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Activity of matrix metalloproteinases (MMP) is regulated by a family of proteins called tissue inhibitors of metalloproteinases (TIMP). Four TIMPs have been cloned, and their molecular weights range from 29,000 to 20,000. By reverse zymography, we have observed a metalloproteinase inhibitor with an apparent molecular weight of 16, 500 from medium conditioned by human brain tumor cells. Antibodies directed against TIMPs failed to react with the 16,500 molecular weight inhibitor, indicating that it was not a truncated form of a known TIMP. The inhibitor was isolated from conditioned medium using affinity and ion exchange chromatography. N-terminal sequences of the inhibitor matched amino acid sequences within the C-terminal domain of a protein known as procollagen C-terminal proteinase enhancer (PCPE). Thus, the inhibitor was named CT-PCPE. Comparison of the N-terminal domain of TIMP with CT-PCPE revealed that both contained six cysteine residues. As in the case of TIMP, reduction and alkylation abolished the inhibitory activity of CT-PCPE. Purified CT-PCPE inhibited MMP-2 with an IC(50) value much greater than that of TIMP-2. This implies that MMPs may not be the physiologic targets for CT-PCPE inhibition. However, these results suggest that CT-PCPE may constitute a new class of metalloproteinase inhibitor.

PA28, one of a series of a positive allosteric regulators of the 20 S proteasome, stimulates the enzyme's peptidase activities in an ATP-independent manner by binding to the terminal rings of the 20 S complex. PA28 has a native molecular mass of 180,000 Da and contains at least six subunits of approximately 28,000 Da. In this study we show that PA28 prepared from bovine heart contains two different subunits separable by reverse phase high performance liquid chromatography and that these subunits occur in approximately equal abundance. The subunits display mass values of 27,290 +/- 3.7 and 28,606 +/- 2.8 Da by electrospray mass spectrometry, showing that they differ in covalent structure. Partial amino acid sequence analysis of the subunits indicates that the subunits are the products of two different but homologous genes. A pair of subunits has also been isolated from rabbit heart, and partial amino acid sequence analysis shows each to be homologous to the corresponding subunit in bovine tissues. This indicates that the genes encoding two different polypeptide components of PA28 have been conserved during evolution and suggests the possibility that the two subunits play functionally distinct roles. Isolation of complexes formed between purified PA28 and the 20 S proteasome using density gradient centrifugation reveals that both PA28 subunits bind to the proteasome, indicating that both are components of functional PA28 molecules. These results are consistent with two alternative models for the subunit structure of PA28. There may exist two different PA28 molecules that are homooligomers of the 27,290- and 28,606-Da subunits, respectively. Alternatively, PA28 oligomers may contain mixtures of the 27,290- and 28,606-Da subunits either of fixed or variable stoichiometry.

PA28 is a protein activator of the 20S proteasome. It has a native molecular weight of approximately 200,000 and is composed of six 28,000-dalton subunits arranged in a ring-shaped complex. Purified preparations of PA28 contain two polypeptides, alpha and beta, which are about 50% identical in primary structure. It has been unclear whether native PA28 consists of two distinct homohexameric proteins or of a single protein containing both alpha and beta subunits. To distinguish between these possibilities, we prepared antibodies that reacted specifically with either the alpha or beta subunit and used these subunit-specific antibodies in two types of experiments designed to elucidate PA28 quaternary structure. In the first experiment, the alpha and beta subunits were completely co-immunoprecipitated by each subunit-specific antibody, indicating that both subunits were part of a single protein complex. In the second experiment, PA28 was chemically cross-linked using bis(sulfosuccinimidyl)suberate. When the cross-linked products were immunoblotted after SDS-polyacrylamide gel electrophoresis, indistinguishable patterns were obtained with each subunit-specific antibody. These results confirm that the alpha and beta subunits were part of the same protein complex. The pattern of cross-linked products also provided insight as to the relative abundance and arrangement of the subunits within the PA28 complex and indicated that the ring-shaped PA28 hexamer may be composed of alternating alpha and beta subunits with a stoichiometry of (alphabeta)3. PA28 was inactivated by treatment with carboxypeptidase Y, which cleaved Tyr and Ile residues from the carboxyl terminus of the alpha subunit but had very little effect on the beta subunit. This selective and limited proteolysis prevented binding of both alpha and beta subunits to the proteasome and therefore provides additional evidence of the heterodimeric nature of PA28. These results indicate that a short carboxyl-terminal sequence of the alpha subunit is critical for binding of native PA28 to the proteasome. To learn about the relative functions of the alpha and beta subunits, PA28alpha was expressed in Escherichia coli and purified to homogeneity. Purified PA28alpha stimulated proteasome activity but required 5-10-fold greater concentrations than the heterodimeric PA28 to achieve a given level of activity. These results suggest that the heterodimeric structure of PA28 is required for maximal proteasome activation.