Thrombospondin fragmentation by alpha-thrombin and resistance to gamma-thrombin.

In the presence of 2mM-Ca2+, alpha-thrombin slowly cleaved thrombospondin (Mr 180 000) into 150 000-Mr and 30 000-Mr fragments. In the absence of Ca2+, the platelet glycoprotein was progressively and completely hydrolysed by 3 units of the enzyme/ml to 130 000-Mr, 95 000-Mr and 65 000-Mr fragments. In contrast, the nonclotting enzyme form, gamma-thrombin, did not hydrolyse the platelet protein either in the presence or in the absence of Ca2+, even at 10-fold higher concentrations of enzyme. Protein-interacting regions removed from the catalytic site, like those required for fibrinogen recognition, are necessary for thrombin proteolysis of thrombospondin.     

Ca2+-binding proteins from bovine brain including a potent inhibitor of protein kinase C.

We have previously described the use of Ca2+-dependent hydrophobic-interaction chromatography to isolate the Ca2+ + phospholipid-dependent protein kinase (protein kinase C) and a novel heat-stable 21 000-Mr Ca2+-binding protein from bovine brain [Walsh, Valentine, Ngai, Carruthers & Hollenberg (1984) Biochem. J. 224, 117-127]. The procedure described for purification of the 21 000-Mr calciprotein to electrophoretic homogeneity has been modified to permit the large-scale isolation of this Ca2+-binding protein, enabling further structural and functional characterization. The 21 000-Mr calciprotein was shown by equilibrium dialysis to bind approx. 1 mol of Ca2+/mol, with apparent Kd approx. 1 microM. The modified large-scale purification procedure revealed three additional, previously unidentified, Ca2+-binding proteins of Mr 17 000, 18 400 and 26 000. The 17 000-Mr and 18 400-Mr Ca2+-binding proteins are heat-stable, whereas the 26 000-Mr Ca2+-binding protein is heat-labile. Use of the transblot/45CaCl2 overlay technique [Maruyama, Mikawa & Ebashi (1984) J. Biochem. (Tokyo) 95, 511-519] suggests that the 18 400-Mr and 21 000-Mr Ca2+-binding proteins are high-affinity Ca2+-binding proteins, whereas the 17 000-Mr Ca2+-binding protein has a relatively low affinity for Ca2+. Consistent with this observation, the 18 400-Mr and 21 000-Mr Ca2+-binding proteins exhibit a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, whereas the 17 000-Mr Ca2+-binding protein does not. The amino acid compositions of the 17 000-Mr, 18 400-Mr and 21 000-Mr Ca2+-binding proteins show some similarities to each other and to calmodulin and other members of the calmodulin superfamily; however, they are clearly distinct and novel calciproteins. In functional terms, none of the 17 000-Mr, 18 400-Mr or 21 000-Mr Ca2+-binding proteins activates either cyclic nucleotide phosphodiesterase or myosin light-chain kinase, both calmodulin-activated enzymes. However, the 17 000-Mr Ca2+-binding protein is a potent inhibitor of protein kinase C. It may therefore serve to regulate the activity of this important enzyme at elevated cytosolic Ca2+ concentrations.     

Characterization of a high-Mr plasma-membrane-bound protein and assessment of its role as a constituent of hyaluronate synthase complex.

