Kenneth K. Tsuboi

Actin and bound-nucleotide stoichiometry

Abstract Since actin alone among the structural proteins of muscle contains adenine nucleotide as an integral bound component, purification, homogeneity and stability were assessed on this basis. Actin extracts of muscle powder, prepared according to Ebashi and Maruyama 4, although still obviously impure, contained bound ATP at ratios of nearly one mole per 60 000 g protein present. Purification by reversible polymerization and gel filtration resulted in an apparent near-homogeneous product containing an average equivalent of 1 mole of bound nucleotide per 45 000 g protein. The bound nucleotide values obtained on analyses of both G- and F-actin samples were supported by a molecular weight estimate of approx. 45 000 obtained for G-actin by analytical gel filtration technique. Further, G-actin samples were found to contain approximately equivalent amounts of both bound Ca and ATP. Examination of bound nucleotide stability revealed an unexpected high degree of instability in the case of actin prepared in the polymerized form (i.e. F-actin).

Sugar hydrolases and their arrangement on the rat intestinal microvillus membrane

SummaryThe arrangement of the sugar hydrolases, sucrase-isomaltase, maltase, and lactase on the microvillus membrane of rat intestine was investigated by immunological technique. The enzymes were purified essentially free of each other to near homogeneity and antisera of high specificity were obtained against each. Microvillus membranes were prepared routinely in high purity from rat intestine and contained an average 61% protein, 20% lipid, and 19% carbohydrate, with the sugar hydrolases comprising an estimated 20–25% of the membrane protein. The immunoreactivity of membrane-bound sucrase-isomaltase, maltase, and lactase was investigated with antisera demonstrating specific reactivity to each, when tested in the presence of other membrane extractives. The membrane-bound enzymes were found in each case to combine with antibody in amounts equivalent to that required to effect precipitation of comparable units of the free enzymes from solution. Preloading membrane vesicles with antibodies to any two of the enzymes did not affect either the immunoreactivity or extractability (by papain or Triton X-100) of the third.The antibody-binding studies indicated an arrangement of these enzymes independent of each other on the membrane surface, in a manner allowing each to maintain a high degree of molecular freedom.

Sugar hydrolases of the infant rat intestine and their arrangement on the brush border membrane

Lactase and maltase, the predominant sugar hydrolases associated with the intestinal brush bordermembrane of the suckling rat, were purified essentially free of the other to near homogeneity (lactase at specific activity 23, maltase at specific activity 58), and their specific physiocochemical properties determined. Antisera prepared to each showed by immunodiffusion a single common precipitin line with pure enzyme and solubilized proteins of the brush border membrane. Brush border membranes were purified 26--35-fold from infant rat intestine. Membranes prepared from 10-day-old rats contained 32% protein, 43% lipid and 25% carbohydrate with lactase and maltase estimated to comprise in excess of 10% and 2%, respectively, of the membrane protein. Immunotitration curves of lactase and maltase showed equivalent antibody binding by the membrane-bound and free enzyme forms. Furthermore, antibody binding to one enzyme did not affect the immunotitration curve or the extractability (by papain or Triton X-100) of the other membrane-bound enzyme. It was concluded that the lactase and maltase molecules are attached singly on the external membrane surface in a spatially independent manner with their antigenic sites as freely available to antibody binding as exhibited by their papain-solubilized counterparts.

Acetaldehyde-Dependent Changes in Hemoglobin and Oxygen Affinity of Human Erythrocytes

In the presence of acetaldehyde, metabolizing human erythrocytes accumulate an altered hemoglobin product showing chromatographic similarity to hemoglobin AIa or AIb. The adduct is stable to overnight dialysis with an intracellular half-life of about 5.5 days. Adduct formation is accompanied by proportional changes in cell oxygen affinity (decrease in P50 of 3 mm Hg/mM adduct). Little unaltered hemoglobin remains after overnight incubation in 15 mM acetaldehyde, with significant adduct formation and marked reduction of cell ATP occurring after prolonged incubation in as little as 0.5 mM acetaldehyde.

