Paul J. Geiger

Protein determination by Lowry's method in the presence of sulfhydryl reagents

Abstract Interference with the Lowry protein determination by sulfhydryl compounds cysteine, DTT, 2-mercaptoethanol, and reduced glutathione has been largely removed by adding H 2 O 2 to the alkaline copper solution containing the protein followed by heating for 10 min at 50° before adding phenol reagent. The destruction of sulfhydryl can also be carried out at room temperature but blanks are somewhat higher than those obtained after heating. In the concentration employed here, H 2 O 2 does not interfere with the development of color by the phenol reagent. Sensitivity of the determination appears to be enhanced in the presence of DTT. Detailed directions are given for estimating proteins in the presence of sulfhydryl compounds.

Compartmentation of hexokinase and creatine phosphokinase, cellular regulation, and insulin action.

Publisher Summary This chapter focuses on two enzymes, hexokinase (HK) and creatine phosphokinase (CPK), and their compartmentation in association with mitochondria. It discusses how hexokinase might be influenced by insulin in the creation or maintenance of a compartment affecting cellular metabolism in a significant way. In the acceptor theory of insulin action, the energy is supplied by enhanced mitochondrial activity. This model is based on the proposition that insulin induces a coupling of hexokinase to an energy-generating site in the mitochondrion. This attachment creates a “compartment” in which the efficiency of two processes, specifically the transfer of adenosine triphosphate (ATP) to hexokinase and the return of adenosine diphosphate (ADP) to the oxidative phosphorylation site, is increased.

Formation of creatine phosphate from creatine and 32P-labelled ATP by isolated rabbit heart mitochondria.

Abstract With ATP [γ- 32 P] we have demonstrated directly that mitochondrial creatine phosphokinase catalyzes the formation of large amounts of creatine phosphate with mitochondria generated ATP as substrate rather than added extramitochondrial ATP.

Intimate coupling of creatine phosphokinase and myofibrillar adenosinetriphosphatase

Abstract ATPase and creatine phosphokinase (CPK) activities of isolated cardiac myofibrils were determined with 32 P γ-labeled ATP alone and with the addition of phosphorylcreatine (PC). With ATP and PC as substrates the label in the inorganic phosphate formed is greatly diluted indicating that the ATP formed by PC through CPK can reach the ATPase active site more readily than labeled ATP from the medium. The tight coupling of the ATPase and CPK activities further strengthens our view that PC serves an important role as high energy carrier between the energy producing sites (mitochondria) and the energy utilizing sites (myofibrils).

Separation and automated analysis of phosphorylated metabolic intermediates

Abstract A chromatographic procedure has been described using a new automated detection system that allows separation at the 10 nmole level of all the glycolytic intermediates as well as AMP, ADP, and ATP in pure samples and samples of skeletal muscle and blood. Separations are carried out on 3 × 500 mm columns packed with modern anion exchange resins of closely sized, fine particles and a linear gradient of ammonium chloride containing borate in order to complex the sugar phosphates. Pressures are moderate, and elutions are complete within 5–8 hr with excellent reproducibility and recovery of each compound. Screening runs can be made in only 90 min with shorter columns and with some sacrifice in resolution for certain compounds.

Isozymes of Phenylalanine Hydroxylase

Three isozymes of phenylalanine hydroxylase exist in adult rat liver. They are chromatographically unique. Partial chracterization suggests that they are similar in chemical properties and differ only in charge. Estimation of the Stokes radii indicates that the isozymes have similar molecular weights of about 200,000. Two isozymes exist in human fetal liver. Alterations of the relative amounts of these isozymes may control the phenotype of the disease phenylketonuria.

The intracellular site of action of insulin: the mitochondrial Krebs cycle.

