stably expressed in SaOS-2 cells. The transfected mutant alpha3(VI) chains associated with endogenous alpha1(VI) and alpha2(VI) to form collagen VI monomers, but dimers and tetramers did not form and the mutant-containing molecules were not secreted. Thus, deletions within the triple helical region of both the alpha1(VI) and alpha3(VI) chains can prevent intracellular dimer and tetramer assembly and secretion. These results provide the first evidence of the biosynthetic consequences of structural collagen VI mutations and suggest that functional protein haploinsufficiency may be a common pathogenic mechanism in Bethlem myopathy.     

Kinked collagen VI tetramers and reduced microfibril formation as a result of Bethlem myopathy and introduced triple helical glycine mutations.

Mutations in the genes that code for collagen VI subunits, COL6A1, COL6A2, and COL6A3, are the cause of the dominantly inherited disorder, Bethlem myopathy. Glycine mutations that interrupt the Gly-X-Y repetitive amino acid sequence that forms the characteristic collagen triple helix have been defined in four families; however, the effects of these mutations on collagen VI biosynthesis, assembly, and structure have not been determined. In this study, we examined the consequences of Bethlem myopathy triple helical glycine mutations in the alpha1(VI) and alpha2(VI) chains, as well as engineered alpha3(VI) triple helical glycine mutations. Although the Bethlem myopathy and introduced mutations that are toward the N terminus of the triple helix did not measurably affect collagen VI intracellular monomer, dimer, or tetramer assembly, or secretion, the introduced mutation toward the C terminus of the helix severely impaired association of the mutant alpha3(VI) chain with alpha1(VI) and alpha2(VI). Association of the three chains was not completely prevented, however; and some non-disulfide bonded tetramers were secreted. Examination of the secreted Bethlem myopathy and engineered mutant collagen VI by negative staining electron microscopy revealed the striking finding that in all the cell lines a significant proportion of the tetramers contained a kink in the supercoiled triple helical region. Collagen VI tetramers from all of the mutant cell lines also showed a reduced ability to form microfibrils. These results provide the first evidence of the biosynthetic consequences of collagen VI triple helical glycine mutations and indicate that Bethlem myopathy results not only from the synthesis of reduced amounts of structurally normal protein but also from the presence of mutant collagen VI in the extracellular matrix.     

Characterization of the tissue form of type V collagen from chick bone

Type V collagen was prepared from acetic acid extracts of lathyritic chick bone. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the extracted material demonstrated two collagenous bands of slower mobility than pepsin-extracted alpha 1(V) and alpha 2(V) chains. Cyanogen bromide peptide maps of these protein bands identified them as forms of alpha 1(V) and alpha 2(V). Segment long spacing (SLS) crystallite banding patterns of the acid-extracted Type V were identical within the triple-helical domain to the SLS banding patterns of pepsin-extracted Type V collagen, supporting the identification of this material. A globular domain at one end of the triple helix of the acid-extracted Type V was visualized by both rotary shadowing and negative staining of SLS crystallites. The molecular weights of the globular terminal peptides were 18,000 and 29,000, respectively, for alpha 1(V) and alpha 2(V), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after bacterial collagenase digestion of the isolated alpha chains. The results presented here indicate that fully processed Type V collagen in chick bone exists as a higher molecular weight form than that from pepsin extracts and retains a globular domain at one end of the triple helix. This is in contrast to the interstitial collagens in which only very small non-triple-helical domains (telopeptides) are retained in the fully processed molecules. In vitro aggregation studies demonstrated the intact fully processed form of Type V collagen forms uniform small-diameter fibrous structures. These results suggest that Type V collagen may be present in fibrous structures within tissues.     

Bovine type I collagen: A study of cross-linking in various mature tissues

The cyanogen bromide peptides from insoluble and pepsin solubilised type I collagen of bovine bone, dentine, meniscus, tendon, skin and cornea were compared by SDS-polyacrylamide gel electrophoresis. In each case alpha 1CB6 was shown to be the only peptide of molecular weight greater than 10 000 involved in cross-linking. The major helical peptides alpha 1CB3, alpha 1CB8, alpha 1CB7 and alpha 2CB4 were not implicated in cross-linking in any tissue either by end overlap or helix-helix interaction. The C-terminal alpha 2 chain peptide alpha 2CB3,5, which contains a large helical region, was not involved in cross-linking to any large peptides, although a slight increase in molecular weight in all tissues examined did suggest a possible interaction(s) with a very small peptide of molecular weight 4--5000.     

