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.     

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.     

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.     

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.     

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.     

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.     

Purification and partial characterization of neuraminidase from type III group B streptococci

Extracellular neuraminidase from a type III fresh clinical isolate of a group B streptococcus was purified by a combination of salt fractionation, affinity chromatography of Affi-Gel blue, ion-exchange chromatography on diethylaminoethylcellulose, and gel filtration on Sephacryl S-200. These procedures yielded enzyme which was purified approximately 1,000-fold compared with the enzyme found in the original supernatant fluid. This type III streptococcal neuraminidase had a molecular weight of approximately 125,000 as estimated by filtration on Sephacryl S-200 and approximately 106,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast to the majority of other bacterial neuraminidases, the type III group B streptococcal enzyme had no effect on colominic acid or N-acetylneuramin-lactose; however, it was quite active on bovine submaxillary mucin.     

Purification and partial characterization of a protease associated with photosystem II particles

A protease was extracted with 1 M NaCl from spinach ( Spinacia oleracea L.) photosystem II (PSII) particles and purified through gel filtration and anion-exchange chromatography. SDS-polyacrylamide gel electrophoresis of the protease revealed a polypeptide with a molecular mass of 43 kDa. The activity of the purified protease was assayed using a 24 kDa water-soluble protein as substrate, visualized through SDS-PAGE. The protease even remained active in the presence of 0.1 and 0.2 M NaCl, although the degradation pattern changed, which indicated that the protease was different from that reported earlier by another group. The presence of 0.3 M NaCl was shown to be inhibitory. The protease was inhibited by 1,10-phenanthroline and EGTA-NaOH (pH 7.0), indicating that the metal ions are essential for activity and that the enzyme is a metal-protease. FTIR spectroscopy was used to examine the conformationally sensitive amide I' bands of the protease. The protease was observed to undergo spectroscopic changes that reflect the conformational changes that take place when Ca²⁺ is bound, which further confirms that the protease is a metal-protease.     

Purification and partial characterization of the gamma-D-glutamyl-L-di-amino acid endopeptidase II from Bacillus sphaericus.

1. A gamma-D-glutamyl-L-di-amino acid endopeptidase II (EC3.4.-.-) active on the peptide moieties of some bacterial peptidoglycans has been purified to homogeneity from the sporulation medium and from the spores of Bacillus sphaericus. 2. Enzyme from both sources showed a single protein band (Mr 28,000) by polyacrylamide gel electrophoresis under denaturing conditions. It is an acidic protein (pI 4.1). Kinetic studies have shown a Km value of 0.24 mM and an apparent Vmax of 8.3 mumol min-1 mg-1 with the pentapeptide L-Ala-gamma-D-Glu-L-Lys-D-[14C]Ala-D-[14C]Ala as substrate. 3. The enzyme was inhibited by p-hydroxymercuribenzoate, a sulfhydryl inhibitor. 4. The 38-residue N-terminal region was sequenced. It may be useful to construct a nucleotide probe for the research of the gene encoding this enzyme.     

Characterization of procollagen synthesized by matrix-free cells isolated from chick embryo tendons.

The genetic type and molecular structure of the precursor forms of collagen synthesized by matrix-free tendon cells isolated from 17-day old chick embryos were examined by chromatographic and electrophoretic techniques. The [14C]proline-labeled collagenous proteins secreted by the cells resolved on diethylaminoethylcellulose into two peaks, A and B. Both peaks contained type I collagenous proteins since on chromatography on carboxymethylcellulose, after limited pepsin proteolysis, both peaks contained alpha1 and alpha2 chains of collagen in a 2:1 ratio, and cyanogen bromide peptide maps of the 14C-labeled protein in both peaks were similar to cyanogen bromide peptide maps derived from authentic type I collagen. Enzymatic digestion with purified mammalian collagenase demonstrated that the collagen precursor in peak B contained noncollagenous peptide extensions at both the amino- and carboxy-terminal ends of the molecule, while peak A had only carboxy-terminal extension peptides. Although both the amino- and carboxy-terminal extensions incorporated radioactive cystine, only the carboxy-terminal extensions contained interchain disulfide bonds. The carboxy-terminal extensions were also shown to incorporate radioactive tryptophan. Since most of the precursor forms of collagen recovered in the incubation medium chromatographed in peak B, it is concluded that matrix-free tendon cells secrete only type I procollagen with extension peptides at both the amino- and carboxy-terminal ends of the molecule.     

Purification and characterization of cystathionine gamma-synthase type II from Bacillus sphaericus

Cystathionine gamma-synthase type II, which catalyzes L-cystathionine synthesis from O-acetyl-L-homoserine and L-cysteine was purified from Bacillus sphaericus (IFO 3536) in seven steps. The purified enzyme appeared to be homogeneous by the results of polyacrylamide electrophoresis and ampholyte electrofocusing. The enzyme is a typical pyridoxal-P dependent enzyme, has a molecular mass of 165 kDa and consists of four subunits identical in molecular mass. The enzyme catalyzed the gamma-replacement reaction and the elimination reaction was hardly detected even when a large amount of enzyme was added. In the replacement reaction, O-acetyl-L-homoserine and the following thiol compounds: L and D-cysteine, L and D-homocysteine, sodium sulfide, various alkyl and aryl mercaptans, acted as the most suitable substrate to produce L-cystathionine and the corresponding S-substituted L-homocysteine derivatives.     

Identification and partial characterisation of the non-collagenous amino- and carboxyl-terminal extension peptides of cartilage procollagen.

Procollagen secreted by embryonic chick cartilage cells has been partially purified by (NH₄)₂SO₄ precipitation and DEAE-cellulose chromatography. Analyses of the products of digestion by human rheumatoid synovial collagenase and bacterial collagenase indicated the presence of non-collagenous peptide sequences at the N- and C-termini. Both regions were found to incorporate [³⁵S]cystine but inter-chain disulphide bonds were restricted to a C-terminal location. Electrophoretic analysis gave apparent molecular weights of 13000 and 36000 daltons for the respective N- and C-terminal extensions.