A high-Mr phosphoprotein (Mr 442,000) was purified from Nonidet-P-40-solubilized plasma membranes of cultured human skin fibroblasts. The protein comprised one 200,000-Mr subunit consisting of 116,000- and 84,000-Mr polypeptides and two identical 121,000-Mr subunits each consisting of 66,000- and 55,000-Mr polypeptides. The 200,000-Mr subunit and its polypeptides contained phosphotyrosine residues and were also [32P]phosphorylated at these residues from [gamma-32P]ATP in vitro by an intrinsic tyrosine kinase activity of the protein molecule in response to the presence of hyaluronate precursors, UDP-glucuronic acid and UDP-N-acetylglucosamine. The 121,000-Mr subunits and their polypeptides contained phosphoserine residues that could not be [32P]phosphorylated during autophosphorylation of the protein in vitro. The protein molecules separated from exponential- and stationary-growth-phase cells were identical in their quaternary structure, but appeared to exist in different proportions with respect to the state of phosphorylation of their 121,000-Mr subunits during different growth phases of the cell. Phosphorylation of polypeptides appeared to predispose in favour of their UDP-glucuronic acid- and UDP-N-acetylglucosamine-binding activities. The phosphorylated 116,000- and 84,000-Mr polypeptides of 200,000-Mr subunits possessed a single binding site for UDP-glucuronic acid and UDP-N-acetylglucosamine respectively. The phosphorylated 200,000-Mr subunit could also cleave the UDP moiety from UDP-glucuronic acid and UDP-N-acetylglucosamine precursors. The phosphorylated 121,000-Mr subunit possessed two binding sites with equal affinity towards UDP-glucuronic acid and UDP-N-acetylglucosamine but did not possess UDP-moiety-cleavage activity. The phosphorylation of 200,000-Mr subunit by an intrinsic kinase activity of the protein molecule appeared to elicit its oligosaccharide-synthesizing activity, whereas phosphorylation of 121,000-Mr subunits, presumably carried out in vivo, abolished this activity of the protein molecule. The oligosaccharides synthesized by the protein were about Mr 5000 and about 12 disaccharide units in length. Neither nucleotide sugars nor glycosyl residues nor newly synthesized oligosaccharides were bound covalently to the protein molecule. The UDP moiety of nucleotide sugar precursors did not constitute a link between protein molecule and oligosaccharide during its synthesis. Although isolated 442,000-Mr protein did not synthesize high-Mr hyaluronate in vitro, this protein molecule can be considered as a constituent of membrane-bound hyaluronate synthase complex because of its observed properties.     

Purification and characterization of glutathione S-transferases of human kidney.

Several forms of glutathione S-transferase (GST) are present in human kidney, and the overall isoenzyme pattern of kidney differs significantly from those of other human tissues. All the three major classes of GST isoenzymes (alpha, mu and pi) are present in significant amounts in kidney, indicating that GST1, GST2 and GST3 gene loci are expressed in this tissue. More than one form of GST is present in each of these classes of enzymes, and individual variations are observed for these classes. The structural, immunological and functional properties of GST isoenzymes of three classes differ significantly from each other, whereas the isoenzymes belonging to the same class have similar properties. All the cationic GST isoenzymes of human kidney except for GST 9.1 are heterodimers of 26,500-Mr and 24,500-Mr subunits. GST 9.1 is a dimer of 24,500-Mr subunits. All the cationic isoenzymes of kidney GST cross-react with antibodies raised against a mixture of GST alpha, beta, gamma, delta and epsilon isoenzymes of liver. GST 6.6 and GST 5.5 of kidney are dimers of 26,500-Mr subunits and are immunologically similar to GST psi of liver. Unlike other human tissues, kidney has at least two isoenzymes (pI 4.7 and 4.9) associated with the GST3 locus. Both these isoenzymes are dimers of 22,500-Mr subunits and are immunologically similar to GST pi of placenta. Some of the isoenzymes of kidney do not correspond to known GST isoenzymes from other human tissues and may be specific to this tissue.     

Mouse alpha-macroglobulin. Structure, function and a molecular model.

