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Antisera were prepared by immunizing a rabbit with procollagen synthesized and secreted by cells from chick embryo tendons. Specific antibodies were then purified from the antisera by using an immunoadsorbent which contained the isolated NHz-terminal extensions of procollagen. The purified antibodies were shown to react specifically with intact procollagen as well as with procollagen in which the interchain disulfide bonds were ruptured by reduction under non-denaturing conditions. The antibodies also reacted with the pro-a1 chain but not the pro-a2 chain isolated from the procollagen. There was no reaction after the intrachain bonds in the pro-a chains of the procollagen were reduced under denaturing conditions and alkylated. In the course of characterizing the antibodies it was shown that the NH2-terminal extensions on the two pro-a1 chains and the one pro-a2 chain of type I procollagen are non-identical. Also, it was shown that the serum of chick embryos contains an antigen which reacts with the specific antibodies to procollagen. Collagen is synthesized by connective tissue cells in a precursor form called procollagen which is larger than the collagen molecule because of peptide extensions on the NH2-terminal end of each of the three a chains (for recent review, see [l]). The amino-acid composition of the extensions differs from that of collagen and it includes cysteine and tryptophan, aminoacids which are not found in collagen itself. A large fraction of the procollagen synthesized and secreted by cells from chick embryo tendons has been shown to consist of pro-a chains linked by interchain disulfide bonds [2]. Interchain disulfide bonds have also been demonstrated in procollagens from cultured fibroblasts [3,4], membranous bone [5,6], and lens cells from chick embryos [7]. Antibodies against the NH2-terminals extensions of procollagen have recently been prepared by using procollagen from three different sources for immunization. Purified antibodies to the NH2-terminal extension of procollagen which was extracted from the skin of cattle with the disease called dermatosparaxis were shqwn to react both with the native procollagen from the skin of the diseased cattle and with the isolated polypeptide chains from this form of procollagen [S]. Enzymes. Collagenase or clostridiopeptidase A (EC

cis,cis-Muconate cycloisomerase (cis,cis-muconate lactonizing enzyme, EC 5.5.1.1.) was purified in crystalline form from Pseudomonas putida. Ultracentrifugation studies, as well as gel filtration chromatography and electrophoresis, indicate that the enzyme is an oligomeric protein of molecular weight 252,000 (s20,w 12.20 × 10−13 s), which is built of six homologous protomers of molecular weight 42,000. Studies of enzyme crystals and enzyme molecules in the electron microscope suggest that the cis,cis-muconate cycloisomerase is a hexamer in which the six protomers are arranged in a dihedral point-group symmetry 32 (D3). Each protomer has a diameter of 42.5Aand six protomers are associated in a structure with a trigonal antiprismatic geometry (a hexamer D3 octahedron). This model could account for the dimensions most frequently observed by negative staining of the enzyme in solution. A model for the three-dimensional structure of enzyme crystals in which each hexameric enzyme molecule is surrounded by eight neighbouring enzyme molecules, is described.

Protocollagen proline hydroxylase was purified from chick embryos with an affinity-column procedure which yielded pure enzyme in a tetramer form. Electron microscopy of the enzyme after it was largely dissociated into monomers indicated that the monomers were rod-shaped with a diameter of 3.31 ± 0.07 nm (S.E.M.) and a length of 6.95 ± 0.07 nm. In preparations of the enzyme in which the protein was partially dissociated, dumb-bell and V-shaped structures were seen which morphologically consisted of two sub-structures each with dimensions similar to those of the isolated monomers. The results suggested that these structures were dimers in which the monomers were joined at one end with an angle between their longitudinal axes. The largest regular protein structures seen in preparations of the enzyme were about 8 × 8 nm and in some of these structures four sub-structures were seen, indicating that they were tetramers. On the basis of the data presented here we propose a model of the tetramer in which two V-shaped dimers were interlocked. Further studies demonstrated that the single-ring and four-ring structures seen by electron microscopy of enzyme preparations obtained by a previous purification procedure were not the enzyme.

Previous reports demonstrated that synthetic polytripeptides of the structure (Pro-Pro-Gly)10, prepared with a specific modification of the Merrifield technique, formed triple helical structures in aqueous solution which were similar to the triple helical structures of tropocollagen. The studies presented here were undertaken in order to answer the question of whether the individual polytripeptide chains within the triple helices were packed in a parallel fashion similar to vertebrate collagen or in an antiparallel fashion similar to the structure which has been suggested for collagen from the cuticle of the round worm, Ascaris lumbricoides. It was found that electrostatic interactions between the COOH-terminal and NH-terminal groups of (Pro-Pro-Gly)10 could be used to probe the detailed structure of triple helices formed by the polytripeptide. Titration of (Pro-Pro-Gly)10 in triple helical conformation indicated that the pK of the NH-terminal group was 0.59 units lower than the pK for the same group in nonhelical (Pro-Pro-Gly)5. The titration of the NH-terminal group of helical (Pro-Pro-Gly)10 was significantly broader than the theoretically predicted curve, and the pK increased after the polytripeptide was heated and quenched. Furthermore, the melting curve for the helix coil transition of (Pro-Pro-Gly)10 was changed by protonation of the NH-terminal or COOH-terminal groups. The Tm for the dipolar ion form was about 10° lower than for either the fully protonated or unprotonated forms of the helical polytripeptide. The results indicate that in the triple helical structure formed by (Pro-Pro-Gly)10 the three chains are in register and parallel.

