Several investigations showed a positive influence of orally administered gelatin on degenerative diseases of the musculo-skeletal system. Both the therapeutic mechanism and the absorption dynamics, however, remain unclear. Therefore, this study investigated the time course of gelatin hydrolysate absorption and its subsequent distribution in various tissues in mice (C57/BL). Absorption of (14)C labeled gelatin hydrolysate was compared to control mice administered (14)C labeled proline following intragastric application. Plasma and tissue radioactivity was measured over 192 h. Additional "gut sac" experiments were conducted to quantify the MW distribution of the absorbed gelatin using SDS-electrophoresis and HPLC. Ninety-five percent of enterally applied gelatin hydrolysate was absorbed within the first 12 h. The distribution of the labeled gelatin in the various tissues was similar to that of labeled proline with the exception of cartilage, where a pronounced and long-lasting accumulation of gelatin hydrolysate was observed. In cartilage, measured radioactivity was more than twice as high following gelatin administration compared to the control group. The absorption of gelatin hydrolysate in its high molecular form, with peptides of 2.5-15kD, was detected following intestinal passage. These results demonstrate intestinal absorption and cartilage tissue accumulation of gelatin hydrolysate and suggest a potential mechanism for previously observed clinical benefits of orally administered gelatin.

1. The isolation of the ADPiATP translocator from beef heart mitochondria as the bongkrekateprotein complex is described, using hydroxyapatite chromatography and gel filtration in Triton X-100 solution.2. The inhibitor is bound to the protein prior to solubilization with detergent for protection against denaturation. Only the intact bongkrekate-protein passes easily through the hydroxyapatite column. Bongkrekate shields the protein in contrast to carboxyatractylate only partially against proteinases present in the crude extract.3. The isolated bongkrekate protein shows the same molecular weights in dodecylsulfate and Triton X-100, the same amino acid composition and the same isoelectric point as the earlier isolated carboxyatractylate-protein complex. It differs by its higher sensitivity against trypsin and thermolysin.4. The identity of both proteins is demonstrated by interconversion of the bongkrekate-protein into the carboxyatractylate-protein. The process requires the catalysis by ADP or ATP, the natural substrates of the protein.5. The formation of the extractable [3H]bongkrekate-protein complex in mitochondria requires the presence of ADP or ATP.6. These data, the immunological studies presented earlier, and the differences in the reactivity of -SH groups of the isolated bongkrekate and carboxyatractylate complexes (to be published) indicate that both proteins represent different conformational states of the translocator protein (m-state and c-state).The isolation of the most active transport carrier of biomembranes in aerobic eukaryotic cell, the mitochondrial ADP/ATP translocator, has been reported by us recently [I -31. The protein was isolated in undenatured form as the carboxyatractylate-protein complex and turned out, in agreement with its eminent role, to be also the most prominent polypeptide of mitochondria and the most abundant membrane protein in heart and probably many other aerobic eukaryotic cells.' A unique feature of this translocator is the existence of two types of tightly binding inhibitors, carboxyatractylate and its homologues and bongkrekate [4,5], the application of which permitted to give a first insight into the molecular mechanism of a translocation process. Thus opposite effects of both ligands on the ADP binding to the carrier were observed: whereas carboxyatractylate removed ADP, bongkrekate appeared to increase the affinity of ADP [6,7]. This effect and a number of other results could be explained by the reorientation mechanism which implies that the translocator exists in two extremes of conformations largely identical with the translocation step [I, 8,9] ; in the 'c-state' where the substrate binding site is turned to the cytosol, and in the 'mstate' where the site is turned to the matrix. Transition between both states occurs only in the presence of ADP or ATP accompanied by the transport of the substrates across the membrane. The c-state can be fixed by binding carboxyatractylate and the m-state by binding bongkrekate.The isolation of the bongkrekate complex of the carri...

The cDNA and protein sequences of the N-terminal half of human basement membrane collagen (type IV) have been determined. Overlapping cDNA clones were constructed by repeated primer extension with synthetic oligonucleotides. They cover 2953 bp, beginning at the 5' end of the corresponding mRNA. At the protein level, the sequence of the cyanogen bromide peptide CB6 adjacent to the 7s domain has been additionally elucidated. The data presented here complete the protein sequence and nearly the entire cDNA sequence of the human al(IV) chain. The amino-terminal half of the al(1V) chain contains 8 cysteine residues involved in intramolecular and intermolecular cross-links. The entire triple-helical domain of al (1V) is interrupted by 21 non-triplet regions.Type IV collagen, the main structural constituent of basement membranes, is a 400-mm-long triple-helical heterotrimer consisting of two al(IV) and one a2(IV) chains [l-41. Both chains are more than 1600 amino acid residues long and contain three functionally distinct domains. These are the two end regions, the N-terminal 7s domain and the C-terminal globular NCI domain and the central triple-helical part. The two terminal domains are responsible for the aggregation of monomeric molecules into a network and the formation of the stabilizing intermolecular cross-links [5]. In the central triple helix, the repetitive tripeptide sequence (Gly-Xaa-Yaa) is frequently interrupted by non-tripeptide regions [6 -81.The amino acid sequences of the 7s domain [9], the NC1 domain [lo, 1 I], and about 900 residues of the central triplehelical part [7] of the human al(1V) chain are known. The NC1 domain and 165 residues at the C-terminus of the triple helix of the murine al(1V) chain have also been published [12]. Sequence studies of the a2(IV) chain are less advanced. The sequences of the 7 s domain of human a2(1V) collagen (B. Siebold, R. Q. Qian, R. W. Glanville, H. Hofmann, R. Deutzmann and K. Kiihn, unpublished results) and the Cterminal 500 residues of the triple-helical area [8] and the NC1 domain of murine collagen are known [13].Continuing investigations of the protein and gene structure of human type IV collagen have elucidated the cDNA sequence of the 5' half of the al(1V) chain. This included the N-terminal signal sequence and the N-terminal region of the triple-helical domain and thus completed the amino acid sequence of the human al(1V) chain. Part of the triple-helical domain sequence was also determined using protein sequencing.

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