Degradation Of Extracellular Proteins Research Articles

Type IV collagenases in invasive tumors.

The matrix metalloproteinases appear to be elevated in tumors with metastatic potential, and may well be involved in penetration of the basement membrane and degradation of extracellular proteins including type IV collagen. An imbalance between the 72 kDa and 92 kDa type IV collagenases and the associated tissue inhibitors of these metalloproteinases (TIMPs) may therefore have a role in the invasive phenotype. Cultured tumor cells with invasive potential secrete both type IV collagenases, though in tumors there is some evidence that the 72 kDa form at least may be produced by stromal cells at the invading tumor front rather than primarily by the tumor cells themselves, while the 92 kDa form may be synthesized in macrophages near the front. These collagenases are elevated in invasive as compared with in situ tumor components, but their specific roles and prognostic significance are not yet established.

Extracellular RGD-binding proteins modulate cell adhesion

Cell/fibronectin adhesion in extracellular matrices is partly mediated by integrin receptor recognition of RGD domains in fibronectin. Since blood contains significant levels of soluble fibronectin we have now investigated the occurrence of extracellular RGD-binding proteins. Attachment assays indicate that extracellular RGD-binding proteins prevent cell adhesion, suggesting their potential as novel secreted modulators of blood-borne cell adhesive interactions. These extracellular RGD-binding proteins also showed electrophoretic changes with reducing agents, suggestive of intrachain disulphide bonds, like those found in RGD-binding integrins. However, they differed from the latter in their electrophoretic profile, which was greatly dependent on the presence of protease inhibitors. Plasma from tumor-bearing mice showed a greater proportion of fast-migrating RGD-binding species under reducing condition compared to similarly treated normal plasma, suggesting that tumor development is associated with a partial degradation of extracellular RGD-binding proteins.

Decreased collagen concentration in rat uterine implantation sites compared with non-implantation tissue at days 6-11 of pregnancy

Collagen concentrations at implantation sites in the rat uterus were found to be significantly decreased compared with concentrations in adjacent non-involved uterine tissue in early pregnancy (75% by Day 11). The decrease in collagen was most marked in primary decidua and was also observed to a lesser extent (20%) in myometrium at the implantation site. There was a decrease of 20% in the concentration of total proteins at Day 7 (as measured by the ninhydrin method) and a slight increase in water content (2%) at Days 6 and 7. The differences in total protein and water content were transient, but the difference in collagen was maintained throughout early pregnancy. The localized changes in collagen content observed in this study, along with previously reported morphological changes in fibrillar and basement-membrane collagens in the uterus, give support to a theory of remodelling in early pregnancy involving simultaneous synthesis and degradation of extracellular proteins during decidualization.

Effect of subunit size and conformation on the rate of lysosomal degradation of extracellular proteins in cultured mouse peritoneal macrophages.

The degradation of nine well-defined proteins was studied in cultured mouse peritoneal macrophages following their uptake by fluid phase pinocytosis. After uptake, approximately one-third of the radioactivity was released into the medium in the form of trichloroacetic acid/phosphotungstic acid-insoluble material. When the time courses for the appearance of trichloroacetic acid/phosphotungstic acid-soluble and -insoluble radioactivities were independently analyzed, identical observed rate constants (kobs) were obtained. This is in agreement with an earlier claim that regurgitated protein and low molecular weight products arise from a common intracellular pool of radiolabeled substrates, presumably within lysosomes, and that the traffic of substrates between the plasma membrane and the lysosome is probably bidirectional (Buktenica, S., Olenick, S. J., Salgia, R., and Frankfater, A. (1987) J. Biol. Chem. 262, 9469-9476). When intrinsic degradation rate constants (kd) were calculated, these were found to vary inversely with protein subunit molecular weights, from 0.0347 h-1 for horse heart cytochrome c to 0.0104 h-1 for rabbit muscle phosphorylase b. The proportion of peptide bonds in a protein which are initially available to the action of lysosomal proteases should be proportional to the fraction of the total potential surface of a protein which remains accessible to solvent after polypeptide folding (AS/AT). In agreement, lysosomal degradation rates were observed to correlate well with known or estimated values of AS/AT, and thermal denaturation, which may expose previously buried amino acid residues, increased the rate of degradation of bovine serum albumin.