Protocollagen proline hydroxylase was purified from chick embryos, and it was examined by electron microscopy with negative staining techniques. Enzyme prepared in Helsinki consisted almost entirely of single ring structures. Enzyme prepared in Philadelphia under essentially the same conditions consisted almost entirely of tubular forms in which four rings were stacked together. Repeated freezing and thawing of the Helsinki preparation markedly increased the number of four-ring structures, but it was not possible to establish reproducible conditions for the interconversion of these two major forms of the enzyme. Each ring in the four-ring structure had a diameter of 100 A, a height of 30 to 35 A, and a central hole of about 20 A. The single ring structure was slightly more distended. The molecular weight of each ring was about 200,000, and therefore the molecular weight of the four-ring structure was about 800,000. The inner two rings of the four-ring structure were closer together than the outer two rings. Since the four rings appeared to be identical, the observations suggested that the rings were held together by a combination of heterologous and isologous bonds. No regular structures with more than four rings were seen, and the simplest bonding sequence consistent with these observations was (AB)—(AB)— (BA)—(BA) in which larger structures were not favored, because the formation of the inner B—B bond altered the A surfaces on the inner rings so that they were not equivalent to the A surfaces on the outer rings.

When matrix-free tendon cells from chick embryos were incubated with [14C]proline and then extracted with sodium dodecyl sulfate and mercaptoethanol, a major part of the newly synthesized 14C-protein was found to consist of collagen polypeptides which were complete in that their content of [14C]hydroxyproline and their size was the same as that of polypeptides of the precursor form of collagen secreted into the medium. When the cells were incubated with 0.3 mm α,α'-dipyridyl so as to inhibit protocollagen proline hydroxylase and protocollagen lysine hydroxylase, secretion of 14C-protein was inhibited and [14C]protocollagen accumulated within the cells. [14C]Protocollagen extracted from the cells was comprised of polypeptides which were of the same size as polypeptides of the intracellular collagen or about 125,000 as estimated by gel filtration in sodium dodecyl sulfate. In contrast, the small amount of peptide-bound 14C still secreted in the presence of α,α'-dipyridyl was shown to consist of small peptides which were in part derived from the intracellular degradation of [14C]protocollagen. The results demonstrated therefore that in freshly isolated tendon cells protocollagen itself is not secreted. Since the rate of protein synthesis in the presence of α,α'-dipyridyl was the same as under control conditions for about 90 min, the data suggested that the intracellular [14C]protocollagen accumulated in some postribosomal compartment. Extraction of control cells with acetic acid solubilized a large fraction of the intracellular collagen, and amino acid analyses indicated that 40 to 50% of the protein in the extracts was collagen. The protein extracted from cells incubated with α,α'-dipyridyl was similar in amino acid composition, but it contained essentially no hydroxylysine and hydroxyproline and was correspondingly rich in lysine and proline. In addition, 40% of the proline in the protein extract was converted to hydroxyproline after incubation with pure protocollagen proline hydroxylase. Experiments involving limited pepsin digestion provided the first demonstration that intracellular collagen and proto-collagen are largely in a native, triple-helical conformation. After acetic acid extracts from control cells and from cells incubated with α,α'-dipyridyl were dialyzed against ATP, segment long spacing aggregates were obtained. The aggregates were similar to those formed by extracellular, fibrillar collagen except that they had a 130-A NH2-terminal extension which was indistinguishable from that seen in aggregates of the precursor form of collagen secreted by the same cells.

An electron microscope investigation of the structure of native vitrosin fibrils and the morphology and interaction properties of their macromolecular building units are described. It is concluded that the morphology, namely the diameter, length, and intramolecular distribution of polar amino acids as revealed by positive staining of vitrosin molecules with phosphotungstic acid, is identical with that of tropocollagen. The packing arrangement of molecular units in the vitrosin fibrils differs from that of other vertebrate collagens investigated. The conclusion is drawn that it is impossible to construct a simple molecular model which faithfully reproduces the vitrosin structure. However, units of higher order, such as dimers with specific properties, are possibly characteristic elements of the native fibrillar structure.