Subunit structure of the purified human placental insulin receptor. Intramolecular subunit dissociation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Insulin receptors purified from human placental membranes by gel-filtration and insulin-agarose affinity chromatography were found to be composed of eight different high molecular weight complexes as identified by nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The subunit stoichiometry of these different high molecular weight forms of the insulin receptor were determined by comparisons of silver-stained gel profiles with the autoradiograms of 125I-insulin specifically cross-linked to the alpha subunit and [gamma-32P]ATP specifically autophosphorylated beta subunit gel profiles. Two-dimensional SDS-polyacrylamide gel electrophoresis in the absence and presence of reductant confirmed the subunit stoichiometries as alpha 2 beta 2, alpha 2 beta beta 1, alpha 2 (beta 1)2, alpha 2 beta, alpha 2 beta 1, alpha 2, alpha beta, and beta, where alpha is the Mr = 130,000 subunit, beta is the Mr = 95,000 subunit, and beta 1 is the Mr = 45,000 subunit. Treatment of the insulin receptor preparations with oxidized glutathione or N-ethylmaleimide prior to SDS-polyacrylamide gel electrophoresis increased the relative amount of the alpha 2 beta 2 complex concomitant with a total disappearance of the alpha 2 beta, alpha 2 beta 1, alpha 2, and free beta forms. The effects of oxidized glutathione were found to be completely reversible upon extensive washing of the treated insulin receptors. In contrast, the effects of N-ethylmaleimide were totally irreversible by washing, consistent with known sulfhydryl alkylating properties of this reagent. The formation of these lower molecular weight insulin receptor subunit complexes was further demonstrated to be due to SDS/heat-dependent intramolecular sulfhydryl-disulfide exchange occurring within the alpha 2 beta 2 complex. These studies demonstrate that the largest disulfide-linked complex (alpha 2 beta 2) is the predominant insulin receptor form purified from the human placenta with the other complexes being generated by proteolysis and by internal subunit dissociation.     

Collagen XXIV,a vertebrate fibrillar collagen with structural features of invertebrate collagens: selective expression in developing cornea and bone

Tissue-specific assembly of fibers composed of the major collagen types I and II depends in part on the formation of heterotypic fibrils, using the quantitatively minor collagens V and XI. Here we report the identification of a new fibrillar-like collagen chain that is related to the fibrillar alpha1(V), alpha1(XI), and alpha2(XI) collagen polypeptides and which is coexpressed with type I collagen in the developing bone and eye. The new collagen was designated the alpha1(XXIV) chain and consists of a long triple helical domain flanked by typical propeptide-like sequences. The carboxyl propeptide is classic, with 8 conserved cysteine residues. The amino-terminal peptide contains a thrombospodin-N-terminal-like (TSP) motif and a highly charged segment interspersed with several tyrosine residues, like the fibril diameter-regulating collagen chains alpha1(V) and alpha1(XI). However, a short imperfection in the triple helix makes alpha1(XXIV) unique from other chains of the vertebrate fibrillar collagen family. The triple helical interruption and additional select features in both terminal peptides are common to the fibrillar chains of invertebrate organisms. Based on these data, we propose that collagen XXIV is an ancient molecule that may contribute to the regulation of type I collagen fibrillogenesis at specific anatomical locations during fetal development.     

The molecular defect in an autosomal dominant form of osteogenesis imperfecta. Synthesis of type I procollagen containing cysteine in the triple-helical domain of pro-alpha 1(I) chains

Synthesis of procollagen was examined in skin fibroblasts from a patient with a moderately severe autosomal dominant form of osteogenesis imperfecta. Proteolytic removal of the propeptide regions of newly synthesized procollagen, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, revealed the presence of type I collagen in which two alpha 1(I) chains were linked through interchain disulfide bonds. Fragmentation of the disulfide-bonded alpha 1(I) dimers with vertebrate collagenase and cyanogen bromide demonstrated the presence of a cysteine residue in alpha 1(I)CB8, a fragment containing amino acid residues 124-402 of the alpha 1(I) collagen chain. Cysteine residues are not normally found in the triple-helical domain of type I collagen chains. The heterozygous nature of the molecular defect resulted in the formation of three kinds of type I trimers: a normal type with normal pro-alpha(I) chains, a type I trimer with one mutant pro-alpha 1(I) chain and two normal chains, and a type I trimer containing two mutant pro-alpha 1(I) chains and one normal pro-alpha 2(I) chain. The presence of one or two mutant pro-alpha 1(I) chains in trimers of type I procollagen was found to reduce the thermal stability of the protein by 2.5 and 1 degree C, respectively. In addition to post-translational overmodification, procollagen containing one mutant pro-alpha 1(I) chain was also cleared more slowly from cultured fibroblasts. The most likely explanation for these disruptive changes in the physical stability and secretion of the mutant procollagen is that a cysteine residue is substituted for a glycine in half of the pro-alpha 1(I) chains synthesized by the patient's fibroblasts.