Mouse alpha-macroglobulin (M-AMG) is believed to be a functional homologue of human alpha 2-macroglobulin (h-alpha 2M). The subunit composition, the tryptic cleavage pattern before and after methylamine incorporation and the two-dimensional tryptic-peptide mapping, however, indicate that these two proteins are structurally distinct. M-AMG is composed of two major types of polypeptides (Mr 163,000 and 35,000) together with a minor polypeptide (Mr 185,000), whereas h-alpha 2M has only one type of polypeptide (Mr 185,000). After incorporation of methylamine, there is no change in the normal tryptic-cleavage pattern of M-AMG; however, tryptic cleavage of h-alpha 2M is severely retarded [Hudson & Koo (1982) Biochim. Biophys. Acta 704, 290-303]. The N-terminal sequence of the 163,000-Mr polypeptide of M-AMG shows sequence homology with the N-terminal sequence of h-alpha 2M. The amino acid compositions of M-AMG and its two major polypeptide chains are compared. Thermal fragmentation studies show that the 163,000-Mr polypeptide is broken down into 125,000-Mr and 29,000-Mr fragments. Trypsin-binding studies show that M-AMG can bind two molecules of trypsin/molecule. Inactivations of the trypsin-binding property of M-AMG and h-alpha 2M with methylamine show similar kinetics of inhibition at 4 degrees C. A structural model of M-AMG is proposed, based on accumulated data.     

The structure of Artemia sp. (brine shrimp) haemoglobins. Purification of a structural unit to homogeneity.

The extracellular haemoglobins (Mr 260 000) of the brine shrimp Artemia sp. were cleaved by limited digestion with subtilisin. Structural units of Mr 16 000, which can bind dioxygen reversibly, were isolated. Analysis of the 16 000-Mr fraction (E) reveals the presence of a limited number of structural units. A single type of structural unit, E1 (Mr 15 800; pI4.8), was purified to homogeneity and characterized.     

Isolation and characterization of six human hepatic isometallothioneins.

Human brain contains one cationic (pI8.3) and two anionic (pI5.5 and 4.6) forms of glutathione S-transferase. The cationic form (pI8.3) and the less-anionic form (pI5.5) do not correspond to any of the glutathione S-transferases previously characterized in human tissues. Both of these forms are dimers of 26500-Mr subunits; however, immunological and catalytic properties indicate that these two enzyme forms are different from each other. The cationic form (pI8.3) cross-reacts with antibodies raised against cationic glutathione S-transferases of human liver, whereas the anionic form (pI5.5) does not. Additionally, only the cationic form expresses glutathione peroxidase activity. The other anionic form (pI4.6) is a dimer of 24500-Mr and 22500-Mr subunits. Two-dimensional gel electrophoresis demonstrates that there are three types of 26500-Mr subunits, two types of 24500-Mr subunits and two types of 22500-Mr subunits present in the glutathione S-transferases of human brain.     

Purification and characterization of the phosphorylated DNA-binding protein from adenovirus-type-2-infected cells.

The virus-coded 72000-Mr DNA-binding protein from adenovirus-type-2-infected cells has been purified to homogeneity by DEAE-cellulose chromatography, selective precipitation and gel filtration. The 72000-Mr DNA-binding protein is phosphorylated and the phosphate is covalently linked predominantly to serine. Analysis of tryptic digests of the 32P-labeled 72000-Mr protein showed that the phosphate residue(s) is present in only one peptide. The DNA-binding fraction contains an additional non-phosphorylated protein with an approximate molecular weight of 45000. Tryptic peptide maps of [35S]methionine-labeled 72000-Mr and 45000-Mr polypeptides are indistinguishable. The amino acid compositions of the 72000-Mr and 45000-Mr polypeptides show closely related distributions. An antiserum produced against the purified 72000-Mr DNA-binding protein precipitates both the 72000-Mr and the 45000-Mr protein from extracts of adenovirus-infected cells. Immunofluorescence studies revealed DNA-binding protein to be accumulated in characteristic structures in nuclei of the infected cells.     

Cleavage of a hydrophilic C-terminal domain increases growth-inhibitory activity of oncostatin M.