An improved procedure was used to conjugate ferritin to antibodies specific for the NH 2 -terminal extensions on the precursor form of collagen known as procollagen. The ferritin-antibody conjugates were then used to determine the localization of procollagen in fibroblasts isolated from chick-embryo tendons. Procollagen was found in the cisternae of the endoplasmic reticulum, indicating that the protein passes into this compartment early in its biosynthesis. Specific labeling of large Golgi vacuoles was also observed, suggesting that this compartment is also involved in the secretory process. When cells were incubated with colchicine so that the secretion of procollagen was delayed, there was an increase of large, smooth-surfaced vacuoles in the cells and these vacuoles were labeled with the ferritin-antibody conjugate.

An improved procedure was employed for linking ferritin to antibodies against prolyl hydroxylase, the enzyme that synthesizes the hydroxyproline in collagen. By electron microscopy, the enzyme was then found to be localized in cisternae of the rough endoplasmic reticulum of embryonic tendon cells; this indicates that hydroxylation of proline occurs while newly synthesized polypeptides are fed into the cisternae.

Highly purified pig renal glutaminase has been subjected to electron microscopy. It is shown that the polymers formed after addition of phosphate-borate to enzyme dissolved in Tris-HCl buffer consist of long, double-stranded helices. Addition of phosphate to the Tris-HCl form of the enzyme probably induces the formation of dimers of the Tris-HCl units. It is further demonstrated that the rate of formation of the phosphate form and phosphate-borate form from Tris-HCl units, closely follows the increase in the catalytic activity of the enzyme.

When cells were isolated from chickembryo tendons and incubated in vitro for 2-6 hr, essentially all the newly-synthesized collagen was recovered from the incubation medium as a transport form larger than tropocollagen. Experiments in which cells were incubated with [ 14 C]cystine suggested that the transport form contained cystine and that it was, in part, stabilized by disulfide bonds. Electron microscopy of segment-long-spacing aggregates prepared from the transport form of collagen showed that the native molecule differed from tropocollagen in that it had an extension of about 13 nm (130 Å) at the NH 2 -terminal end.

Highly purified pig renal glutaminase has been examined by electron microscopy. It is demonstrated that the Tris·HCl form of the enzyme contains cylindrical particles with a diameter of about 60 A and a length of about 82 A. Treatment of the Tris·HCl form of the enzyme with sodium dodecyl sulphate and mercapto-ethanol, followed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate, shows that the enzyme contains two types of sub units with molecular weights of 53,000 and 61,000. In agreement with earlier data, it is further demonstrated that the phosphate form is a simple dimer of the Tris·HCl form. Evidence that major conformational changes are involved in the formation of large, helical polymers of the enzyme described earlier, is also presented.

The present paper describes an electron microscope investigation of the ultrastructure of native collagen fibrils with negative and positive staining techniques.

The native and subunit molecular weights of the pH6 acetolactate-forming enzyme from Aerobacter aerogenes have been investigated by sedimentation equilibrium analysis and gel electrophoresis. The values obtained are 220 000 for the native enzyme and 58 000 for its subunits. The native enzyme is dissociated into four equal-sized subunits by exposure to guanidine hydrochloride, urea, or sodium dodecylsulfate. Electron micrographs of enzyme molecules, negatively stained, show that they appear as spherical particles with a diameter of about 6 nm forming dimeric structures of two particles close together. The molecular weight as calculated from the diameter of the particles, are consistent with the values obtained by sedimentation equilibrium and gel electrophoresis. The native enzyme molecule is visualized as a morphological dimer with each particle containing two polypeptide chains of similar size.

A soluble fraction was isolated from anterior lens capsule basement membrane by treatment with a dilute, acidic solution of sodium citrate. This fraction had an amino acid and carbohydrate composition similar to that of the intact basement membrane. Upon electron microscopy, this fraction was found to contain two morphologically distinct components : thin strands measuring 2-3 nm in diameter and globular units measuring 5-20 nm diameter. Treatment with pepsin destroyed the globular component leaving the filaments intact. The amino acid composition of the pepsin-treated extract was similar to that of basement membrane collagen. Treatment with bacterial collagenase resulted in the destruction of the filamentous components leaving the globular components intact. These data indicate that the filamentous structure was collagenous whereas the globular component was not. Reduction of disulfide bonds resulted in an apparent dissociation of the globular unit from the filamentous component. The data indicate that the protein extracted with sodium citrate represents a structural subunit of the lens capsule basement membrane.

The problem whether the molecular size of horseradish peroxidase is significantly altered when the enzyme is injected into the blood stream in tracer studies, has been studied by molecular sieve chromatography (gel filtration). The results obtained rule out the possibility that the horseradish peroxidase molecule (containing about 20% carbohydrates) is significantly degraded by carbohydrate-splitting enzymes or by proteases in the circulation into a smaller active unit. Furthermore, significant binding of peroxidase to plasma proteins or polymerization of the enzyme, has not been detected. It is concluded, therefore, that conclusions about the size of functional pores based on the known molecular weight (40000 daltons) are permitted, when the enzyme is used in permeability studies.