Transient state isoelectric focusing: effects of zone load and carrier ampholyte concentration on the kinetics of defocusing and refocusing in sucrose density gradients

The kinetics of focusing, defocusing, and refocusing of l-histidyl-l-tyrosine in a sucrose density gradient have been studied utilizing a special apparatus for repetitive scanning of the isoelectric focusing column, employing uv absorption optics and a digital data acquisition system. Starting from a uniform or triangular concentration profile, the band is “focused” and a nearly linear pH gradient is formed during initial focusing. The electrical field is then abolished and free diffusion occurs. The electrical field is then reapplied (“refocusing”) and the band is allowed to sharpen, presumably reapproaching a steady state. The band width was measured quantitatively as the second moment about the mean (square of the standard deviation, σ²). In theory, measurements of σ² versus time permit the estimation of the apparent diffusion coefficient (D) and the isoelectric focusing parameter (pE). If the electrical field strength E and the pH gradient, $\text{d(}\text{pH}\text{)}\text{dx}$, were also measured, then one could calculate the slope of the pH mobility curve of the protein $\text{dM}\text{d(}\text{pH}\text{)}$ evaluated at the isoelectric point. D can be measured during the defocusing stage, and $\text{pE,}\text{D}\text{pE}\text{,}\text{or}\text{D}$ can be measured during focusing or refocusing. Several limitations and difficulties in the verification of this theory have been encountered: First, the apparent diffusion coefficient depends on zone load in approximately a linear fashion. Accordingly, it is necessary to measure D at several zone loads, and then extrapolate to zero load by linear regression techniques. Second, the ampholyte concentration has a marked effect on both D and pE. Here we have no a priori reason to extrapolate to zero ampholyte concentration. Also, at present we have no satisfactory method for measurement of E. These preliminary studies should be helpful in indicating further directions for experimental refinement and for generalization of theory.     

Pitfalls of immunogold labeling: analysis by light microscopy, transmission electron microscopy, and photoelectron microscopy

The immunogold method is widely used to localize, identify, and distinguish cellular antigens. There are, however, some pitfalls that can lead to nonspecific binding, particularly in cytoskeletal studies with gold probes prepared from small gold particles. We present a list of suggestions for minimizing nonspecific binding, with particular attention to two problems identified in this study. First, we find that the method used to prepare the colloidal gold particles affects the degree of nonspecific binding. Second, the standard BSA-stabilized small gold probes evidently possess exposed regions that bind to the proteins of cytoskeletal preparations. This was investigated in whole-mount cytoskeletal preparations of cultured cells by use of light microscopy, transmission electron microscopy, and photoelectron microscopy of silver-enhanced specimens. Gold probes were made from approximately 5-nm particles generated by reduction of HAuCl4 with three different reducing agents: white phosphorus, sodium borohydride, and citrate-tannic acid. All three preparations stabilized in the conventional way showed significant levels of nonspecific binding, which was highest with citrate-tannic acid. This problem was largely solved with all three types of probes by including fish gelatin in the probe buffer, by substituting fish gelatin for the BSA stabilizer used to prepare the probes, or by pre-adsorption methods. Application of these techniques resulted in clear immunogold labeling patterns with minimal nonspecific background.     

Nonmarket Labor Processes in an Advanced Capitalist Economy

In advanced capitalist economies, the treatment of labor as a commodity suggests that labor's cost and availability depend on market mechanisms. Although neoclassical economic thought recognizes the influence of collective bargaining and labor legislation on the cost and availability of labor, economists generally pay scant attention to the ways informal and formal relations of power, along with myths of sexuality, ethnicity, and nationality, combine within labor processes in advanced capitalist economies to influence the cost and availability of labor. This article presents data on the labor processes in the North Carolina seafood processing industry and compares them to the labor processes accompanying the annual, seasonal importation of legal alien farm labor by U.S. agricultural producers. The labor processes of the former rest on kinship and informal social relations while those of the latter rest on formal political authority. The analysis suggests that, even in advanced capitalist economies, employers faced with labor supply problems do not rely on market mechanisms but instead tap formal and informal systems of authority to assure supplies of labor, and support their behaviors with myths of sexuality, ethnicity, and nationality. The article concludes with a discussion of these behaviors in the context of post-World War II international divisions of labor.     

