Binding of the Factor IX γ-Carboxyglutamic Acid Domain to the Vitamin K-dependent γ-Glutamyl Carboxylase Active Site Induces an Allosteric Effect That May Ensure Processive Carboxylation and Regulate the Release of Carboxylated Product

Propeptides of the vitamin K-dependent proteins bind to an exosite on gamma-glutamyl carboxylase; while they are bound, multiple glutamic acids in the gamma-carboxyglutamic acid (Gla) domain are carboxylated. The role of the propeptides has been studied extensively; however, the role of the Gla domain in substrate binding is less well understood. We used kinetic and fluorescence techniques to investigate the interactions of the carboxylase with a substrate containing the propeptide and Gla domain of factor IX (FIXproGla41). In addition, we characterized the effect of the Gla domain and carboxylation on propeptide and substrate binding. For the propeptide of factor IX (proFIX18), FIXproGla41, and carboxylated FIXproGla41, the Kd values were 50, 2.5, and 19.7 nM and the koff values were 273 x 10(-5), 9 x 10(-5), and 37 x 10(-5) s(-1), respectively. The koff of proFIX18 is reduced 3-fold by FLEEL and 9-fold by the Gla domain (residues 1-46) of FIX. The pre-steady state rate constants for carboxylation of FIXproGla41 was 0.02 s(-1) in enzyme excess and 0.016 s(-1) in substrate excess. The steady state rate in substrate excess is 4.5 x 10(-4) s(-1). These results demonstrate the following. 1) The pre-steady state carboxylation rate constant of FIXproGla41 is significantly slower than that of FLEEL. 2) The Gla domain plays an allosteric role in substrate-enzyme interactions. 3) Carboxylation reduces the allosteric effect. 4) The similarity between the steady state carboxylation rate constant and product dissociation rate constant suggests that product release is rate-limiting. 5) The increased dissociation rate after carboxylation contributes to the release of product

OPTIMAL INBOUND/OUTBOUND PRICING MODEL FOR REMANUFACTURING IN A CLOSED-LOOP SUPPLY CHAIN

The paper presents a model for optimizing inbound and outbound pricing for closed-loop supply chains that remanufacture reusable products. Remanufacturers create reusable products from returned used products and sell the products “as new” to manufacturers or consumers. By implementing a return subsidy, remanufacturers can encourage the consumer to return used products. Demand for the as-new components often depends on the selling price and inventory. The available inventory increases as the subsidy increases and as the price decreases. Our model can determine the optimal subsidy and selling price for used and remanufactured products, respectively. Our model uses the Karush–Kuhn–Tucker conditions to solve its nonlinear problem. Sensitivity analysis reveals how different parameters affect profit under model-optimized conditions

The Propeptides of the Vitamin K-dependent Proteins Possess Different Affinities for the Vitamin K-dependent Carboxylase

The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the modification of specific glutamates in a number of proteins required for blood coagulation and associated with bone and calcium homeostasis. All known vitamin K-dependent proteins possess a conserved eighteen-amino acid propeptide sequence that is the primary binding site for the carboxylase. We compared the relative affinities of synthetic propeptides of nine human vitamin K-dependent proteins by determining the inhibition constants (Ki) toward a factor IX propeptide/gamma-carboxyglutamic acid domain substrate. The Ki values for six of the propeptides (factor X, matrix Gla protein, factor VII, factor IX, PRGP1, and protein S) were between 2-35 nM, with the factor X propeptide having the tightest affinity. In contrast, the inhibition constants for the propeptides of prothrombin and protein C are approximately 100-fold weaker than the factor X propeptide. The propeptide of bone Gla protein demonstrates severely impaired carboxylase binding with an inhibition constant of at least 200,000-fold weaker than the factor X propeptide. This study demonstrates that the affinities of the propeptides of the vitamin K-dependent proteins vary over a considerable range; this may have important physiological consequences in the levels of vitamin K-dependent proteins and the biochemical mechanism by which these substrates are modified by the carboxylase

