Interchain disulfide bonds at the COOH-terminal end of procollagen synthesized by matrix-free cells from chick embryonic tendon and cartilage.

Matrix-free cells from tendons and cartilage of chick embryos were incubated in suspension, and the procollagens secreted by the cells were isolated in the presence of protease inhibitors. Tendon procollagen was shown to contain both NH_2? and COOH-terminal extensions and interchain disulfide bonds were located in the COOH-terminal region. A disulfide-linked fragment previously isolated after digestion of the molecule with bacterial collagenase was shown to originate from the COOH-terminal end. Cartilage procollagen was also shown to contain interchain disulfide bonds in the COOH-terminal region.     

Purification and characterization of collagenase activator protein synthesized by articular cartilage

We have isolated an activator of collagenase from medium conditioned with articular cartilage. The activity is contained in an acidic protein appearing as a doublet band of Mr 57,000 and 56,000 on sodium dodecyl sulfate polyacrylamide gels. Both components of the doublet have identical isoelectric points as demonstrated by gel electrophoresis. Purified synovial collagenase has a high dependence on the presence of this factor for activity. Other known activators of latent proteolytic enzymes such as trypsin and mercurials will stimulate collagenase but only if activator protein is present. The activator protein is itself a latent metalloprotease because in the presence of p-aminophenylmercuric acetate and calcium it will digest casein. The caseinase activity and collagenase activation activity have identical heat inactivation profiles, both being stable to a temperature of 60 degrees C and partially inactivated at 80 degrees C. The synthesis of the activator is localized in the superficial zone of articular cartilage.     

Isolation and partial characterization of the amino-terminal propeptide of type II procollagen from chick embryos

The amino-terminal propeptide from type II procollagen was isolated from organ cultures of sternal cartilages from 17-day-old chick embryos. The procedure provided the first isolation of the propeptide in amounts adequate for chemical characterization. The propeptide had an apparent molecular weight of 18000 as estimated by gel electrophoresis in sodium dodecyl sulfate. It contained a collagen-like domain as demonstrated by its amino acid composition, circular dichroism spectrum, and susceptibility to bacterial collagenase. One residue of hydroxylysine was present, the first time this amino acid has been detected in a propeptide. The peptide contained no methionine and only two residues of half-cystine. Antibodies were prepared to the propeptide and were used to establish its identity. The antibodies precipitated type II procollagen but did not precipitate type II procollagen from which the amino and carboxy propeptides were removed with pepsin. Also, they did not precipitate the carboxy propeptide of type II procollagen. The data demonstrated th at the type II amino propeptide was similar to the amino propeptides of type I and type III procollagens in that it contained a collagen-like domain. It differed, however, in that it lacked a globular domain as large as the globular domain of 77-86 residues found at the amino-terminal ends of the pro alpha 1 chains of type I and type III procollagens.     

Synthesis of type I and type II collagen by embryonic chick cartilage

Embryonic chick articular and keel cartilage was found to synthesize two types of collagen. The amount of Type I collagen synthesis decreased from 60% to nearly 10% during the embryonic period studied, thus suggesting not only coexistence of both collagen types in the same tissue, but also a developmental transformation from predominantly Type I synthesis to Type II synthesis with cartilage development and maturation. Radioautographs suggested that all chondrocytes were equally active in collagen synthesis and failed to show any significant non-cartilagenous tissue contamination. Therefore variation in collagen type synthesis must be a product of some unknown genetic regulatory mechanism within the cartilage tissue.     

Isolation and characterization of proteoglycans synthesized in ovo by embryonic chick cartilage and new bone

Cartilage proteoglycans have been well characterized in a number of developing systems, both in vitro and in vivo, but the newly synthesized molecules have been analyzed only from culture material. Because of potential culture artifacts, an attempt was made to characterize the proteoglycans newly synthesized in ovo in chick embryo sternum, tibial epiphysis, and tibial shaft. These in ovo synthesized proteoglycans share many structural features with chick proteoglycans synthesized by chondrocytes in culture including average monomer size, chondroitin sulfate chain size, keratan sulfate chain size, and the ability to aggregate with hyaluronic acid. Moreover, the newly synthesized in ovo proteoglycans, notably those of the tibial epiphysis, display reproducible changes in their structure as a function of embryonic age. These changes correlate with similar changes documented for chick cartilage proteoglycans synthesized in culture. Finally, the proteoglycans synthesized in ovo in the day 17 tibial shaft include, in addition to cartilage proteoglycans, one proteoglycan which seems to be characteristic of bone.     

Purification and characterization of the N-terminal propeptide of human type III procollagen.

