Calcium regulation of myosin-I tension sensing

Myo1b is a myosin that is exquisitely sensitive to tension. Its actin-attachment lifetime increases > 50-fold when its working stroke is opposed by 1 pN of force. The long attachment lifetime of myo1b under load raises the question: how are actin attachments that last >50 s in the presence of force regulated? Like most myosins, forces are transmitted to the myo1b motor through a light-chain binding domain that is structurally stabilized by calmodulin, a calcium-binding protein. Thus, we examined the effect of calcium on myo1b motility using ensemble and single-molecule techniques. Calcium accelerates key biochemical transitions on the ATPase pathway, decreases the working-stroke displacement, and greatly reduces the ability of myo1b to sense tension. Thus, calcium provides an effective mechanism for inhibiting motility and terminating long-duration attachments.     

Phosphorylation of brush border myosin by brush border calmodulin-dependent myosin heavy and light chain kinases

Calmodulin-dependent myosin light chain kinase isolated from chicken intestinal brush border phosphorylates brush border myosin at an apparently single serine identical to that phosphorylated by smooth muscle myosin light chain kinase. Phosphorylation to 1.8 mol phosphate/mol myosin activated the myosin actin-activated ATPase about 10-fold, to about 50 nmol/min per mg. Myosin phosphorylated on its light chains could then be further phosphorylated to a total of 3.2 mol phosphate per mol by brush border calmodulin-dependent heavy chain kinase. Heavy chain phosphorylation did not alter the actin-activated ATPase of either myosin prephosphorylated on its light chains or of unphosphorylated myosin.     

Mechanisms of cytoskeletal regulation: modulation of membrane affinity in avian brush border and erythrocyte spectrins

The spectrins isolated from chicken erythrocytes and chicken intestinal brush border, TW260/240, share a common alpha subunit and a tissue-specific beta subunit. The ability of these related proteins to bind human erythrocyte inside out vesicles (IOVs) and human erythrocyte ankyrin in vitro have been quantitatively compared with human erythrocyte spectrin. Chicken erythrocyte spectrin binds human IOVs and human ankyrin with affinities nearly identical to that for human erythrocyte spectrin. TW260/240 does not significantly bind to either IOVs or ankyrin. These results demonstrate a remarkable tissue preservation of ankyrin-binding capacity, even between diverse species, and confirm the role of the avian beta-spectrins in modulating this functionality. Avian brush border spectrin may represent a unique spectrin which serves primarily as a filament cross-linker and which does not interact strongly with membrane-associated proteins.     

