The ontogenetic transition of collagen deposition in rat skin.

Minimizing the morbidity of in utero surgery or, perhaps more important, capturing the unique characteristics of scarless remodeling, as is the fetal response to injury, demands better elucidation of the observed variations from adults in whom the normal progression of wound healing leads to fibrosis. Species-dependent fetal phlogistic responses and wound scar formation represent a temporal continuum before the onset of adult patterns. We have analyzed skin collagen synthesis and content in Sprague-Dawley rats as one possible factor in this evolution showing that the fetal characteristics of a high percentage of type III collagen relative to type I and low total collagen content are maintained as long as the first 10 to 15 days postpartum. Although extrapolation of such a crucial "golden period" to justify the delay of human surgical procedures while still capturing the benefits of the fetal milieu remains speculative, anecdotal observations of minimal scar formation lend some credibility for performing less invasive maneuvers in the neonate.

Analysis of collagen content in the fetal wound.

The absence of apparent scar formation following the creation of surgical wounds in utero appears to be a phenomenon peculiarly privileged as a sequela of fetal wound healing. Little information exists to explain this disparity from our knowledge of adult wound healing. Therefore, following creation of surgical wounds in fetal rats, at different intervals the healing wounds were harvested and analyzed for collagen content and types. The average proportion of type III collagen was elevated in normal (26.5%) as well as wounded fetal skin (33.8%) when compared with normal levels for the adult (15%). The total collagen content was markedly diminished in the fetal wound. Although embryonal collagen synthesis apparently does exist in fetal reparative processes, the relationship to the lack of gross scarring remains undetermined.

Type I and type III collagen content of healing wounds in fetal and adult rats.

Full-thickness, dermal wounds were surgically created on the dorsa of fetal rats on the 17th day of gestation. The granulation tissue which developed after 2 days (19 days of gestation) was harvested from six to nine animals and pooled and the collagen was extracted with 0.5 M acetic acid and acetic acid plus pepsin. The ratio of type III:type I collagen was estimated from densitometer scans of electrophoretically separated alpha-chains. Full-thickness (to fascia depth) wounds were also produced on the dorsa of adult rats and granulation tissue which had developed for different periods of time up to 30 days was excised. Relative proportions of type III and type I collagen were assessed in normal and granulation tissues taken from the adult rats. Both fetal and adult granulation tissues have elevated type III collagen content but normal fetal tissue has a much higher content of type III than does normal adult tissue.

Improved purification and some molecular and kinetic properties of sn-glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae.

The purification procedure for isolating sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) from Saccharomyces cerevisiae was improved by the introduction of an ion-exchange step. Enzyme yields were doubled and the specific activity was increased as compared to the original procedure. A new value of 42,000 was obtained for the molecular weight by several denaturing methods. By native gel chromatography the molecular weight appears to be 31,000 as reported earlier. Michaelis constants were found to be 0.37 mM with dihydroxyacetone phosphate as the variable substrate and 0.018 mM for NADH as the variable substrate.

Collagenolytic activity of Vibrio vulnificus: potential contribution to its invasiveness.

Vibrio vulnificus (lactose-positive vibrio) produced collagenase when grown in 2% synthetic sea salts supplemented with hydrolyzed casein. The addition of collagen or peptone to the medium increased the level of collagenase production. Collagenase activity was inhibited by EDTA but not by fetal calf serum.

Nereis cuticle collagen. Proteolysis by marine vibrial and clostridial collagenases.

Proteolysis of Nereis cuticle collagen by two bacterial collagenases was investigated using viscosimetry, enzyme kinetics, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and ion exchange chromatography of collagenolytic peptides. Collagenase of the marine Vibrio B-30 completely degrades native cuticle collagen at 7 degress C with a turnover number 50 times greater than that of the clostridial collagenase. Although turnover numbers for the two enzymes are comparable when using denatured cuticle collagen as substrate, the vibrial collagenase appears to cleave twice as many peptide bonds per mg of cuticle collagen as does the clostridial enzyme. Sodium dodecyl sulfate gel electrophoresis of collagenase-digested native cuticle collagen reflects the resistance of the collagen to clostridial collagenase; however, the vibrial enzyme completely degrades the cuticle collagen with the formation of one transient intermediate (Mr 400,000). Peptide analysis of fully digested denatured cuticle collagen reveals that the two enzymes have a number of qualitative and quantitative similarities. Despite these, however, only the vibrial collagenase seems capable of extensively degrading native cuticle collagen.