The Putative Vitamin K-dependent γ-Glutamyl Carboxylase Internal Propeptide Appears to Be the Propeptide Binding Site

The vitamin K-dependent gamma-glutamyl carboxylase binds an 18-amino acid sequence usually attached as a propeptide to its substrates. Price and Williamson (Protein Sci. (1993) 2, 1997-1998) noticed that residues 495-513 of the carboxylase shares similarity with the propeptide. They suggested that this internal propeptide could bind intramolecularly to the propeptide binding site of carboxylase, thereby preventing carboxylation of substrates lacking a propeptide recognition sequence. To test Price's hypothesis, we created nine mutant enzyme species that have single or double mutations within this putative internal propeptide. The apparent K(d) values of these mutant enzymes for human factor IX propeptide varied from 0.5- to 287-fold when compared with that of wild type enzyme. These results are consistent with the internal propeptide hypothesis but could also be explained by these residues participating in propeptide binding site per se. To distinguish between the two alternative hypotheses, we measured the dissociation rates of propeptides from each of the mutant enzymes. Changes in an internal propeptide should not affect the dissociation rates, but changes to a propeptide binding site may affect the dissociation rate. We found that dissociation rates varied in a manner consistent with the apparent K(d) values measured above. Furthermore, kinetic studies using propeptide-containing substrates demonstrated a correlation between the affinity for propeptide and V(max). Taken together, our results indicated that these mutations affected the propeptide binding site rather than a competitive inhibitory internal propeptide sequence. These results agree with our previous observations, indicating that residues in this region are involved in propeptide binding

Effect of Vitamin K-dependent Protein Precursor Propeptide, Vitamin K Hydroquinone, and Glutamate Substrate Binding on the Structure and Function of γ-Glutamyl Carboxylase

The γ-glutamyl carboxylase utilizes four substrates to catalyze carboxylation of certain glutamic acid residues in vitamin K-dependent proteins. How the enzyme brings the substrates together to promote catalysis is an important question in understanding the structure and function of this enzyme. The propeptide is the primary binding site of the vitamin K-dependent proteins to carboxylase. It is also an effector of carboxylase activity. We tested the hypothesis that binding of substrates causes changes to the carboxylase and in turn to the substrate-enzyme interactions. In addition we investigated how the sequences of the propeptides affected the substrate-enzyme interaction. To study these questions we employed fluorescently labeled propeptides to measure affinity for the carboxylase. We also measured the ability of several propeptides to increase carboxylase catalytic activity. Finally we determined the effect of substrates: vitamin K hydroquinone, the pentapeptide FLEEL, and NaHCO3, on the stability of the propeptide-carboxylase complexes. We found a wide variation in the propeptide affinities for carboxylase. In contrast, the propeptides tested had similar effects on carboxylase catalytic activity. FLEEL and vitamin K hydroquinone both stabilized the propeptide-carboxylase complex. The two together had a greater effect than either alone. We conclude that the effect of propeptide and substrates on carboxylase controls the order of substrate binding in such a way as to ensure efficient, specific carboxylation

Transmembrane segments of nascent polytopic membrane proteins control cytosol/ER targeting during membrane integration

Vastly different folded transmembrane segments of nascent multispanning membrane proteins each induce structural changes in the ribosome tunnel and translocon that target the loops of the growing polypeptide alternately into the cytosol or ER lumen

Polytopic membrane protein folding at L17 in the ribosome tunnel initiates cyclical changes at the translocon

Interaction between L17 in the ribosome tunnel and folded nascent chain transmembrane segments during multi-spanning membrane protein synthesis triggers structural rearrangements in the ribosome that cause switching between cytosolic and ER lumenal targeting of the growing polypeptide