The histochemical demonstration of brush border endopeptidase

Summary A histochemical method for the demonstration of a brush border endopeptidase is described based on results of biochemical and histochemical experiments. The substrate of choice is Glut-Ala-Ala-Ala-MNA which displays a very good localization ability and suitable kinetic properties. K_m estimated in rat kidney homogenate amounts to 2.35×10^–4 M. pH optimum of this endopeptidase associated with the brush border membrane is in the alkaline range. The activity is dependent on the buffer used. In phosphate and cacodylate buffers of pH 7.2 about 30% lower activity in rat kidney and about 25% lower activity in rat small intestine than in Tris-HCl buffer of the same pH was found. The most suitable diazonium salt for the detection in situ is Fast Blue B. It inhibits the endopeptidase activity of rat kidney by about 85% at pH 7.2 and by about 55% at pH 6.0. The best results are obtained in cryostat sections adherent to semipermeable membranes treated with chloroform-acetone before the incubation. A microdensitometric evaluation of the reaction product is possible and results are in good agreement with those of the biochemical determination. When Suc-Ala-Ala-Ala-INA is used as substrate hexazonium-p-rosaniline is the most suitable coupling agent although it inhibits more than Fast Blue B. The reaction using acylated trialanyl naphthylamides as substrates runs in two steps. Endopeptidase sets free Ala-NA which is attacked by aminopeptidase M. Aminopeptidase M is not reaction rate or localization limiting factor because its activity in the brush border is very high and the enzyme is anchored to the cell membrane very closely to endopeptidase. In homogenates of rat kidney and jejunal mucosa the endopeptidase activity was inhibited by EDTA (2×10^–3M) by 75% in the kidney and by 68% in the jejunum, by DFP (10^–3M) by 41% in the kidney and by 35% in the intestine, by Mn²⁺ (5×10^–3M) by 25% in the kidney and by 30% in the intestine. No inhibition was exerted by E 600. In sections the results were similar. 1,10-phenanthroline (10^–2M) caused a substantial inhibition.Endopeptidase activity was detected in the brush border of cells of proximal convuluted tubules of the kidney and in the brush border of differentiated enterocytes of the small intestine. In the same species enterocytes display a lower activity than kidney tubular cells. There are species differences in the distribution pattern of endopeptidase in the kidney. In the rabbit and man the positive reaction occurs in the whole cortex. It is distributed unevenly, however. In the rat the tubules of the inner cortex display a very high activity. In the outer cortex straight portions react strongly. In the rabbit kidney cells of the parietal layer of Bowman's capsule display a weak reaction as well. No sex differences were found in the distribution pattern of endopeptidase in the rat kidney. In the intestine of all species examined a proximo-distal gradient was found. The reaction appears in the duodenum, reaches its maximum in the jejunum and declines in the aboral direction. In patients suffering coeliac sprue the endopeptidase is the most seriously affected enzyme of the brush border peptidases which can be demonstrated in situ. There follow in decreasing order -glutamyl transferase, dipeptidyl (amino) peptidase IV, aminopeptidase A and aminopeptidase M.     

Molecular organization of the intestinal brush border

The brush border of enterocytes represents one of the more specialized apical poles of epithelial cells. It is formed by particularly well-developed apical plasma membrane microvilli, whose shape is ensured by a highly organized cytoskeleton. The molecular organization of the cytoskeleton is described. Whereas several cytoskeleton proteins are ubiquitous, villin is highly specific for intestinal cells and can be used as a differentiation marker of these cells. The major glycoproteins, in particular hydrolases, of the brush border membrane have been characterized. They have many common structural features, in particular their mode of integration into the membrane by their N-terminal hydrophobic sequences that also plays the role of the 'signal peptide' responsible for their co-translational insertions into the endoplasmic reticulum. Studies on the biosynthesis and intracellular pathway of aminopeptidase N strongly suggest that sorting of apical and basolateral glycoproteins could occur after their integration into the basolateral domain.     

Influence of age on duodenal brush border membrane and specific activities of brush border membrane enzymes in Wistar rats.

To examine age-related changes in the morphology of intestinal brush border membrane (BBM; microvilli) and specific activities of intestinal BBM enzymes including alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (gamma-GT), and disacchridase, four groups of Wistar rats were sacrificed at 2.5 wk, 5 wk, 5 mon and 23 mon. In an electron microscopic examination, morphologically a less dense BBM structure in the duodenum of rats aged 23 mon was observed than that of rats aged 5 mon. Specific activity of ALP in the duodenum from 5-mon-old rats was significantly higher than from rats aged 2.5 wk and 23 mon. The mucosal tissues from 5-wk-old rats had significantly higher specific activity of gamma-GT than did tissues from the other ages. In sucrase and maltase specific activities, 5-mon-old rats had higher activities of these enzymes than other age groups, especially 2.5-wk- and 23-mon-old rats. There was also a significant effect of site on intestinal BBM enzyme activities in post-weanling rats. Regional gradients of ALP and gamma-GT along the entire small intestine (duodenum > jejunum > ileum) were remarkable. Disaccharidase activities peaked in the jejunum and declined toward both the duodenum and ileum. Taken together the result obtained here suggested that 5-mon-old rats had the most elevated intestinal function. This result also strongly indicated that the structure of the intestinal BBM and development of intestinal BBM enzymes in Wistar rate were markedly influenced by age during the postnatal period.     