The N-terminal propeptide of type III procollagen was purified from human ascitic fluid by using (NH4)2SO4 precipitation, DEAE-Sephacel chromatography at pH 8.6, Sephacryl S-300 chromatography and another DEAE-Sephacel chromatography at pH 4.5. The Mr of the human peptide was about 42 000, which corresponds in size to the propeptide released by the specific N-proteinase during the extracellular processing of collagen. Bacterial-collagenase digestion of the human peptide produced three fragments, which could be separated on a Bio-Gel P-10 column. The human propeptide and its collagenase-derived fragments, an N-terminal non-collagenous domain Col 1, a C-terminal non-helical domain Col 2 and a collagenous domain Col 3, resembled those derived from the N-terminal segment of bovine type III procollagen in their amino acid composition. The human peptide was found to contain sulphate, which may explain its extremely low isoelectric point (3.1). Antibodies against the human N-terminal propeptide reacted similarly with both the purified human peptide and a corresponding segment of bovine type III procollagen. The human propeptide could be used in developing radioimmunoassays for monitoring fibrotic processes.     

Identification of a disulfide-bonded 70 Kd type X procollagen in embryonic chick sternum cartilage

Chondrocytes from the presumptive calcification region of 20 day old embryonic chick sternum were found to synthesize a 70 Kd Type X procollagen precursor in addition to the previously described 59 Kd Type X collagen molecules. The 70 Kd molecules exhibited an additional cyanogen bromide peptide, contained a disulfide-bonded domain, and were converted into the 59 Kd moieties during pulse-chase experiments. The conversion of the 70 Kd to the 59 Kd Type X collagen was prevented upon microtubular transport inhibition with colchicine and resulted in tissue accumulation of the 70 Kd Type X procollagen.     

Quantitation of type II procollagen mRNA levels during chick limb cartilage differentiation

A single-stranded DNA probe complementary to chicken type II procollagen mRNA has been used to quantitate levels of that mRNA present in chicken limb mesenchyme during cartilage differentiation. Excess labeled probe prepared from a cDNA template cloned in M13mp9 was hybridized to completion to increasing amounts of total RNA and assayed by protection from S1 nuclease digestion. Estimates of the absolute levels of type II procollagen RNA were determined using the M13mp9 template containing the coding strand as a standard. RNA complementary to the probe increased from 20 copies per diploid genome in stage 24 limb to approximately 2000 copies per diploid genome in stage 24 limb mesenchyme which had differentiated to cartilage in culture. Similar levels were found in cartilage from stage 31 limb. Sternal cartilage from 17-day embryos contained approximately 10,000 copies per diploid genome suggesting that the level of expression of this gene is different in limb growth cartilage compared with sternal cartilage. Low but detectable levels of RNA complementary to the probe were observed in limb at stages 20-24. Since a large fraction of the type II procollagen RNA in these early limbs is associated with polysomes, the type II procollagen gene appears to be expressed at a low level prior to phenotypic differentiation and prior to the accumulation of immunologically detectable levels of type II collagen.     

Purification of procollagen type II by covalent chromatography with activated thiol-sepharose 4B.

Procollagen type II was isolated from Swarm chondrosarcoma of rat by NaCl-extraction and DEAE-cellulose chromatography and further purified from remaining type II collagen using activated thiol-Sepharose 4B for covalent chromatography. The purity of the separated collagen fractions was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results demonstrate that this method is a useful tool for separating procollagen from other collagenous material, which does not contain thiol groups.     

Characterization of a type IV procollagen synthesized by human amniotic fluid cells in culture.

Fetal epithelioid cells, isolated from human amniotic fluid, synthesize and secrete a type IV-like procollagen characterized by a unique pattern of cyanogen bromide (CNBr)-produced peptides. The procollagen is disulfide-bonded and, after reduction, migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a doublet between collagen beta components and pro-alpha 1(I) chains. No conversion of the procollagen to collagen or to procollagen intermediates is observed in cell culture. The procollagen was purified by salt fractionation and ion exchange chromatography; its amino acid composition resembles that of collagenous proteins extracted from basement membranes, with a high 3- and 4-hydroxyproline and hydroxylysine content and low levels of alanine and arginine. The major products obtained after limited proteolytic digestion of the protein retain interchain disulfide bonds and, after reduction, migrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis near intact pro-alpha 1(I) chains. The procollagen is secreted efficiently by amniotic fluid cells despite almost complete inhibition of peptidyl hydroxylation but, unlike type I procollagen, the secreted underhydroxylated chains lack interchain disulfide bonds. Since these cells also secrete fibronectin and elaborate an extensive extracellular matrix, the system should prove useful in the study of cell-matrix interactions.     