A Study on the Deaf-Mute in Formosa, Especially on their hearing

334 students of the School for the Deaf-Mute in Taipei, Taiwan have been dealt with in the present study. 201 out of 334 or 60.2% were males, while 133 or 39.8% were females. 81 out of 334 or 22.7% were congenitally deaf, while acquired deafness was found in 220 or 67.4%. The ratio of congenital deafness to acquired deafness was not significantly different between 2 sexes. Attempts were made to determine the cause of deafness by studying questionnaires answered by the parents. Heredity was the cause in 35 or 43.2% of 81 congenitally deaf cases. Prenatal quinine intoxication and birth injuries were the causes in small number of cases. Causes were unknown in 41 or 18.6%. Febrile diseases, meningitis and measles were most prominent as the cause of acquired deafness, each accounting for 19.09%, 18.64% and 13.18% of the entire acquired deafness cases. Otitis media was the fourth in ranking. It is a remarkable fact that T. B. Meningitis together with Streptomycin intoxication caused deafness in approximately 10% of the acquired deafness cases. More than half of the acquired deafness cases had lost hearing before or at the age of 3. Of the total ears numbering 668 on which pure tone audiometry was done, 40% were totally deaf, while the ears with residual hearing were 60%. Of the total cases, 50% had residual hearing binaurally, 20% had residual hearing monaurally and the remaining 30% showed binaural total deafness. The incidence of binaural residual hearing was about equal in congenital and acquired deafness group, but binaural total deafness was more frequent in the latter group. A study on the incidence of residual hearing for each test frequency revealed the highest incidence for 500 cps., decreasing in order of 250-, 1,000-, 125-, 3,000-, 2,000-, 4,000- and 8,000 cps. in total ears as well as in acquired deafness group. The incidence of residual hearing is higher in congenital than in acquired deafness group throughout the entire frequency range, the difference being most marked over the middle tone range. The order of incidence of residual hearing for each test frequency is almost similar in both groups except that the incidence is very slightly higher at 2, 000cps. than at 3,000cps. in congenital group. Test of vestibular function was done by observing nystagmus elicited by caloric stimulation on 263 cases, i. e. 526 ears. There was no response whatsever in 40% of the total ears thus tested, 23% of congenital group and 50% of the acquired deafness group. The incidence of positive response was higher in the ears with residual hearing than in totally deaf ears. In the present series of cases, there were comparatively more students skilled in lipreading in the group of cases whose deafness started during 6 to 9 years of age than in the group of cases born deaf or whose deafness began at or before the age of 5. The difference, however, has no definite statistical significance. There were more good lip-readers in the group of cases with residual hearing for 2 or 3 speech frequencies than in the group of cases with residual hearing limited to 1 speech frequency and the cases without any hearing for all speech frequencies. In order to clarify the indications for the use of hearing aid by the present series of cases, 2 factors were considered; firstly the average value of the hearing loss at 3 and at times 2 speech frequencies, and secondly the shape of audiogram. Of the total 334 cases use of hearing aid was not indicated in 113 cases who were totally deaf at 3 speech frequencies and 70 cases with residual hearing only at 1 speech frequency; both together constituting 54.8% of total cases. The remaining 221 cases were considered to be candidates for the use of hearing aid. They were divided into the following 3 classes according to the degree of hearing loss. 1. Ideal cases regarding the use of hearing aid. Hearing loss of less than 60 db

Built environment effects on leisure travel for children: Trip generation and travel mode

This study empirically analyzed the effects of built environment on leisure travel among children. Students of three elementary schools, namely Yangmingshan, Sanyu and Shilin, all located in the Shilin District of Taipei, were chosen to provide sample data. The negative binomial regression model and multinomial logit model were used to analyze trip generation and travel mode, respectively. This study reached the following empirical findings: (1) mixed land use, employment density, walkway quality, leisure facility supply and leisure travel distance encouraged generation of leisure trips for children; (2) intersection density, building density, employment density and walkway quality encouraged a child to use transit systems or non-motorized travel modes for leisure travel; and (3) vehicle density and leisure travel distance discouraged walking and biking but encouraged the use of transit systems for leisure travel involving children. Local government can use the empirical findings of this study to develop urban planning strategies to encourage children to perform leisure activities outside the home using transit systems or non-motorized travel modes.Built environment Child Leisure travel Negative binomial regression model Multinomial logit model