Oncostatin M is a polypeptide cytokine, produced by normal and malignant hematopoietic cells, that has several in vitro activities, including the ability to inhibit growth of cultured carcinoma cells. Here we present a structural and functional comparison of two oncostatin M-related proteins (Mr 36,000 and 32,000) secreted by COS cells transfected with oncostatin M cDNA. The smaller of these forms lacked a hydrophilic C-terminal domain comprising predominantly basic amino acids. This domain was also absent from native oncostatin M produced by U937 cells. The 32,000-Mr form of oncostatin M was not produced by cells transfected with plasmids (G195 and G196) in which a potential trypsinlike cleavage site within the hydrophilic C-terminal domain was altered by site-directed mutagenesis. A 32,000-Mr fragment was produced by trypsin treatment of the 36,000-Mr form of oncostatin M. These observations suggest that the 32,000-Mr form of oncostatin M was derived from the 227-amino-acid propeptide by proteolytic cleavage at or near the paired basic residues at positions 195 and 196. Pro-oncostatin M was equally active in radioreceptor assays as the processed form but was 5- to 60-fold less active in growth inhibition assays. Likewise, nonprocessed mutant protein encoded by plasmid G196 was equally active in the radioreceptor assays as the processed form but was five- to ninefold less active in growth inhibition assays. Thus, the highly charged C-terminal domain of pro-oncostatin M is not required for receptor binding or growth-inhibitory activity but may alter the functional properties of the molecule. Propeptide processing of oncostatin M may be important for regulating in vivo activities of this cytokine.     

Adenovirus E1B 55-Mr polypeptide facilitates timely cytoplasmic accumulation of adeno-associated virus mRNAs.

Adenovirus provides helper functions that facilitate replication of adeno-associated virus (AAV). Both the adenovirus E1B 55-Mr and E4 34-Mr polypeptides are required for efficient and timely accumulation of AAV mRNA, proteins, and DNA. The E1B 55-Mr polypeptide is also required for rescue of the integrated AAV genome in Detroit 6-D5 cells in a normal time frame. All of these effects probably result from a single, primary delay in AAV mRNA accumulation. The AAV helper function provided by the E1B 55-Mr and E4 34-Mr polypeptides appears to closely parallel their normal role in the adenovirus replication cycle.     

Structural homology among calf thymus alpha-polymerase polypeptides.

A sample of highly purified calf thymus alpha-polymerase contained an abundant 118,000 Mr polypeptide as well as five lower molecular weight polypeptides in the range of 54,000- to 64,000-Mr. This 118,000-Mr polypeptide was capable of DNA polymerase activity, as revealed by in situ assay after SDS-polyacrylamide gel electrophoresis. Tryptic peptide mapping indicated that the 118,000-Mr polypeptide shared extensive primary structure homology with 57,000-, 58,000- and 64,000-Mr polypeptides and some limited homology with 54,000- and 56,000-Mr polypeptides. This is the first evidence that lower and higher Mr polypeptides of purified calf thymus alpha-polymerase share sequence homology; these results are interpreted in the context of a model that predicts the existence of a common precursor with molecular weight greater than 140,000.     

G-proteins of fat-cells. Role in hormonal regulation of intracellular inositol 1,4,5-trisphosphate.

Pertussis toxin abolishes hormonal inhibition of adenylate cyclase, hormonal stimulation of inositol 1,4,5-trisphosphate accumulation in rat fat-cells, and catalyses the ADP-ribosylation of two peptides, of Mr 39,000 and 41,000 [Malbon, Rapiejko & Mangano (1985) J. Biol. Chem. 260, 2558-2564]. The 41,000-Mr peptide is the alpha-subunit of the G-protein, referred to as Gi, that is believed to mediate inhibitory control of adenylate cyclase by hormones. The nature of the 39,000-Mr substrate for pertussis toxin was investigated. The fat-cell 39,000-Mr peptide was compared structurally and immunologically with the alpha-subunits of two other G-proteins, Gt isolated from the rod outer segments of bovine retina and Go isolated from bovine brain. After radiolabelling in the presence of pertussis toxin and [32P]NAD+, the electrophoretic mobilities of the fat-cell 39,000-Mr peptide and the alpha-subunits of Go and Gt were nearly identical. Partial proteolysis of these ADP-ribosylated proteins generates peptide patterns that suggest the existence of a high degree of homology between the fat-cell 39,000-Mr peptide and the alpha-subunit of Go. Antisera raised against purified G-proteins and their subunits were used to probe immunoblots of purified Gt, Gi, Go, and fat-cell membrane proteins. Although recognizing the 36,000-Mr beta-subunit band of Gt, Gi, Go and a 36,000-Mr fat-cell peptide, antisera raised against Gt failed to recognize either the 39,000- or the 41,000-Mr peptides of fat-cells or the alpha-subunits of Go and Gi. Antisera raised against the alpha-subunit of Go, in contrast, recognized the 39,000-Mr peptide of rat fat-cells, but not the alpha-subunit of either Gi or Gt. These data establish the identity of Go, in addition to Gi, in fat-cell membranes and suggest the possibility that either Go or Gi alone, or both, may mediate hormonal regulation of adenylate cyclase and phospholipase C.     

