The secretion of plasminogen activators has been implicated in the controlled extracellular proteolysis that accompanies cell migration and tissue remodeling. We found that the human plasminogen activator urokinase (Uk) (Mr 55,000 form) binds rapidly, specifically, and with high affinity to fresh human blood monocytes and to cells of the monocyte line U937. Upon binding, Mr 55,000 Uk was observed to confer high plasminogen activator activity to the cells. Binding of the enzyme did not require a functional catalytic site (located on the B chain of the protein) but did require the noncatalytic A chain of Mr 55,000 Uk, since P/r 33,000 Uk did not bind. These results demonstrate the presence of a membrane receptor for Uk on monocytes and show a hitherto unknown function for the A chain of Uk: binding of secreted enzyme to its receptor results in Uk acting as a membrane protease. This localizes plasminogen activation near the cell surface, an optimal site to facilitate cell migration.
Summary In this prospective 10-year study in elderly aged 60 years and over, there was a 1.3% per year reduction in the standardized incidence of hip fracture in women but not in men. This decrease was mainly due to changes in the standardized incidence of hip fracture in institutiondwelling women.
A binding protein for y-butyrobetaine was purified from osmotic shock fluid of an Agrobacterium sp. It was a monomeric protein with an apparent molecular weight of 52,000 or 53,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. The isoelectric point was 4.3, as determined by isoelectric focusing. Amino acid analysis of the protein showed that Asx and Glx were predominant components and that the protein contained no cysteine. The dissociation constant of this protein for 'y-butyrobetaine was found to be 0.7 ,uM by equilibrium dialysis. Attempts to sequence the amino-terminal end with the Edman method failed, suggesting that this region of the protein is blocked.Agrobacterium sp. was isolated from soil by selective aerobic culture with -y-butyrobetaine (4-trimethylaminobutyrate; see inset of Fig. 5) as the sole source of carbon and nitrogen. The metabolism of -y-butyrobetaine in various bacteria has been reviewed recently (5). This quaternary ammonium base is involved in the biosynthesis of carnitine (3-hydroxy-4-trimethylaminobutyrate) or other related betaines (5). In the Agrobacterium sp., the biosynthetic pathway of -y-butyrobetaine leads to the production of the L-isomer of carnitine. This synthesis occurs in two steps:first, oxidation of y-butyrobetaine, which produces the ac4 unsaturated compound trans-crotonobetaine; and second, addition of a molecule of water to the double bond, which results in the formation of L-carnitine. L-Carnitine can be further metabolized to produce intermediates of the Krebs cycle.In mammals, L-carnitine is a normal constituent of plasma and tissues. It comes both from the diet and from biosynthesis in certain tissues, such as the liver and the kidney (12). The principal function of L-carnitine appears to be that of a carrier of long-chain fatty acids across the inner mitochondrial site of P-oxidation. This important function of Lcarnitine is underscored by skeletal muscle weakness, cardiomyopathy, and the accumulation of lipid droplets in tissues from patients diagnosed as having a carnitine deficiency. Such diseases can be treated by administration of L-carnitine.We believe that the Agrobacterium sp. could be a suitable organism for microbial biosynthesis of L-carnitine. For example, L-carnitine can be obtained from the culture medium of mutants blocked in the catabolism of this molecule. However, the first step of microbiological biosynthesis involves the transport of the substrate into the living cell. Since the lipid bilayer cytoplasmic membrane that forms the limit between the cytoplasm and the external medium is essentially impermeable to most hydrophilic substances, cells have evolved specific processes, catalyzed by proteins, that promote the transport of these substances across membranes. These transport processes, which can be the ratelimiting step of biosynthesis, are tightly coupled to the intracellular metabolism. They are subject to regulation by * Corresponding author. metabolic processes, and in turn, ...
The photoaffinity reagent S‐(p‐azidophenacyl)thiocarnitine (PAP‐TC) has been synthesized according to Mauro et al. [(1986) Biochem. J. 237, 533–540]. This compound, originally designed for a structure‐function study of carnitine acetyl‐transferase, was used to analyze the Agrobacterium sp. γ‐butyrobetaine transport system. PAP‐TC appears to be a reagent specific to the transport system since it showed a competitive inhibition (K i = 70 μM) of γ‐butyrobetaine transport. UV irradiation of periplasmic proteins in the presence of [14C]PAP‐TC resulted in the irreversible labeling of the γ‐butyrobetaine‐binding protein. The addition of 1 mM γ‐butyrobetaine in the mixture significantly decreased the incorporation of the reagent, showing that this compound reacts specifically with the binding protein.
Recent cell biological and biochemical studies on the urokinase-type plasminogen activator (u-PA) have revealed an unsuspected property of this protein: it binds with high affinity and specificity to the plasma membrane of a number of cell types. Hence, while the interaction of tissue-type plasminogen activator (t-PA) with fibrin suggests a preferred role for this enzyme in the maintenance of fluidity of the extracellular milieu, the cellular binding of u-PA results in the focalisation of plasmin generation to the close environment of the cell surface; this appears as an optimal configuration if u-PA is to participate in the enzymatic events required for cell migration.The available information on the cellular binding of u-PA can be summarized as follows:1. Human monocytes-macrophages, monocyte-like cell lines, fibroblasts, and a variety of other cell lines all express u-PA binding sites. The number of u-PA binding sites on a given cell type may vary as a function of the functional state of the cells. In some cases all sites are occupied by “endogenous” u-PA.2. Binding does not require u-PA activity, and prou-PA binds with the same affinity as does the active enzyme.3. The Kd for u-PA binding is between 1 and 10×10-10 M. The binding site appears to be specific for u-PA.4. Binding requires the presence of the A chain of u-PA; the growth factor module of the A chain is involved in this interaction.5. Bound enzyme does not dissociate readily, nor is it rapidly endocytosed; most importantly, it retains catalytic activity.Studies in progress are aimed at further defining the u-PA determinants responsible for binding. In this context it is noteworthy that there is a tight species specificity of binding: human and murine u-PA, for instance, bind only to cells of the homologous species. Characterization of the u-PA binding site suggests that it is an integral membrane protein that includes at least one Mf 50.000 polypeptide chain.In addition to allowing for the peri-cellular focalisation of u-PA catalysed proteolysis, expression of the u-PA binding site provides a mecanism whereby one cell type can acquire membrane-bound u-PA activity following secretion of the (pro)enzyme by another cell population. A striking example of this is the binding of u-PA, synthesized by the epithelial layer of the male genital tract, to the head region of murine spermatozoa.
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