Proteomic analysis of the enterocyte brush border

The brush border domain at the apex of intestinal epithelial cells is the primary site of nutrient absorption in the intestinal tract and the primary surface of interaction with microbes that reside in the lumen. Because the brush border is positioned at such a critical physiological interface, we set out to create a comprehensive list of the proteins that reside in this domain using shotgun mass spectrometry. The resulting proteome contains 646 proteins with diverse functions. In addition to the expected collection of nutrient processing and transport components, we also identified molecules expected to function in the regulation of actin dynamics, membrane bending, and extracellular adhesion. These results provide a foundation for future studies aimed at defining the molecular mechanisms underpinning brush border assembly and function.     

Alpha-Glutamyl-beta-naphthylamide hydrolase of rabbit small intestine. Localization in the brush border and separation from other brush border peptidases.

About 70% of the total mucosal enzymatic activity hydrolyzing beta-L-glutamyl-beta-naphthylamide in the rabbit small intestine is present in the brush border; the specific activity in this subcellular fraction is 7 times higher than that of the homogenate. Similar results are obtained for L-leucyl beta-naphthylamide hydrolase. The enzyme activity is efficiently solubilized by papain digestion and is clearly separated from L-leucyl-beta-naphthylamide hydrolase by chromatography on concanavalin A-Sepharose. It probably represents a digestive peptidase, different from the other known peptide hydrolases of the digestive surface of the small intestine.     

Ion pathways in renal brush border membranes

The absorbance change of the weak base dye probe, Acridine orange, was used to monitor alterations of pH gradients across renal brush border membrane vesicles. The presence of Na⁺/H⁺ or Li⁺/H⁺ exchange was demonstrated by diluting Na₂SO₄ or Li₂SO₄ loaded vesicles into Na⁺- or Li⁺-free solutions, which caused dye uptake. About 20% of the uptake was abolished by lipid permeable cations such as valinomycin-K⁺ or tetraphenylphosphonium, indicating perhaps the presence of a finite Na⁺ conductance smaller than electroneutral Na⁺/H⁺ exchange. The protonophore tetrachlorosalicylanilide raised the rate of dye uptake under these conditions, hence the presence of an Na⁺ conductance greater than the H⁺ conductance was suggested. K⁺ gradients also induced changes of pH, at about 10% of the Na⁺ or Li⁺ rate. Partial inhibition (21%) was seen with 0.1 mM amiloride indicating that K⁺ was a low affinity substrate for the Na⁺/H⁺ exchange. Acceleration both by tetrachlorosalicylanilide (2-fold) and valinomycin (4-fold) suggested the presence of 2 classes of vesicles, those with high and those with low K⁺ conductance. The larger magnitude of the valinomycin dependent signal suggested that 75% of the vesicles had a low K⁺ conductance. Inward Cl^? gradients also induced acidification, partially inhibited by the presence of tetraphenylphosphonium, and accelerated by tetrachlorosalicylanilide. Thus both a Cl^? conductance greater than the H⁺ conductance and a Cl^?/OH^? exchange were present. The rate of Na⁺/H⁺ exchange was amiloride sensitive with a pH optimum of 6.5 and an apparent K_m for Na⁺ or Li⁺ of about 10 mM and an E_A of 14.3 kcal per mol. A 61-fold Na₂SO₄ gradient resulted in a pH gradient of 1.64 units which increased to 1.8 with gramicidin. An equivalent NaCl gradient gave a much lower ΔpH even in the presence of gramicidin showing that the H⁺ and Cl^? pathways could alter the effects of the Na⁺/H⁺ exchange.     