The synthesis and origin of chloroplast low-molecular-weight ribosomal ribonucleic acid in spinach.

Chloroplasts isolated from young spinach leaves incorporate [3H]uridine into RNA species which co-electrophorese with 5-S rRNA and tRNA, but show very little incorporation into 4.5-S rRNA. Chloroplast 4.5-S rRNA is labelled in vivo after a distinct lag period relative to 5-S rRNA and tRNA. The kinetics of labelling in vivo of chloroplast 5-S rRNA are similar to those of the immediate precursors to the 1.05 x 10(6)-Mr and 0.56 x 10(6)-Mr rRNAs, whereas the kinetics of labelling of the 4.5-S rRNAare similar to those of mature 1.05 x 10(6)-Mr and 0.56 x 10(6)-Mr rRNAs. Chloramphenicol inhibits the labelling of chloroplast 4.5-S rRNA in vivo, and concomitantly inhibits the processing of the immediate precursors to the 1.05 x 10(6)-Mr and 0.56 x 10(6)-Mr rRNAs, but has little effect on the appearance of label in chloroplast 5-S rRNA. DNA/RNA hybridization using 125I-labelled RNAs suggests that chloroplast DNA contains a 2--3-fold excess of 4.5-S and 5-S rRNA genes relative to the high-molecular-weight rRNA genes. Competition hybridization experiments show that the immediate precursor to the 1.05 x 10(6)-Mr rRNA effectively competes with 125I-labelled 4.5-S rRNA for hybridization with chloroplast DNA, and is therefore a likely candidate for a common precursor to both the 1.05 x 10(6)-Mr and 4.5-S rRNAs.     

Phosphorylation in vitro of membrane fragments from Torpedo marmorata electric organ. Effect on membrane solubilization by detergents

Acetylcholine receptor-rich membrane fragments purified from Torpedo marmorata electric organ were phosphorylated, in vitro, by endogenous protein kinases. The 40 000-Mr chain, which carries the acetylcholine receptor site, was never labelled; on the other hand, protein bands of apparent molecular weights 43 000, 50 000 and 66 000, which are present in the acetylcholine receptor-rich membranes, were repeatedly phosphorylated. The phosphorylation of these three peptides required the presence of divalent cations, such as Mg2+ or Mn2+, and was, in addition, stimulated up to 3--5-fold by K+. The effect of Na+ ions appeared less specific since Na+ ions reduced the labelling of all the polypeptides susceptible to phosphorylation. Cholinergic agonists and antagonists, local anesthetics and cyclic nucleotides did not affect the phosphorylation of the receptor-rich membranes. Phosphorylation selectively modified the solubilization of several polypeptides by nondenaturing detergents: phosphorylated 43 000-Mr, 50 000-Mr and 66 000-Mr polypeptides were solubilized at lower concentrations of detergent than their non-phosphorylated counterparts. Two-dimensional gels revealed the existence of a charge heterogeneity of the 40 000-Mr and 43 000-Mr chains. The microheterogeneity of the 43 000-Mr chain, but not that of the 40 000-Mr chain, might result from a selective phosphorylation of this particular chain.     