Publisher Summary This chapter discusses the intracellular site of action of insulin involving the mitochondrial Krebs cycle. To begin with, there is at present no coherent model that accounts, through the process of partial phosphorylation or dephosphorylation of enzymes or other proteins, for all of the manifold actions of insulin on cells. It is known that insulin stimulates the uptake of glucose most effectively in fat cells in which mitochondria are close to the plasma membrane. Also, much evidence has accumulated to show that insulin reverses the net catabolism of protein, glycogen, and fat and promotes their build up and storage. Furthermore, insulin stimulates the incorporation into protein of primarily those pyruvate carbons that are converted to glutamic acid through the Krebs cycle. Insulin has a far greater stimulatory effect on the incorporation into protein of the methylene carbons of succinate than the carboxyl carbons. This can be attributed to the fact that carbons 2 and 3 of succinate either reenter the cycle as acetyl-CoA or remain in the mitochondrial Krebs cycle for a second or third opportunity to be converted to glutamate by the transamination of α-KG.

Prevalence of Corynebacteria in Diabetic Foot Infections

OBJECTIVE Microbiological flora of diabetic foot infections are usually polymicrobial and frequently include bacteria of the Corynebacterium sp. (diphtheroids). The purpose of this study was to determine the prevalence of these bacteria in both deep and superficial cultures in diabetic patients with foot infections. RESEARCH DESIGN AND METHODS The charts of 50 patients of successive admissions to the Orthopedic-Diabetes Service at our hospital were reviewed to obtain the following data: age, sex, ethnic origin, method of treatment of diabetes, blood glucose level, prior antibiotics, and reports of cultures taken from bedside and intraoperative sites. Data were analyzed to compare the prevalence of diphtheroids in reliable versus nonreliable cultures and the influence of other parameters on the presence of these organisms. RESULTS Fourteen of 19 (74%) of the intraoperative specimens grew diphtheroids compared with 25 of 65 (39%) of the bedside cultures, a highly significant difference. In addition, there was a somewhat greater occurrence of diphtheroids in women compared with men. The likelihood that contamination is the cause for the presence of diphtheroids is highly unlikely, because one arm of the study included cultures derived from deep tissue at the time of the surgical procedure (i.e., the intraoperative cultures). Cultures always grew at least one other organism in addition to the diphtheroid. CONCLUSIONS Corynebacteria, commonly known as diphtheroids, are present as a part of the polymicrobial flora in a large percentage of diabetic patients with foot infections. Because the diphtheroids were identified in culture material taken in the operating room or at the time of incision and drainage in a higher percentage of patients than in specimens from superficial cultures, it is highly unlikely that they are contaminants.

Acid-soluble precursors and derivatives of phospholipids increase after stimulation of quiescent swiss 3T3 mouse fibroblasts with serum

Abstract Automated phosphate analysis of acid-soluble pools of phosphate esters was employed to reveal possible biochemical changes during the transition of Swiss 3T3 mouse fibroblasts from quiescence to active replication of DNA. After 12 hours of stimulation with 10% fetal bovine serum the most notable were 3-fold increases in pools of phospholipid precursors and derivatives. These included glycerophosphocholine, glycerophosphoethanolamine, phosphocholine and phosphoethanolamine. Concurrent but less dramatic increases in pools of ATP, CTP and fructose 1,6-diphosphate were also obtained.

Kinetic properties and functional role of creatine phosphokinase in glycerinated muscle fibers--further evidence for compartmentation.

The following phenomena were observed when relative contraction and relaxation effects of ATP and creatine phosphate (CP) were studied in rabbit psoas muscle glycerinated fiber bundles containing native creatine phosphokinase (CPK) and ATPase activities: (1) nucleotide was absolutely necessary for contraction; (2) in the presence of a small amount of ADP (250 microM), physiological concentration of CP (10 mM) produced faster and stronger contraction and faster, more complete, relaxation than equimolar or higher concentrations of ATP; (3) if the nucleotide was in the form of ATP, the nucleotide Km for contraction was about 1.5 mM; (4) if the nucleotide was in the form of ADP, the nucleotide Km for contraction at physiological concentration of CP (10 mM) was 0.076 to 1.18 mM depending upon the order of addition of ADP and CP; (5) the apparent Km for CP for contraction was 2.67 mM independent of sequence of addition of ADP and CP.