Detection of gastric contents in tracheal fluid of infants by lactose assay

We designed an in vitro assay to detect the presence of lactose in the tracheal aspirates of premature, ventilator-dependent infants. This method was employed to identify recurrent, unrecognized aspiration, which could prolong the requirements for ventilator support and contribute to the development of chronic lung disease. One hundred five determinations of lactose were performed on the tracheal fluid obtained from 42 ventilator-dependent infants who were receiving enteral feedings. There was a wide range of lactose levels (0 to 3,270 nmol lactose/ml tracheal aspirate). Six infants had samples that were highly suggestive of aspiration (greater than 200 nmol lactose/ml tracheal aspirate). Twenty infants had questionably positive samples (25 to 200 nmol lactose/ml tracheal aspirate), and 16 infants had samples that were considered negative for aspiration (less than 25 nmol lactose/ml tracheal aspirate).

A proposed mechanism of normal intestinal lactase decline in the postweaned mammal

Abstract Intestinal lactase declines to low levels in the postweaned mammal as an adaptation to normal termination of lactose ingestion. Although a well known phenomenon and presumably the basis of milk intolerance among most human adults, the mechanism of this decline has remained obscure. Evidence is presented to support a proposal that temporally related cytokinetic changes, which effect a generally corresponding reduction in enterocyte life-span, serve as the causal basis of lactase decline in the postweaned mammal.

The nature of maturational decline of intestinal lactase activity.

We have examined the nature of the decline of lactase (EC 3.2.1.23) activity in the maturing rat intestine. It was established in an initial study that the activity decline reflected a proportional reduction in the concentration of the enzyme protein. Accumulation patterns of label into lactase, total intestinal proteins and sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10) were compared, 4 h following administration of a tracer dose of [3H]leucine to weanling rats exhibiting a wide range of lactase decline. Accumulation of increasing amounts of label in total intestinal proteins and sucrase-isomaltase pools was found to accompany the lactase decline, in contrast to accumulation of a constant amount of label in the declining lactase pools. The pattern of increased label accumulation in total intestinal proteins was shown in a corollary study to reflect a corresponding acceleration of total protein synthesis. On this basis, the finding of a constant amount of label in the declining lactase pools suggested a constant synthesis of lactase. We proposed earlier that associated reductions in enterocyte life-span (leading to correspondingly less lactase accumulation) rather than suppressed synthesis may provide the primary causal basis of lactase decline in the postweaned mammal.

Enzymes of the human erythrocyte. V. Pentose phosphate isomerase, purification and properties

Abstract Pentose phosphate isomerase has been prepared from the human erythrocyte in a highly purified form. The procedures, which are described in detail, essentially involved chromatography on cellulose columns to remove hemoglobin, followed by ammonium sulfate, heat, and calcium phosphate gel fractionations. Based upon the analyses of its reaction products, the purified enzyme preparation was found to contain only a negligible amount of epimerase activity and to be free of transketolase, phosphoribomutase, phosphatase, and hexose phosphate isomerase activities.

Concentrative accumulation (active transport) of 2-deoxy-D-glucose in primate fibroblasts

Incorporation of 2-deoxy-D-glucose into cultured rhesus diploid cells includes transport and subsequent phosphorylation with resultant accumulation of both free and phosphorylated sugar. Accumulation of the free sugar proceeds to a maximal limit of 4–5 mM which is determined by intrinsic cell factors and is independent of medium 2-deoxy-D-glucose up to 5 mM concentration. Concentrative accumulation (active transport) pf the free sugar is readily demonstrable on maintaining medium concentrations of 2-deoxy-D-glucose at less than the maximal accumulation potential of the cells (i.e. <4–5 mM). Cellular concentrations of the free sugar in excess of 30-times medium concentrations are demonstrable on incubation of the cells in the presence of <0.05 mM 2-deoxy-D-glucose. In contrast, accumulation of 2-deoxy-D-glycose is not demonstrable in the human erythrocyte.

Relationship of solute permeability to erythrocyte glycolysis

Abstract Erythrocyte suspensions demonstrate remarkable changes in glycolytic rate in response to the permeability characteristics of the solutes present. Erythrocytes when placed in electrolyte or nonelectrolyte media of limited permeability produce lactate at accelerated rates, attributable to a stimulation of ATP degradation. The mechanism(s) of increased turnover of ATP and the purpose to which the excess energy is directed remain to be clarified.