Heterogeneity of insulin-like growth factor binding proteins between structure and affinity. 2. Forms released by human and rat liver in culture

Since the liver is considered to be the major source both of circulating insulin-like growth factors (IGFs) and of their specific binding proteins (BPs), human and rat liver explants were cultured in serum-free medium with a view to characterizing the binding proteins released into the medium and to comparing them with serum binding proteins. In the culture media, as in the serum, IGFs are associated with their binding proteins in the form of complexes. In gel filtration experiments the liver IGF-BP complexes eluted as a single, homogeneous peak with a relative molecular mass of about 40,000, which is similar to that of the 'small' complex of serum. Their sedimentation coefficient, estimated from sucrose gradient centrifugation, was 2.9 S. Polyacrylamide gel electrophoresis (SDS-PAGE) of human liver culture media, in which the binding proteins were cross-linked to 125I-labelled IGF I revealed molecular heterogeneity. Three specific bands corresponding to Mr 46,000, 40,000 and 37,000 were observed, which resemble those of the serum small complex, but none of the higher-Mr bands seen in serum. SDS-PAGE followed by transfer onto nitrocellulose and incubation with 125I-labelled IGF I (western blot) led to the identification in human liver culture media of five molecular forms of binding protein with Mr of 41,500, 38,500, 34,000, 30,000 and 24,000, identical to those seen in serum. The relative concentrations of the 41,500 and 38,500-Mr forms varied from one medium to another, but the 34,000 and 30,000-Mr forms were regularly more abundant in the liver culture media than in normal serum. The binding proteins produced by the liver therefore represent the native forms in the circulation (although this does not exclude other sources). The absence of high-Mr IGF-BP complexes in the liver culture media, and yet the presence of the 41,500 and 38,500-Mr forms, which are the only binding units of the serum 'large' complex (150,000 Mr), indicates that these two binding proteins are capable of binding IGFs to form 'monomeric' IGF-BP complexes. Western-blot analysis of rat binding proteins revealed a certain analogy with the human proteins, three forms having their Mr between 43,000 and 39,000 and three between 32,000 and 24,000. Liver binding proteins in human adults and foetuses were found to be identical, whereas in the case of serum the 41,500 and 38,500-Mr forms were more abundant in the adult and the 34,000 and 30,000-Mr forms more abundant in the foetus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of the divergent tricarboxylate transport operon (tctI) of Salmonella typhimurium.

Membrane-associated gene products of shock-sensitive bacterial transport operons are often difficult to detect. A 4.5-kilobase DNA fragment, known to completely encode the Salmonella typhimurium tctI operon, was cloned in both orientations behind the T7 phage promoter phi 10 and expressed by using the T7 polymerase-promoter system of Tabor and Richardson (S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci. USA 82:1074-1078, 1985). Under these conditions, five proteins were clearly demonstrated. One DNA strand was shown to encode the periplasmic (29,000-Mr) C protein (as a 31,000-Mr precursor), a 19,000-Mr protein, and a 40,000- to 45,000-Mr protein which ran as a diffuse band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The opposite strand carried the information for two additional proteins of 29,000 and 14,000 Mr. By Tn5 mutagenesis, subcloning of Tn5 insertions, and subcloning of various deletion mutants it was shown that the tctI system is divergently transcribed. The periplasmic binding protein (C protein) is the first product of one operon, followed by the 19,000-Mr and 45,000-Mr integral inner membrane proteins. On the opposite strand only the 29,000-Mr protein was essential for tctI function, and it was found to be weakly attached to the inner membrane. Thus tctI encodes four proteins, one periplasmic, two integral, and one peripheral to the cytoplasmic membrane, with the genes arranged as tctA tctB tctC tctD.     

A novel type of putrescine (diamine)-acetylating enzyme from the nematode Ascaris suum.