Labeling of bovine heart cytochrome c oxidase with analogues of phospholipids. Synthesis and reactivity of a new cardiolipin benzaldehyde probe

The syntheses of two new radioactive probes derived from cardiolipin and phosphatidylcholine are reported. These probes are derivatives of natural lipids and contain an amine-specific benzaldehyde in the head-group region. This functional group allows a choice of timing of the reaction (e.g., after equilibration and detergent removal) because an irreversible covalent bond is formed only upon the addition of reducing agent. These probes, as well as a benzaldehyde analogue of phosphatidic acid, and a water-soluble benzaldehyde reagent were covalently attached to bovine heart cytochrome c oxidase. After reconstitution into vesicles, the lipid-benzaldehyde probes selectively incorporated into the smaller polypeptides of the enzyme, while the remaining subunits (I-IV) exhibited little incorporation of label. The accessibility of amine groups labeled under the conditions used here was independent of the structural and charge differences between the benzaldehyde probes. This suggests that all three lipid probes react with polypeptides of the cytochrome c oxidase complex at general contact sites for membrane phospholipids. A water-soluble benzaldehyde reagent predominantly labeled subunits IV, Va, and Vb and polypeptides of VII-VIII. A comparison of these results facilitates a more refined view of the disposition of polypeptides of cytochrome c oxidase in respect to the lipid and aqueous phases.     

Multiple equilibria binding treatment of lipid and detergent interactions with membrane proteins. Application to cytochrome c oxidase solubilized in cholate

A modified multiple binding equilibria treatment is presented that allows determination of thermodynamic parameters of the interaction of phospholipids with integral membrane proteins solubilized in excess detergent. Lipid binding is modeled as a series of exchange reactions between lipid molecules and detergent molecules at the hydrophobic protein surface. A general equation is derived which expresses a relative association constant (K) and the total number of contact sites at the lipid-protein interface (N) in terms of experimentally measurable variables. A useful simplification of the general equation occurs when the amount of detergent is high relative to the total number of lipid binding sites in the sample. Computer simulations show that in cases we have examined there appears to be an experimentally accessible range of detergent to protein molar ratios where the approximation at high detergent is useful for analyzing experimental data. This model is used to examine the competition between cholate and spin-labeled phospholipids for the hydrophobic surfaces of bovine heart cytochrome c oxidase. We find, for example, that K = 12 +/- 2 for phosphatidylcholine relative to cholate (i.e., the cholate molecules are relatively easily displaced by membrane lipids). This helps to explain the experimental observation that cholate is an effective detergent both for solubilizing cytochrome c oxidase and for reconstituting this protein into a defined lipid bilayer environment. An excess of cholate readily displaces almost all of the native phospholipids, and the protein is dispersed in cholate micelles. However, when phospholipids are added back, the cholate molecules at the protein surface are replaced because of the higher relative binding of the phospholipids. Observed differences between the behavior of phosphatidylcholine and phosphatidylglycerol suggest that reconstitution in cholate is a selective process in which detergent molecules in localized areas on the protein surface are more readily displaced by certain phospholipids.     

Low temperature delays development of photosystem II activity in winter rye leaves

The ability of leaves to acclimate photosynthetically to low temperature was examined during leaf development in winter rye plants ( Secale cereale L. cv. Puma) grown at 20°C or at 6°C. All leaves grown at 6°C exhibit increased chlorophyll (Chl) levels per leaf area, higher rates of uncoupled, light-saturated photosystem I (PSI) electron transport, and slower increases in photosystem II (PSII) electron transport capacity, when compared with 20°C leaves. The stoiehiometry of PSI and PSII was estimated for each leaf age class by quantifying Chl in elcctrophorctic separations of Chl-protein complexes. The ratio of PSII/PSI electron transport in 20°C leaves is highly correlated with the ratio of core Chl a -proteins associated with PSII (CPa) to those associated with PSI (CP1). In contrast, PSII/PSI electron transport in 6°C leaves is not as well correlated with CPa/CP1 and is related, in part, to the amount and organization of light-harvesting Chl a/b -proteins associated with PSII. CPa/CP1 increases slowly in 6°C leaves, although the ratio of CPa/CP1 in mature 20°C and 6°C leaves is not different. The results suggest that increased PSI activity at low temperature is not related to an increase in the relative proportion of PSI and may reflect, instead, a regulatory change. Photosynthetic acclimation to low environmental temperature involves increased PSI activity in mature leaves shifted to 6°C. In leaves grown entirely at 6°C, however, acclimation includes both increased PSI activity and modifications in the rate of accumlation of PSII and in the organization of LHCII.     