Induction of collagenase production in Vibrio B-30.

The inducible nature of an extracellular collagenase produced by a marine Vibrio (Vibrio B-30, ATCC 21250) was demonstrated by observing the increase in extracellular collagenase activity after the addition of collagen to cell cultures in the latter part of the exponential growth phase. When collagenase-hydrolyzed collagen was added, the lag time required before collagenase production was detected decreased significantly compared with cultures receiving collagen. Cells preinduced to synthesize collagenase did not produce the enzyme when collagen was removed from the culture medium. Incorporation of penicillin G had no effect on final collagenase activity levels in suspensions of Vibrio B-30 in complete medium supplemented with collagen. However, chloramphenicol and tetracycline inhibited collagenase production, indicating that de novo protein synthesis was necessary for the appearance of activity. Attempts to isolate the inducing substance(s) involved filtering hydrolyzed collagen through a series of ultrafiltration membranes. The lowest-molecular-weight fraction of collagen hydrolysate with inducing ability was between 1,000 and 10,000. Gel filtration of this fraction on Sephadex G-50 resulted in the appearance of three protein peaks, two of which were capable of inducing collagenase production. Results from amino acid composition and N-terminal amino acid analysis suggest that the inducing substance originates from the polar helical portion of the collagen molecule.

Purification and characterization of a marine bacterial collagenase.

A true collagenase was isolated from the culture fluid of a marine bacterium which has been designated Vibrio B-30 (ATCC 21250). Collagenase production was obtained only in media containing collagen or certain degradation products of collagen. Partial purification on DEAE-cellulose and Sephadex G-200 columns produced active enzyme which was free of nonspecific proteases but which contained two collagenases. The two collagenases have the same apparent molecular size, and evidence is presented to support the theory that one collagenase is derived from the other. Vibrio B-30 collagenase appears to be a tetramer with a molecular weight of about 105 000 composed of two different subunits (mol wt 24 000 and 28 000). Some of the properties of the Vibrio collagenase are compared with those of Clostridium histolyticum collagenase. Molecular weights, subunit structures, specificity and mode of collagen hydrolysis, insensitivity to diisopropyl fluorophosphate and calf serum, and sensitivity to certain metal ion complexing agents and isopropyl alcohol are similar for the collagenases from both organisms. However, Vibrio B-30 collagenase and Clostridium collagenase differ immunologically and electrophoretically.

Collagenolytic activity of some marine bacteria.

Reconstituted, acid-extracted collagen was used to prepare a medium to screen proteolytic marine bacteria for their ability to elaborate collagenolytic enzymes. The medium was resistant to solubilization by trypsin, hyaluronidase, chondroitinase ABC, and various marine proteinases, but was readily hydrolyzed by commercial Clostridium collagenases. Eighty-seven marine isolates collected in the vicinity of Bermuda, Oahu (Hawaii), and Stone Harbor and Cape May, N. J., were screened. Approximately 44 per cent of the isolates were capable of elaborating enzymes that hydrolyzed reconstituted collagen gels. Several cultures produced collagenolytic enzymes only when grown in the presence of collagen or degradation products of collagen, and with very few exceptions the presence of collagen in the medium greatly enhanced collagenolytic enzyme production. The enzymes from a collagenolytic Bermuda marine isolate were studied in more detail to illustrate that the enzymes capable of hydrolyzing reconstituted collagen were separable from nonspecific proteinases by zone electrophoresis and that these enzymes were true collagenases by virtue of their ability to hydrolyze native bovine Achilles'tendon obtained from three different sources.