Component polypeptides of both the bovine lens and pituitary multicatalytic proteinase complexes demonstrate different immunoreactivities with a polyclonal antiserum raised against the purified pituitary enzyme. Four (Mr 24000, 26000, 34000 and 38000) of eight bands that have been resolved by SDS/polyacrylamide-gel electrophoresis are stained in immunoblot experiments. Monospecific antibodies obtained from this antiserum by affinity purification from the 38000- and 34000-Mr bands of the lens enzyme bound equally well to either band, but showed little or no binding to the 26000- and 24000-Mr bands upon immunoblotting. Antibody affinity-purified from the 24000-Mr band showed comparable binding to the 24000-, 34000- or 38000-Mr band. One explanation of these results is that the 24000-Mr polypeptide is derived from the higher-Mr polypeptide(s) and has lost some of the common immunodeterminants.     

Synthesis of a truncated Mr 46,000 mannose 6-phosphate receptor that is secreted and retains ligand binding.

On purification, human fibroblast collagenase breaks down into two major forms (Mr22,000 and Mr 27,000) and one minor form (Mr 25,000). The most likely mechanism is autolysis, although the presence of contaminating enzymes cannot be excluded. From N-terminal sequencing studies, the 22,000-Mr fragment contains the active site; differential binding to concanavalin A shows the 25,000-Mr fragment is a glycosylated form of the 22,000-Mr fragment. These low-Mr forms can be separated by Zn2+-chelate chromatography. An activity profile of this column, combined with data from substrate gels, indicates no activity against collagen in the 22,000-Mr and 25,000-Mr forms, but rather, activity casein and gelatin. The 27,000-Mr form has no activity. The 22,000/25,000-Mr form can act as an activator for collagenase in a similar way to that reported for stromelysin. The activity of the 22,000/25,000-Mr form is not inhibited by the tissue inhibitor of metalloproteinases (TIMP). The 27,000-Mr C-terminal part of the collagenase molecule therefore appears to be important in maintaining the substrate-specificity of the enzyme, and also plays a role in the binding of TIMP.     

Conversion of a mitochondrial precursor polypeptide into subunit 1 of cytochrome oxidase in the mi-3 mutant of Neurospora crassa.

1. The cytochrome-alpha alpha 3-deficient mi-3 cytoplasmic mutant of Neurospora crassa synthesizes a mitochondrial translation product which crossreacts with antibodies specific to subunit 1 of cytochrome oxidase. The immunoprecipitated polypeptide migrates more slowly during gel electrophoresis than the authentic 41 000-Mr subunit 1 of the wild-type enzyme. An apparent molecular weight of about 45 000 was estimated for the mutant product. 2. Radioactive labelling experiments in vivo show that the crossreacting material found in the mutant is relatively stable and does not form complexes with other subunits of the oxidase. 3. After induction of a functional cytochrome oxidase in the mutant cells with antimycin A, the 45 000-Mr polypeptide is converted to a 41 000-Mr component, which exhibits the same electrophoretic mobility as subunit 1 of the oxidase. Pulse-chase labelling kinetics reveal a typical precursor product relationship. 4. The converted polypeptide becomes assembled with other enzyme subunits to form a protein complex which has the immunological characteristics of cytochrome oxidase. A possible physiological role of the post-translational processing of the mitochondrially synthesized component is discussed.     

Endocytosis of a small dermatan sulphate proteoglycan. Identification of binding proteins.

Endosomal preparations from human osteosarcoma cells and from fibroblasts contain 51,000- and 26,000-Mr proteins which bind a small dermatan sulphate proteoglycan after SDS/polyacrylamide-gel electrophoresis and Western blotting. Binding can be inhibited by unlabelled proteoglycan core protein. The proteins co-precipitate with a proteoglycan core protein-antibody complex. Scatchard analysis of immobilized endosomal proteins yielded a KD of about 37 nM for the proteoglycan. In intact cells proteins of the same size can be found. They are sensitive to trypsinization. A 51,000-Mr protein is the predominant membrane protein with strong binding to immobilized dermatan sulphate proteoglycan. There are additional proteoglycan-binding proteins with Mr values of around 30,000 and 14,000 which are insensitive to trypsin treatment. In contrast with the 51,000- and 26,000-Mr proteins, they resist deoxycholate/Triton X-100 extraction several days after subcultivation.