Purification and partial characterization of ATP pyrophosphohydrolase from fetal bovine epiphyseal cartilage

ATP pyrophosphohydrolase was partially purified from fetal bovine epiphyseal cartilage. The purification was about 10- and 100-fold over the enzyme activities of matrix vesicle fraction and cell homogenate, respectively. The pyrophosphohydrolase and alkaline phosphatase were separated by a sequential application of Sepharose CL-6B and DEAE-cellulose column chromatographies. The purified enzyme migrated as a single band corresponding to the molecular weight of 230,000 in sodium dodecyl sulfate-polyacrylamide disc gel by electrophoresis. The enzyme absolutely required Zn2+ for its activity and appeared to bind Zn2+ strongly with an apparent affinity of p[Zn2+]0.5 = 13.4. The apparent Km for ATP was 0.18 mM. The enzyme was also reactive toward various nucleoside triphosphates including GTP, CTP, and UTP. In contrast, various phosphodiesters including RNA, UDP-glucose, NAD, and bis-p-nitrophenylphosphate were 5% or less as reactive as the nucleoside triphosphates. The pyrophosphohydrolase was inactive toward adenosine 3':5'-monophosphate or various phosphonates. UDP-glucose (1 mM), NAD (1 mM), or RNA (1 mg/ml) failed to inhibit the ATP pyrophosphohydrolase activity. These observations suggest that the ATP pyrophosphohydrolase of the cartilage is probably not a phosphodiesterase I. The matrix vesicle fraction, which probably also included some plasma membrane vesiculated during collagenase digestion, contained the highest specific activity of the enzyme as compared to other subcellular fractions of either epiphyseal or articular cartilage.     

Isolation and partial characterization of high-buoyant-density proteoglycans synthesized in ovo by embryonic chick skeletal muscle and heart

Proteoglycans, a major component of the extracellular matrix, are produced in many tissues. A report from this laboratory describes the proteoglycans synthesized in culture by chick embryonic skeletal muscle myotubes. To extend this study to in vivo conditions, chick embryos were radiolabeled in ovo and the newly synthesized high-buoyant-density proteoglycans from skeletal muscle analyzed. In both leg muscle and pectoral muscle, three major high-density proteoglycans are synthesized. One is small and is similar to the proteoglycans synthesized in culture by muscle fibroblasts. The other two proteoglycans are large. The larger of these shares structural features with the proteoglycan synthesized by skeletal muscle cells in culture. It has large chondroitin sulfate chains (estimated molecular weight of 70,000) with a high proportion of chondroitin 6-sulfate (approximately 90%). The smaller of the two large proteoglycans is distinct (chondroitin sulfate of estimated molecular weight 24,000 and approximately 60% 6-sulfated disaccharides) and is not detected in muscle cultures; evidence suggests it is not made by myoblasts. Whole hearts synthesize proteoglycans with some structural similarities, and also differences, to those made in skeletal muscle. These data indicate that the proteoglycans synthesized in muscle cultures are likewise made in developing muscle in ovo but that another distinct strictly in ovo proteoglycan is also produced.     

Molecular cloning, characterization and localization of chicken type II procollagen gene

Chicken type II procollagen (ccol2a1) has become as an important oral tolerance protein for effective treatment of rheumatoid arthritis. However, its molecular identity remains unclear. Here, we reported the full-length cDNA and nearly complete genomic DNA encoding ccol2a1. We have determined the structural organization, evolutional characters, developmental expression and chromosomal mapping of the gene. The full-length cDNA sequence spans 4837 bp containing all the coding region of the ccol2a1 including 3' and 5' untranslation region. The deduced peptide of ccol2a1, composed of 1420 amino acids, can be divided into signal peptide, N-propeptide, N-telopeptide, triple helix, C-telopeptide and C-propeptide. The ccol2a1 genomic DNA sequence was determined to be 12,523 bp long containing 54 exons interrupted by 53 introns. Comparison of the ccol2a1 with its counterparts in human, mouse, canine, horse, rat, frog and newt revealed highly conserved sequence in the triple helix domain. Chromosomal mapping of ccol2a1 locates it on 4P2. While the ccol2a1 mRNA was expressed in multiple tissues, the protein was only detected in chondrogenic cartilage, vitreous body and cornea. The ccol2a1 was found to contain two isoforms detected by RT-PCR. The distribution of the ccol2a1 lacking exon 2wasfrequently detected in chondrogenic tissues, whereas the exon 2-containing isoform was more abundant in non-chondrogenic tissues. These results provide useful information for preparing recombinant chicken type II collagen and for a better understanding of normal cartilage development.     

Purification and partial characterization of myosin II from rat testis

The intent, in this work, was to isolate rat testis myosin II. Testis 40,000 x g x 40' supernatant was frozen at -20 degrees C for 48 h and, after it was thawed and centrifuged. The precipitate, after washed twice, was enriched in three polypeptides bands: p205, p43 and one that migrated together with the front of the gel. These polypeptides were solubilized in pH 10.8 at 27 degrees C and separated in Sephacryl S-400 column. Three low weight polypeptides co-eluted together with p205. The p205 was marked with anti-myosin II, possess actin-stimulated Mg-ATPase activity and co-sedimented with F-actin in the absence, but not in the presence, of ATP. In the present study, we have been developing a method for purification of myosin II from rat testis.     