Degradation and regurgitation of extracellular proteins by cultured mouse peritoneal macrophages and baby hamster kidney fibroblasts. Kinetic evidence that the transfer of proteins to lysosomes is not irreversible.

The uptake and degradation of bovine serum albumin (BSA), bovine liver catalase, and rabbit muscle enolase have been studied in cultured mouse peritoneal macrophages (MPM) and baby hamster kidney fibroblasts (BHK cells). Rates constant for the uptake of the three proteins by MPM were similar. In addition, BSA accumulation was independent of BSA concentration in the uptake medium and was not inhibited by a large excess of serum, suggesting that protein accumulation was by fluid phase pinocytosis. Following an overnight uptake, 20-30% of the accumulated protein was subsequently regurgitated into the medium in a trichloroacetic acid/phosphotungstic acid-precipitable form. This material co-migrated with the authentic protein during molecular sieve chromatography on Sephadex G-50. The rates of appearance of trichloroacetic acid/phosphotungstic acid-insoluble products were greater than expected for cell death and leakage. The observed first order rate constants, kobs, for the appearance of trichloroacetic acid/phosphotungstic acid-soluble and trichloroacetic acid/phosphotungstic acid-insoluble products in the culture medium were identical, indicating that both products were released in parallel from MPM and BHK cells. The kobs for intracellular BSA degradation and regurgitation were independent of the initial BSA concentration in the uptake medium, but were decreased about 35% when degradation was allowed to proceed in the presence of high concentrations of serum. Degradation was also inhibited by chloroquine and pepstatin. Inhibition of degradation was accompanied by an increase in the total amount of regurgitated protein appearing in the medium. Remarkably, however, these inhibitors also decreased kobs for regurgitation, thereby preserving the similarity in the observed rate constants for the appearance of trichloroacetic acid/phosphotungstic acid-soluble and trichloroacetic acid/phosphotungstic acid-insoluble products. These and other results were inconsistent with desorption of proteins from the surface of the culture dish or the surface of cells as the source of the trichloroacetic acid/phosphotungstic acid-insoluble label appearing in the medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Human gingival proteases. I: Extraction and preliminary characterization of trypsin-like and elastase-like enzymes.

In order to characterize neutral proteases in gingiva, tissue specimens from periodontitis patients were subjected to different extraction procedures. In addition to collagenase and gelatinase, serine proteases having trypsin‐like and elastase‐like properties were detected by utilizing synthetic aminoacid substrates. High salt concentration was required for the optimal extraction of these enzymes. They also needed a certain ionic strength for their activity. The pH optimum for the trypsin‐like activity was 7.5 and for the elastase‐like activity 8.0. A synthetic plasmin substrate was also degraded by the extracts but no activity against native fibrin was found. These neutral proteases may play a significant part in the extracellular protein degradation in periodontium during inflammation.

Incorporation of nitrogen into rumen bacterial fractions of steers given protein- and urea-containing diets. Ammonia assimilation into intracellular bacterial amino acids.