Renal brush border membrane bound intrinsic factor

A highly active receptor for intrinsic factor (IF)-cobalamin (Cbl) complex has been detected and reported in mammalian kidney earlier (Seetharam, B., et al. (1988) J. Biol. Chem. 263, 4443-4449). The physiological role of this receptor in normal Cbl homeostasis is not known. In addition to binding of exogenously added IF-[57Co]Cbl, the renal apical membranes contain endogenous IF or IF-Cbl. Washing with pH 5/EDTA buffer enhanced the binding of exogenously added IF-[57Co]Cbl to renal apical but not basolateral membranes. The pH 5/EDTA extract from renal apical membranes bound [57Co]Cbl. The complex also bound to rat ileal brush border membrane and promoted ileal transport of [57Co]Cbl. On immunoblots using monospecific antiserum to IF a 62 kDa protein was identified in renal and intestinal apical membranes, serum and in tissue extracts of unperfused rat liver, kidney and heart. The 62 kDa band was eliminated from the renal apical membranes following pH 5/EDTA wash. Rat urine demonstrated unsaturated [57Co]Cbl binding (0.2 to 0.4 pmol/day) of which only 30-40% was immunoprecipitated with anti IF and could be identified on immunoblots. The identification of IF in rat renal apical membranes (160-200 ng/mg protein) and secretion of only traces of IF in urine suggest that the renal IF-Cbl receptor may play a role in sequestering IF/IF-Cbl and prevent urinary loss of Cbl.     

The histochemical demonstration of brush border endopeptidase.

A histochemical method for the demonstration of a brush border endopeptidase is described based on results of biochemical and histochemical experiments. The substrate of choice is Glut-Ala-Ala-Ala-MNA which displays a very good localization ability and suitable kinetic properties. Km estimated in rat kidney homogenate amounts to 2.35 X 10(-4) M. pH optimum of this endopeptidase associated with the brush border membrane is in the alkaline range. The activity is dependent on the buffer used. In phosphate and cacodylate buffers of pH 7.2 about 30% lower activity in rat kidney and about 25% lower activity in rat small intestine than in Tris-HCl buffer of the same pH was found. The most suitable diazonium salt for the detection "in situ" is Fast Blue B. It inhibits the endopeptidase activity of rat kidney by about 85% at pH 7.2 AND BY ABOUT 55% AT PH 6.0. The best results are obtained in cryostat sections adherent to semipermeable membranes treated with chloroform-acetone before the incubation. A microdensitometric evaluation of the reaction product is possible and results are in good agreement with those of the biochemical determination. When Suc-Ala-Ala-Ala-INA is used as substrate hexazonium-p-rosaniline is the most suitable coupling agent although it inhibits more than Fast Blue B. The reaction using acylated trialanyl naphthylamides as substrates runs in two steps. Endopeptidase sets free Ala-NA which is attacked by aminopeptidase M. Aminopeptidase M is not reaction rate or localization limiting factor because its activity in the brush border is very high and the enzyme is anchored to the cell membrane very closely to endopeptidase. In homogenates of rat kidney and jejunal mucosa the endopeptidase activity was inhibted by EDTA (2X10(-3) M) by 75% in the kidney and by 68% in the jejunum, by DFP (10(-3) M) by 41% in the kidney and by 35% in the intestine, by Mn2+ (5X10(-3) M) by 25% in the kidney and by 30% in the intestine. No inhibition was exerted by E 600. In sections the results were similar. 1,10-phenanthroline (10(-2) M) caused a substantial inhibition. Endopeptidase activity was detected in the brush border of cells of proximal convuluted tubules of the kidney and in the brush border of differentiated enterocytes of the small intestine. In the same species enterocytes display a lower activity than kidney tubular cells. There are species differences in the distribution pattern of endopeptidase in the kidney. In the rabbit and man the positive reaction occurs in the whole cortex. It is distributed unevenly, however. In the rat the tubules of the inner cortex display a very high activity. In the outer cortex straight portions react strongly. In the rabbit kidney cells of the parietal layer of Bowman's capsule display a weak reaction as well. No sex differences were found in the distribution pattern of endopeptidase in the rat kidney. In the intestine of all species examined a proximo-distal gradient was found...     

Detergent-induced proteolysis of rabbit intestinal brush border vesicles

Proteolysis of brush border vesicle proteins is induced by detergent solubilisation. This proteolysis is selective in that some of the proteins are more susceptible than others. The rate of induced proteolysis is decreased by decreasing the temperature, has a minimum of about pH 6 and is effectively prevented by a combination of the protease inhibitors, EDTA, diisopropylfluorophosphate and iodoacetamide.