Deoxythymidine kinase induced in HeLa TK- cells by herpes simplex virus type I and type II. II. Purification and characterization.

Deoxythymidine kinase activities were induced in HeLa TK- (deoxythymidine kinase-deficient) cells infected with either herpes simplex virus type I or herpes simplex virus type II. The herpes simplex virus type I-induced enzyme was found in the cytoplasmic and nuclear fractions of the infected cells, whereas the herpes simplex type II-induced deoxythymidine kinase could only be found in the cytoplasm. Herpes simplex virus type I and II specific deoxythymidine kinases were purified by affinity column chromatography. Both purified deoxythymidine kinases retained the deoxycytidine kinase activity present in the crude preparation. The purified herpes simplex virus type I deoxythymidine kinase had a different mobility on electrophoresis, but the same sedimentation rate on a glycerol gradient as the corresponding unpurified enzyme, whereas the purified herpes simplex virus type II deoxythymidine kinase had the same mobility and sedimentation rate as the corresponding unpurified enzyme. In the presence of Mg2+ATP and dithiothreitol, herpes simplex virus type II deoxythymidine kinase was more stable than herpes simplex virus type I deoxythymidine kinase at both 45 degrees and 4 degrees. The deoxycytidine kinase activity present in the purified preparations was inactivated at the same rate as the deoxythymidine kinase activity. In the presence of the other substrate, deoxythymidine, herpes simplex virus type I deoxythymidine kinase was more stable than herpes simplex virus type II kinase. The purified herpes simplex virus type I and II deoxythymidine kinase had different activation energies when Mg2+ATP and deoxythymidine were used as substrates, but showed the same sensitivity toward ammonium sulfate inhibition.     

Purification and partial characterization of membrane-associated type II (cGMP-activatable) cyclic nucleotide phosphodiesterase from rabbit brain

Membrane-associated, Type II (cGMP-activatable) cyclic nucleotide phosphodiesterase (PDE) from rabbit brain, representing 75% of the total homogenate Type II PDE activity, was purified to apparent homogeneity. The enzyme was released from 13,000 x g particulate fractions by limited proteolysis with trypsin and fractionated using DE-52 anion-exchange, cGMP-Sepharose affinity and hydroxylapatite chromatographies. The enzyme showed 105 kDa subunits by SDS-PAGE and had a Stokes radius of 62.70 A as determined by gel filtration chromatography. Hydrolysis of cAMP or cGMP showed positive cooperativity, with cAMP kinetic behavior linearized in the presence of 2 microM cGMP. Substrate concentrations required for half maximum velocity were 28 microM for cAMP and 16 microM for cGMP. Maximum velocities were approx. 160 mumol/min per mg for both nucleotides. The apparent Kact for cGMP stimulation of cAMP hydrolysis at 5 microM substrate was 0.35 microM and maximal stimulation (3-5-fold) was achieved with 2 microM cGMP. Cyclic nucleotide hydrolysis was not enhanced by calcium/calmodulin. The purified enzyme can be labeled by cAMP-dependent protein kinase as demonstrated by the incorporation of 32P from [gamma-32P]ATP into the 105 kDa enzyme subunit. Initial experiments showed that phosphorylation of the enzyme did not significantly alter enzyme activity measured at 5 microM [3H]cAMP in the absence or presence of 2 microM cGMP or at 40 microM [3H]cGMP. Monoclonal antibodies produced against Type II PDE immunoprecipitate enzyme activity, 105 kDa protein and 32P-labeled enzyme. The 105 kDa protein was also photoaffinity labeled with [32P]cGMP. The purified Type II PDE described here is physicochemically very similar to the isozyme purified from the cytosolic fraction of several bovine tissues with the exception that it is predominantly a particulate enzyme. This difference may reflect an important regulatory mechanism governing the metabolism of cyclic nucleotides in the central nervous system.     

Isolation and characterization of genomic clones corresponding to the human type II procollagen gene.

A recombinant human DNA library was screened using probes corresponding to the chick alpha 1 (II) procollagen gene. This resulted in the isolation of 2 different genomic clones, LgHCol(II)a and LgHCol(II)b. LgHCol(II)a was identified as corresponding to the alpha 1(II) gene by comparative hybridization and DNA sequence analysis. DNA sequence established that LgHCol(II)a extends at least from amino acid 694 of the triple helix through 54 amino acids of the COOH-propeptide. Hybridization with a probe containing only the exon at the 3' end of the chicken gene suggests that the clone contains the 3' end of the human gene. Thus LgHCol(II)a contains approximately 40% of the coding sequences of the human type II collagen gene.