Nitrogen Assimilating Enzyme Activities and Enzyme Protein during Development and Senescence of Effective and Plant Gene-Controlled Ineffective Alfalfa Nodules

Effective (N₂-fixing) alfalfa (Medicago sativa L.) and plant-controlled ineffective (non-N₂-fixing) alfalfa recessive for the in₁ gene were compared to determine the effects of the in₁ gene on nodule development, acetylene reduction activity (ARA), and nodule enzymes associated with N assimilation and disease resistance. Effective nodule ARA reached a maximum before activities of glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT), asparagine synthetase (AS), and phosphoenolpyruvate carboxylase (PEPC) peaked. Ineffective nodule ARA was only 5% of effective nodule ARA. Developmental profiles of GS, GOGAT, AAT, and PEPC activities were similar for effective and ineffective nodules, but activities in ineffective nodules were lower and declined earlier. Little AS activity was detected in developing ineffective nodules. Changes in GS, GOGAT, AAT, and PEPC activities in developing and senescent effective and ineffective nodules generally paralleled amounts of immunologically detectable enzyme polypeptides. Effective nodule GS, GOGAT, AAT, AS, and PEPC activities declined after defoliation. Activities of glutamate dehydrogenase, malate dehydrogenase, phenylalanine ammonia lyase, and caffeic acid-o-methyltransferase were unrelated to nodule effectiveness. Maximum expression of nodule N-assimilating enzymes appeared to require the continued presence of a product associated with effective bacteroids that was lacking in in₁ effective nodules.     

NG-methylarginine, an inhibitor of endothelium-derived nitric oxide synthesis, is a potent pressor agent in the guinea pig: does nitric oxide regulate blood pressure in vivo?

Nitric oxide is a major endothelium-derived vascular smooth muscle relaxing factor; its synthesis from L-arginine is selectively inhibited by L-NG-methylarginine. To assess whether basal nitric oxide release contributes to blood pressure regulation in vivo, we have investigated the cardiovascular effects of L-NG-methylarginine in the anesthetized guinea pig. L-NG-methylarginine (0.1-10 mg/kg, i.v. bolus) elicited a sustained, dose-dependent, increase in arterial pressure and a moderate bradycardia. L-arginine (30 mg/kg i.v.) prevented or reversed the pressor effect of L-NG-methylarginine, while atropine (2 mg/kg) abolished the associated bradycardia. In contrast, L-arginine did not attenuate the pressor effect of norepinephrine or angiotensin. Our findings suggest that basal nitric oxide production is sufficient to modulate peripheral vascular resistance; hence nitric oxide may play a role in arterial pressure homeostasis.     

In vivo and in vitro phosphorylation of murine lymphocyte differentiation antigen CD5

Ly-1, the murine lymphocyte differentiation antigen CD5, is phosphorylated constitutively in vivo. This phosphorylation is enhanced by phorbol 12-myristate 13-acetate (PMA) treatment, but not by concanavalin A, Ca2+ ionophore or dibutyryl cAMP. Prolonged PMA treatment abolished PMA-induced Ly-1 phosphorylation but not constitutive phosphorylation, suggesting that protein kinase C (PKC) is responsible for this enhanced phosphorylation, but not the basal phosphorylation of Ly-1. Ly-1 is phosphorylated by PKC added to membranes, further supporting a role for protein kinase C in the in vivo phosphorylation of Ly-1.