Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, catalyzes the conversion of tyrosine to DOPA, Cyclic AMP-dependent protein phosphorylation conditions alter tyrosine hydroxylase activity in rat striatal homogenates. In agreement with other laboratories, we find that short-term pre-incubation (3 min) of extracts under phosphorylating conditions (Mg . ATP, cAMP) increases enzyme activity two- to tenfold over control as measured during a subsequent 15-min assay. We now report that preincubation under phosphorylating conditions for longer periods (30 min) results in a loss of activity to levels equal to or below that of the control enzyme. Addition of purified bovine brain protein kinase catalytic subunit and Mg . ATP enhances activation and increases the rate of inactivation. To demonstrate that inactivation is not associated with proteolytic degradation or irreversible denaturation, the inactivated form of the enzyme can be reactivated. The protein kinase inhibitor protein decreases the activation process and prevents inactivation of the enzyme to below control values. The sedimentation coefficient is not changed by phosphorylation conditions (S = 8.8 +/- 0.1). Although the apparent Km of the enzyme for the 6-methyltetrahydropterine (6-MPH4) cofactor is reduced (0.86 mM, control; 0.32 mM, activated), it is also reduced in the inactivated form (0.38 mM). The Ki for dopamine is increased from 4.5 microM for the control to 28 microM for the activated enzyme, whereas the inactivated form of the enzyme exhibits a Ki of 10 microM. Removal of catecholamines by gel filtration fails to alter activity and the apparent cofactor Km. Moreover, both the activated and the inactivated states persist following gel filtration. It therefore appears that the activation-inactivation process is not mediated solely by the modulation of enzyme feedback inhibition or changes in the Km for 6-MPH4. We also describe a coupled decarboxylase assay in which labeled dopamine is resolved from the precursors tyrosine and DOPA by low-voltage paper electrophoresis.

This study was undertaken to help clarify the relationship between capillary hemodynamic events and the tissue uptake of circulating immune complexes (IC). In each of 23 dogs, bovine serum albumin (BSA) and rabbit antiBSA soluble IC labeled with 125I were given by constant i.v. infusion, and IC uptake by a normally perfused kidney was compared to that of the contralateral kidney in which renal blood flow (RBF) was changed by renal artery constriction or raised ureteral pressure. In these same animals, IC uptake in 15 other major organ systems was also measured simultaneously. During IC infusion microspheres of 85Sr were injected to measure cardiac output and tissue blood flow, and red cells labeled with 51Cr were infused to mark tissue vascular volume. At completion of the IC infusion, tissue samples were taken from the kidneys and the 15 other major organs systems. From the isotope content of each tissue, we determined IC content, blood flow rate, vascular transit time, and fractional uptake of IC (FIC). In addition, glomeruli were isolated from renal cortex to assess IC uptake in glomerular versus renal nonglomerular tissue. We found that 1) for kidney, IC delivery rate, capillary hydrostatic pressure, and capillary ultrafiltration rate are less important than the plasma IC concentration in determining IC uptake; 2) for each organ studied, the principal determinant of IC uptake per gram of tissue, at any given PIC, is vascular volume per gram of tissue; 3) tissue vascular volume per gram of tissue may determine IC uptake per gram of tissue because tissue vascular volume determines the capillary surface area in contact with circulating IC or because tissue vascular volume determines tissue vascular transit time, at any given tissue blood flow rate.

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