Person:

Olsen, Bjorn

Three distinct antiprocollagen preparations were characterized and used in immunocytochemical staining of chick embryo corneal and tendon cells. The several ferritin-conjugated antibody preparations permitted similar location of procollagen in the cisternae of the rough endoplasmic reticulum and in Golgi elements in both cell types. The ability to demonstrate and interpret specific ferritin staining was dependent on the extent of membrane breakage in each of those organelles, coupled with adequate retention of cell morphology. Corneal fibroblasts appeared to suffer more extensive intracellular membrane damage under controlled conditions of homogenization than tendon fibroblasts, facilitating the identification of procollagen in Golgi vacuoles of these cells. None of the labeled material appeared to by cytoplasmic in origin since ferritin was observed in the cytoplasm only in the vicinity of Golgi elements that were extensively broken. This study extends previous immunological evidence for the presence of procollagen in the Golgi complex and calls attention to the problems to be encountered in locating the antigen in small Golgi vesicles and lamellae.

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.

Morphological studies were carried out on fibroblasts from chick embryo tendons, cells which have been used in a number of recent studies on collagen biosynthesis. The cells were relatively rich in endoplasmic reticulum and contained a well-developed Golgi complex comprised of small vesicles, stacked membranes, and large vacuoles. Techniques were then devised for preparing cell fragments which were penetrated by ferritin-antibody conjuates but which retained the essential morphological features of the cells. Finally, the new procedures were employed to develop further information as to how collagen is synthesized. As reported elsewhere, preliminary studies with ferritin- labeled antibodies showed that prolyl hydroxylase was found in the endoplasmic reticulum of freshly isolated fibroblasts and that procollagen is found in both the cisternae of the endoplasmic reticulum and the large Golgi vacuoles. In the experiments described here, the cells were manipulated so that amino acids continued to be incorporated into polypeptide chains but assembly of the molecule was not completed because hydroxylation of prolyl and lysyl residues was prevented. The results indicated that these manipulations produced no change in the distribution of prolyl hydroxylase. Examination of the cells with ferritin conjugated to antibodies which reacted with protocollagen, the unhydroxylated form of procollagen, demonstrated that protocollagen was retained in the cisternae of the endoplasmic reticulum during inhibition of the prolyl and lysyl hydroxylases. Assays for prolyl hydroxylase with an immunologic technique demonstrated that although the enzyme is found within the endoplasmic reticulum, it is not secreted along with procollagen. The observations provided further evidence for a special role for prolyl hydroxylase in the control of collagen biosynthesis.

Two improved procedures were developed for activating ferritin so that the ferritin could be covalently linked to antibodies. One procedure involved use of a water-soluble carbodiimide and N-hydroxysuccinimide to prepare ferritin-containing activated esters. The other involved activation of the ferritin with excess glutaraldehyde. The ferritin- antibody conjugates prepared with the two procedures were shown to have a number of properties which made them suitable for locating antigenic components in cells.

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.