Experiments were carried out in vivo to investigate the pathways of ammonia incorporation into rumen bacteria, bacterial fractions and free amino acids within the bacteria. Steers were alternately given two isoenergetic, isonitrogenous diets containing the nitrogen mainly as either urea or decorticated groundnut meal (DCGM). At the end of each period on a given diet, a solution of 15NH4Cl was infused into the rumen and samples of rumen contents were removed at 2, 10, 20 and 90 min and 5, 10 and 24 h afterwards. Concentrations of ammonia and its 15N enrichment were determined and samples of mixed rumen bacteria were prepared. Bacteria were disrupted ultrasonically and separated into bacterial protein, cell wall and protein-free cell supernatant fractions. Amino acids were separated after hydrolysis and their 15N contents determined. A rumen fluid circulation pump was developed so that representative samples could be taken at very short time intervals after the introduction of the 15N label. Rumen pH changes, rumen fluid dilution rates and patterns of rumen ammonia concentrations were consistent with normal rumen metabolism. Net bacterial synthesis (as calculated from the net outflow of bacteria from the rumen) was significantly (P less than 0.05) greater with the DCGM diet (12.4 g bacterial N/d) than with the urea diet (9.24 g bacterial N/d). With both diets the 15N label rapidly left the rumen ammonia pool and entered the rumen bacteria. Analysis of the bacterial fractions indicated that the label appeared rapidly in the protein-free cell supernatant fraction and more slowly in the bacterial protein and cell wall fractions. With the DCGM diet bacteria apparently utilized intracellular label less efficiently than with the urea diet. The proportion of N in the protein-free cell supernatant was higher with the DCGM diet, suggesting increased levels of intracellular amino acids and peptides, following extracellular protein degradation. Levels of enrichment of the amino acids alanine and glutamate in the protein-free cell supernatant fraction suggested that the enzymes alanine dehydrogenase (EC 1.4.1.1) and glutamate dehydrogenase (EC 1.4.1.2 and 1.4.1.4) may be the major enzymes for assimilating ammonia when concentrations of soluble carbohydrate and rumen ammonia are high in the rumen. The high levels of intracellular alanine are discussed with reference to published work on the excretion of alanine by rumen bacteria.

Synthesis and secretion of C-reactive protein by rabbit primary hepatocyte cultures

The synthesis and secretion of the acute-phase protein C-reactive protein by rabbit primary hepatocyte cultures was investigated. Hepatocytes prepared from animals that had received inflammatory stimuli 18-24 h before cell isolation were found to incorporate radiolabelled amino acids into C-reactive protein throughout the 48 h culture period. Intracellular C-reactive protein was found to be in steady state and there was no significant degradation of extracellular C-reactive protein, permitting direct estimation of rate of synthesis from rate of extracellular accumulation. Both C-reactive protein and total secreted protein were synthesized at constant rates for at least 24 h in culture. Mean rate of accumulation of newly synthesized total proteins in medium of cultures from six stimulated animals was 40% greater than was found in cultures from nine control (unstimulated) animals; this difference did not achieve statistical significance (0.05 less than P less than 0.10). Mean rate of C-reactive-protein synthesis represented 3.9% of total secreted-protein synthesis in cultures prepared from stimulated animals compared with 0.3% in cultures from control animals (P less than 0.001). Further, there was a correlation between C-reactive-protein synthesis by cultured hepatocytes and serum C-reactive-protein concentration at time of hepatocyte isolation (P less than 0.001). Rates of C-reactive-protein synthesis by hepatocyte cultures from stimulated animals were in good agreement with those previously measured in isolated perfused livers and those calculated from results of studies in vivo.

Degradation of extracellular protein by the isolated perfused rat liver: A biochemical and autoradiographic study

The isolated perfused rat liver was used to study the degradation of 125I-labelled protein supplied in the perfusion medium. Formaldehyde-denatured proteins (human serum albumin, bovine serum albumin and especially rat liver phosphoenolpyruvate carboxykinase (GTP)) were taken up by the liver and degraded at high rates. Native human serum albumin was not degraded at significant rates by the perfused liver, while native phosphoenolpyruvate carboxykinase (GTP) was catabolised at about one-fourth the rate of the denatured enzyme. The degradation rate of denatured human serum albumin increased markedly as protein was added up to 0.7 mg, and more gradually with further increases in added protein. The biphasic nature of concentration dependence probably reflects the contribution of different cell types in the liver. Autoradiographic examination of serial biopsies taken during perfusion of the liver with formaldehyde-denatured, 125I-labelled bovine serum albumin showed that at the cellular level the radioactivity was located predominantly in Kupffer and other non-parenchymal cells; and at the subcellular level the radioactivity was largely in endocytic vesicles, lysosomes and occasionally in the sinusoidal spaces. No significant radioactivity was found associated with other cytoplasmic organelles or the nucleus. It is concluded that lysosomes of the non-parenchymal cells are primarily responsible for the degradation of denatured extracellular protein that enters the liver.